Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Evolution of Trypanosoma cruzi: clarifying hybridisations,mitochondrial introgressions and phylogenetic relationships between major lineages

Several different models of Trypanosoma cruzi evolution have been proposed. These models suggest that scarce events of genetic exchange occurred during the evolutionary history of this parasite. In addition, the debate has focused on the existence of one or two hybridisation events during the evolution of T. cruzi lineages. Here, we reviewed the literature and analysed available sequence data to clarify the phylogenetic relationships among these different lineages. We observed that TcI, TcIII and TcIV form a monophyletic group and that TcIII and TcIV are not, as previously suggested, TcI-TcII hybrids. Particularly, TcI and TcIII are sister groups that diverged around the same time that a widely distributed TcIV split into two clades (TcIV_S and TcIV_N). In addition, we collected evidence that TcIII received TcIV_S kDNA by introgression on several occasions. Different demographic hypotheses (surfing and asymmetrical introgression) may explain the origin and expansion of the TcIII group. Considering these hypotheses, genetic exchange should have been relatively frequent between TcIII and TcIV_S in the geographic area in which their distributions overlapped. In addition, our results support the hypothesis that two independent hybridisation events gave rise to TcV and TcVI. Consequently, TcIV_S kDNA was first transferred to TcIII and later to TcV and TcVI in TcII/TcIII hybridisation events.     

Single-nucleotide polymorphisms of the Trypanosoma cruzi MSH2 gene support the existence of three phylogenetic lineages presenting differences in mismatch-repair efficiency

We have identified single-nucleotide polymorphisms (SNPs) in the mismatch-repair gene TcMSH2 from Trypanosoma cruzi. Phylogenetic inferences based on the SNPs, confirmed by RFLP analysis of 32 strains, showed three distinct haplogroups, denominated A, B, and C. Haplogroups A and C presented strong identity with the previously described T. cruzi lineages I and II, respectively. A third haplogroup (B) was composed of strains presenting hybrid characteristics. All strains from a haplogroup encoded the same specific protein isoform, called, respectively, TcMHS2a, TcMHS2b, and TcMHS2c. The classification into haplogroups A, B, and C correlated with variation in the efficiency of mismatch repair in these cells. When microsatellite loci of strains representative of each haplogroup were analyzed after being cultured in the presence of hydrogen peroxide, new microsatellite alleles were definitely seen in haplogroups B and C, while no evidence of microsatellite instability was found in haplogroup A. Also, cells from haplogroups B and C were considerably more resistant to cisplatin treatment, a characteristic known to be conferred by deficiency of mismatch repair in eukaryotic cells. Altogether, our data suggest that strains belonging to haplogroups B and C may have decreased mismatch-repair ability when compared with strains assigned to the haplogroup A lineage.     

Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis

Genetic characterisation of Trypanosoma cruzi variants is of foremost importance, due to considerable genetic and biological heterogeneity in the parasite populations. Two major phylogenetic lineages, each highly heterogeneous, have been previously described within this species. Here we characterised a geographically and ecologically diverse sample of stocks representative of the breadth of the known clonal diversity of each major lineage, using random amplified polymorphic DNA with 20 primers and multilocus enzyme electrophoresis at 22 loci. Molecular hybridisation experiments were performed to control the homology of randomly amplified DNA markers. Both sets of data were highly consistent and supported the existence of two major lineages. Additionally, we found that lineage 2 appeared further partitioned into five sharply delineated phylogenetic clusters, each comprising one of the following reference strains: CanIII cl1 (Z3 reference), M5631 cl5, Esmeraldo cl3 (Z2 reference), CL Brener, and MN cl2. The two first clusters were found mainly in sylvatic environments, whereas the three latter were restricted to domestic transmission cycles and were only collected South to the Amazon Basin. In contrast, lineage 1, which included Miles' Z1 reference strain X10 cl1, was not further subdivided and was encountered across the entire endemic area, in both domestic and sylvatic cycles. Thus, T. cruzi appeared to be subdivided into six discrete typing units, or DTUs, exhibiting distinct geographic and ecological ranges. Reliable diagnostic markers for the two major lineages and the five smaller DTUs of lineage 2 are described, and correspondence with previous classifications of T. cruzi genotypes is given in order to help communication on T. cruzi phylogenetic diversity.     

Inference of phylogenetic relationships among key angiosperm lineages using a compatibility method on a molecular data set

Phylogenetic relationships among the five key angiosperm lineages,Ceratophyllum,Chloranthaceae,eudicots,magnoliids,and monocots,have resisted resolution despite several large-scale analyses sampling taxa and characters extensively and using various analytical methods.Meanwhile,compatibility methods,which were explored together with parsimony and likelihood methods during the early development stage of phylogenetics.have been greatly under-appreciated and not been used to analyze the massive amount of sequence data to reconstruct thye basal angiosperm phylogeny.In this study,we used a compatibility method on a data set of eight genes (mitochondrial atp1,matR,and nad5,plastid atpB,marK,rbcL,and rpoC2,and nuclear 18S rDNA)gathered in an earlier study.We selected two sets of characters that are compatible with more of the other characters than a random character would be with at probabilities of pM＜0.1 and p＜0.5 respectively.The resulting data matrices were subjected to parsimony and likelihood bootstrap analyses.Our unrooted parsimony analyses showed that Ceratophyllum was immediately related to eudicots,this larger lineage was immediately related to magnoliids,and monocots were closely related to Chloranthaceae.All these relationships received 76%-96% bootstrap support.A likelihood analysis of the 8 gene pM＜0.5 compatible site matrix recovered the same topology but with low support.Likelihood analyses of other compatible site matrices produced different topologies that were all weakly supported.The topology reconstructed in the parsimony analyses agrees with the one recovered in the previous study using both parsimony and likelihood methods when no character was eliminated.Parts of this topology have also been recovered in several earlier studies.Hence,this topology plausibly reflects the true relationships among the five key angiosperm lineages.     

Toward a comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l. (Lamiales)

Acanthaceae (Asteridae; Lamiales) include ～4000 species and encompass a range of morphological diversity, habitats, and biogeographic patterns. Although they are important components of tropical and subtropical habitats worldwide, inadequate knowledge of the family's phylogenetic framework has impeded comparative research. In this study, we sampled all known lineages of Acanthaceae including Andrographideae. Also included were eight of 13 genera whose relationships remain enigmatic. We used sequence data from nrITS and four chloroplast noncoding regions, and parsimony and Bayesian methods of analysis. Results strongly support most aspects of relationships including inclusion of Avicennia in Acanthaceae. Excepting Neuracanthus, newly sampled taxa are placed with strong support; Kudoacanthus is in Justicieae, Tetramerium lineage, and the remaining enigmatic genera are in Whitfieldieae or Barlerieae, and Andrographideae are sister to Barlerieae. This last result is unanticipated, but placement of Andrographideae based on structural characters has been elusive. Neuracanthus is monophyletic but placement relative to (Whitfieldieae (Andrographideae + Barlerieae)) is weakly supported. Many clades have clear morphological synapomorphies, but nonmolecular evidence for some remains elusive. Results suggest an Old World origin with multiple dispersal events to the New World. This study informs future work by clarifying sampling strategy and identifying aspects of relationships that require further study.     

Trypanosoma cruzi: subtractive hybridization as a molecular strategy to generate new targets to distinguish groups and hybrids

RAPD analysis and sequences of the mini-exon and ribosomal genes show that Trypanosoma cruzi can be clustered into two phylogenetic groups-T. cruzi I and II. Herein, the Representational Difference Analysis (RDA) method was used, providing new targets specific for each group. After three rounds of RDA hybridizing F strain (tester) with Y strain (driver) and vice-versa, an F-specific (F#30) and Y-specific (Y#22) clone were obtained specifically recognizing isolates from Amazonas (T. cruzi I) and Piauí (T. cruzi II). These segments corresponded to an unspecified protein (F#30) and a trans-sialidase (Y#22). Analysis of the F#30 sequence in T. cruzi I, T. cruzi II and zymodeme 3 samples displayed negligible specific differences that distinguished each group. In addition this F#30 gene has great potential as a hybrid marker.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Risk of Trypanosoma cruzi infection among travellers visiting friends and relatives to continental Latin America

BackgroundChagas disease (CD) is regarded as a possible risk for travellers to endemic areas of continental Latin America (LA). The aim of the study is to determine the risk of Trypanosoma cruzi (TC) infection among travellers to CD endemic areas and to identify risk factors for acquiring TC infection.Methods/Principal findingWe designed a multicenter cross-sectional study among travellers in Spain (Badalona, Barcelona and Madrid). All available adults with laboratory confirmed proof of absence of TC infection from January 2012 to December 2015 were contacted. Participants referring a trip to LA after the negative TC screening were offered to participate. We performed a standardized questionnaire of travel related factors and measurement of TC antibodies in serum. A total of 971 participants with baseline negative TC serology were selected from the microbiology records. After excluding participants not meeting inclusion criteria, eighty participants were selected. Sixty three (78.8%) were female, and the median age was 38 (IQR 34–47) years. The reason to travel was visiting friends and relatives in 98.8% of the participants. The median duration of travel was 40 (IQR 30–60) days, with 4911 participants-day of exposure. Seventy seven cases (96.25%) participants had two negative TC serology tests after the travel, two cases (2.5%) had discordant serology results (considered false positive results) and one case was infected before travelling to LA. According to our data, the upper limit of the 95% confidence interval of the incidence rate of TC acquisition in travellers is 0.8 per 1000 participant-days.Conclusions/SignificanceAmong 79 non-CD travellers to TC endemic areas, we found no cases of newly acquired TC infection. The incidence rate of TC acquisition in travellers to endemic countries is less than or equal to 0.8 per 1000 traveller-days.     

Lack of correlation between in vitro susceptibility to Benznidazole and phylogenetic diversity of Trypanosoma cruzi, the agent of Chagas disease

Chagas disease remains an important health problem in Central and South America. Nitroimidazole derivative drugs like Benznidazole are commonly used to treat Trypanosoma cruzi infection. Natural variation of drug susceptibility between various T. cruzi stocks has been proposed as a possible explanation of treatment failure. Thus, the aim of this work was to determine potential correlations between in vitro Benznidazole susceptibility of different T. cruzi stocks and their genetic diversity. For this purpose, 16 natural stocks representing the overall genetic diversity of the parasite were analysed. Genetic characterisation was assessed by both random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) analyses. Drug activity was determined by two complementary methods, the MTT-PMS micro-method and FACs analysis. The 50% inhibitory concentrations (IC(50)s) were determined. Important variation of IC(50) values (7.3-16.9 microM) among stocks belonging to different discrete typing units (DTUs) was recorded. Further, correlation analysis showed that natural susceptibility to Benznidazole in T. cruzi expressed as IC(50) level was not related with its genetic structure represented by the different DTUs. These results are discussed in relation with the proposed hypothesis establishing a link between genetic diversity and biological behaviour in T. cruzi.     

Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support

Four New World genera of dwarf boas (Exiliboa, Trachyboa, Tropidophis, and Ungaliophis) have been placed by many systematists in a single group (traditionally called Tropidophiidae). However, the monophyly of this group has been questioned in several studies. Moreover, the overall relationships among basal snake lineages, including the placement of the dwarf boas, are poorly understood. We obtained mtDNA sequence data for 12S, 16S, and intervening tRNA-val genes from 23 species of snakes representing most major snake lineages, including all four genera of New World dwarf boas. We then examined the phylogenetic position of these species by estimating the phylogeny of the basal snakes. Our phylogenetic analysis suggests that New World dwarf boas are not monophyletic. Instead, we find Exiliboa and Ungaliophis to be most closely related to sand boas (Erycinae), boas (Boinae), and advanced snakes (Caenophidea), whereas Tropidophis and Trachyboa form an independent clade that separated relatively early in snake radiation. Our estimate of snake phylogeny differs significantly in other ways from some previous estimates of snake phylogeny. For instance, pythons do not cluster with boas and sand boas, but instead show a strong relationship with Loxocemus and Xenopeltis. Additionally, uropeltids cluster strongly with Cylindrophis, and together are embedded in what has previously been considered the macrostomatan radiation. These relationships are supported by both bootstrapping (parametric and nonparametric approaches) and Bayesian analysis, although Bayesian support values are consistently higher than those obtained from nonparametric bootstrapping. Simulations show that Bayesian support values represent much better estimates of phylogenetic accuracy than do nonparametric bootstrap support values, at least under the conditions of our study.     

Epitope mapping of a single repetitive unit of the B13 Trypanosoma cruzi antigen as fusions to Escherichia coli LamB protein

B13, one of the immunodominant antigens of Trypanosoma cruzi, is composed of repeats of a 12-amino-acid motif. Using synthetic peptides, the sequence FGQAAAGDK was previously shown to contain the B13 immunodominant epitope recognized by chagasic patients sera. To investigate the effects of neighboring sequences in the immunodominance, we tested serum recognition of two B13 sequences fused to LamB. GDKPSPFGQAAA-LamB and FGQAAAGDKPSP-LamB were recognized, respectively, by 15% and 80% of 80 sera reactive to B13 antigen. Recognition of FGQAAAGDKPSP-LamB was inhibited by AAAGDK-containing synthetic peptides. FGQAAAGDKPSP-LamB competed with a B13 recombinant protein containing 16.6 repeats for binding to chagasic antibodies. These results strengthen previous conclusions on the immunodominant epitope of B13 and provide a comparison of two methods for epitope mapping.     

Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States.

Thirty U.S. Trypanosoma cruzi stocks isolated mainly from wild mammals were characterized by multilocus enzyme electrophoresis at 22 genetic loci and random amplification of polymorphic DNA for 10 primers. Two main phylogenetic clusters, separated by large genetic distances, were discriminated by both methods, corresponding, respectively, to the formerly described zymodemes I and III. Two stocks isolated from indigenous human cases were identified as zymodeme I. Genetic diversity of the U.S. T. cruzi isolates was considerable, comparable to that scored in similarly sized samples from South America. These results favor the hypothesis that T. cruzi U.S. stocks were not imported at a historical time and are indigenous to the native fauna of the United States. The population structure of these stocks appeared to be basically clonal, as previously reported in South America, and no evidence of hybrid genotypes was found in the United States.     

Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes

A rabbit antibody to the neuraminidase of the infective form of Trypanosoma cruzi identifies a subpopulation of trypomastigotes that expresses neuraminidase. Complement-mediated lysis by the antibody selectively destroys 30 to 40% of the trypomastigotes, supporting the conclusion that the immune antibody binds to a subset of parasites. The trypomastigotes that react with the immune antibody are the only ones expressing neuraminidase because the trypomastigotes that survive complement-mediated lysis are depleted of neuraminidase activity. The enzyme seems to negatively modulate infection in vitro, since infection of host cells by trypomastigotes is enhanced when neuraminidase activity is blocked by antineuraminidase antibody; infection is also enhanced when the infecting trypomastigotes have been depleted of parasites that express neuraminidase. Addition of exogenous neuraminidase (from Vibrio cholerae) to trypomastigotes treated with immune antibody, reverts the enhancement observed when infection takes place in the presence of antibody to T. cruzi neuraminidase only. Addition of V. cholerae neuraminidase in the absence of immune antibodies has no effect on infection. These results show that T. cruzi neuraminidase depresses infection and also suggest that sialic acid is involved in the parasite-host cell interaction. The antibody to T. cruzi neuraminidase recognizes on the surface of live trypomastigotes a set of proteins with high m.w. (165,000 to 200,000) and also two antigens of 79,000 to 82,000. The high m.w. proteins appear to be associated with neuraminidase activity as shown by renaturation experiments of released enzyme fractionated on a sodium dodecyl sulfate-polyacrylamide gel.     

Long-term impact of a ten-year intervention program on human and canine Trypanosoma cruzi infection in the Argentine Chaco

BackgroundInterruption of domestic vector-borne transmission of Trypanosoma cruzi is still an unmet goal in several American countries. In 2007 we launched a long-term intervention program aimed to suppress house infestation with the main domestic vector in southern South America (Triatoma infestans) and domestic transmission in Pampa del Indio, a resource-constrained, hyperendemic municipality with 1446 rural houses inhabited by Creole and indigenous people, in the Argentine Chaco ecoregion. Here, we assessed whether the 10-year insecticide-based program combined with community mobilization blocked vector-borne domestic transmission of T. cruzi to humans and dogs.MethodsWe carried out two municipality-wide, cross-sectional serosurveys of humans and dogs (considered sentinel animals) during 2016–2017 to compare with baseline data. We used a risk-stratified random sampling design to select 273 study houses; 410 people from 180 households and 492 dogs from 151 houses were examined for antibodies to T. cruzi using at least two serological methods.ResultsThe seroprevalence of T. cruzi in children aged <16 years was 2.5% in 2017 (i.e., 4- to 11-fold lower than before interventions). The mean annual force of child infection (λ) sharply decreased from 2.18 to 0.34 per 100 person-years in 2017. One of 102 children born after interventions was seropositive for T. cruzi; he had lifetime residence in an apparently uninfested house, no outside travel history, and his mother was T. cruzi-seropositive. No incident case was detected among 114 seronegative people of all ages re-examined serologically. Dog seroprevalence was 3.05%. Among native dogs, λ in 2016 (1.21 per 100 dog-years) was 5 times lower than at program onset. Six native adult dogs born after interventions and with stable lifetime residence were T. cruzi-seropositive: three had exposure to T. infestans at their houses and one was an incident case.ConclusionsThese results support the interruption of vector-borne transmission of T. cruzi to humans in rural Pampa del Indio. Congenital transmission was the most likely source of the only seropositive child born after interventions. Residual transmission to dogs was likely related to transient infestations and other transmission routes. Sustained vector control supplemented with human chemotherapy can lead to a substantial reduction of Chagas disease transmission in the Argentine Chaco.     

Effects of human colony-stimulating factor on the uptake and destruction of a pathogenic parasite (Trypanosoma cruzi) by human neutrophils

The ability of granulocyte-macrophage colony-stimulating factor (CSF-H) to modulate human neutrophil functions was studied by using an in vitro system in which this cell type interacted with intracellular (amastigote [AMA]) forms of Trypanosoma cruzi. The presence of CSF-H during the 30-min period of neutrophil incubation with the AMA markedly enhanced parasite internalization. This effect was evidenced by significant increases in both the percentage of neutrophils incorporating AMA and the average number of AMA per 100 neutrophils with respect to mock-treated neutrophils. Pretreatment of the neutrophils with CSF-H reproduced the enhancement effect, whereas pretreatment of the AMA had no detectable consequence. The minimal neutrophil CSF-H pretreatment period required to significantly increase the number of AMA per 100 neutrophils was 20 min--suggesting that CSF-H induced time-dependent events ultimately leading to the manifestation of the noted effect--but neutrophil treatment with CSF-H for longer periods of time (up to 60 min) caused a much greater enhancement. Consistent with the notion of a regulatory action of CSF-H on neutrophils was the fact that the enhancing effect subsided gradually after removal of the factor and was no longer detectable after 16 hr. When 3H-labeled AMA were used, CSF-H-treated neutrophils released greater amounts of radiolabeled substances than mock-treated cells, indicating a stimulatory effect of CSF-H on the killing capacity of neutrophils. This was confirmed by the fact that untreated neutrophils that had internalized 3H-AMA killed the parasites at a faster rate when subsequently incubated with CSF-H. Catalase, but not superoxide dismutase, mannitol, benzoate, or histidine, inhibited neutrophil killing of the 3H-AMA whether the granulocytes had been exposed to CSF-H or not. This indicated that the cytotoxic mechanism involved the production of hydrogen peroxide in both cases, but possibly at a higher rate in the CSF-H-treated neutrophils. These results point to a regulatory effect of CSF-H on neutrophils that promotes cellular activities that might be relevant to the mechanisms of clearance of T. cruzi in vivo.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.