Ana M. Celentano

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE2 involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE2 serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE2 release per cell by CD8+; values of PGE2 release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE2 (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE2 could play a role in resistance in mice infected with K98.

TRYPANOSOMA CRUZI: EFFECT OF PARASITE SUBPOPULATION ON MURINE PREGNANCY OUTCOME

C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi–infected newborns.

Different Trypanosoma cruzi strains promote neuromyopathic damage mediated by distinct T lymphocyte subsets.

The proliferative response of CD4 and CD8 T lymphocytes obtained from C3H/HeN mice chronically infected with Trypanosoma cruzi strains that differ in virulence, tropism and immunogenicity, was assayed against skeletal muscle, sciatic nerve and spinal cord homogenates. Although both CD4 and CD8 T lymphocytes from mice infected with the RA strain strongly proliferated against the nervous system, no response against skeletal muscle antigens was detected. CD4 and CD8 T lymphocytes from mice infected with the K‐98 clone (from CA‐I strain) showed low proliferative response against all the antigens assayed. To determine whether the proliferation patterns showed correlation with T cell‐mediated neuromuscular damage, passive cell transfer studies were performed. Fifteen days after transfer of CD4 T cells from RA‐infected donors (CD4‐RA), normal syngeneic recipients displayed exclusively nervous tissue damage, such as perineural, endoneural and/or meningeal inflammatory infiltrates, with predominance of CD4 T cells. Fifteen days after transfer of CD4 T lymphocytes from mice infected with K‐98 (CD4‐K98), recipients showed inflammatory infiltrates only in skeletal muscle, where CD4 T lymphocytes and macrophages were predominant cells. Recipients of CD8 T cells from RA‐infected mice (CD8‐RA) showed lesions in both spinal cord and sciatic nerves. Higher percentages of CD8 T cells were observed in comparison with the recipients of CD4‐RA or CD4‐K98. In contrast, CD8 T cells from K‐98‐infected donors (CD8‐K98) did not induce tissue damage. These results provide evidence that mice infected with T. cruzi populations that differ in their biological characteristics show diverse immune mechanisms that may be involved in the pathogenesis of peripheral nervous system damage.

Induction of macrophage activation and opsonizing antibodies by Trypanosoma cruzi subpopulations

Summary Macrophage activation and production of opsonizing antibodies were studied in mice either infected with a lethal and reticulotropic Trypanosoma cruzi strain, RA, or with a non lethal and myotropic strain, CA‐I, as well as with a clone, K98 (derived from CA‐I), similar to the parental strain. Measurement of macrophage respiratory burst by chemiluminiscence disclosed that T. cruzi infection induced an enhancement of the respiratory burst, no matter the parasite subpopulation employed. But, while in mice surviving RA infection the respiratory burst was higher than during the acute period and parasitaemia was efficiently controlled, in mice infected with K98 enhanced respiratory burst coexisted with measurable levels of parasitaemia either at acute or chronic infection periods. Macrophage activation was also proved by enhanced trypanocidal activity in macrophages derived from mice infected with any of the parasite subpopulations. Sera from RA mice opsonized and lysed T. cruzi bloodstream forms efficiently, whereas sera from CA‐I or K98 mice neither lysed nor opsonized this parasite stage. All three subpopulations assayed here showed IgG bound to their membranes in vivo and similar capping kinetics, but only antibodies bound to RA parasites invariably triggered lysis. Therefore, the role played by macrophage activation in resistance and control of Pm levels is related to some features of each T. cruzi subpopulation, such as its capacity to invade macrophages and to elicit opsonizing antibodies.

In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations

The macrophage function was investigated in mice infected with Trypanosoma cruzi. Two subpopulations of the parasite were utilized, RA and K98. Strain RA is efficiently internalized by macrophages and is lethal for mice, and clone K98 is poorly phagocytosed by macrophages and is not lethal. Treatment with silica enhanced parasitemia and mortality in mice infected with both parasite subpopulations. Parasitemia kinetics, however, were affected only in mice infected with RA, which suggests that macrophage effector mechanisms may play a more relevant role in this experimental group than in mice infected with K98. Resistance to Salmonella typhimurium infection and bactericidal activity of macrophages depended upon the T. cruzi subpopulation utilized and the infection period. Infection with K98 induced only a trend towards enhanced resistance to bacterial challenge during both the acute and chronic phases, whereas a significantly enhanced bactericidal activity of spleen and liver phagocytes was observed. Mice acutely infected with RA showed significantly enhanced susceptibility to S. typhimurium infection and lower bactericidal activity. Mice surviving infection with this aggressive strain, however, showed significantly enhanced resistance and bactericidal activities. Mice acutely infected with the RA strain displayed a dissociation between macrophage capacities to control S. typhimurium and T. cruzi. A similar phenomenon was also observed in other parasitoses (schistosomiasis, African trypanosomiasis). This fact may be due to differences in the lethal mechanisms through which macrophages control these parasites and S. typhimurium.

Inhibition of HIV-1 Replication in Human Monocyte- Derived Macrophages by Parasite Trypanosoma cruzi

Background Cells of monocyte/macrophage lineage are one of the major targets of HIV-1 infection and serve as reservoirs for viral persistence in vivo. These cells are also the target of the protozoa Trypanosoma cruzi, the causative agent of Chagas disease, being one of the most important endemic protozoonoses in Latin America. It has been demonstrated in vitro that co-infection with other pathogens can modulate HIV replication. However, no studies at cellular level have suggested an interaction between T. cruzi and HIV-1 to date. Methodology/Principal Findings By using a fully replicative wild-type virus, our study showed that T. cruzi inhibits HIV-1 antigen production by nearly 100% (p<0.001) in monocyte-derived macrophages (MDM). In different infection schemes with luciferase-reporter VSV-G or BaL pseudotyped HIV-1 and trypomastigotes, T. cruzi induced a significant reduction of luciferase level for both pseudotypes in all the infection schemes (p<0.001), T. cruzi-HIV (>99%) being stronger than HIV-T. cruzi (∼90% for BaL and ∼85% for VSV-G) infection. In MDM with established HIV-1 infection, T. cruzi significantly inhibited luciferate activity (p<0.01). By quantifying R-U5 and U5-gag transcripts by real time PCR, our study showed the expression of both transcripts significantly diminished in the presence of trypomastigotes (p<0.05). Thus, T. cruzi inhibits viral post-integration steps, early post-entry steps and entry into MDM. Trypomastigotes also caused a ∼60-70% decrease of surface CCR5 expression on MDM. Multiplication of T. cruzi inside the MDM does not seem to be required for inhibiting HIV-1 replication since soluble factors secreted by trypomastigotes have shown similar effects. Moreover, the major parasite antigen cruzipain, which is secreted by the trypomastigote form, was able to inhibit viral production in MDM over 90% (p<0.01). Conclusions/Significance Our study showed that T. cruzi inhibits HIV-1 replication at several replication stages in macrophages, a major cell target for both pathogens.

Co-infection with Trypanosoma cruzi (Chagas' disease agent) decreases HIV-1 transcription in human placenta

Address: 1National Reference Centre for AIDS, Microbiology Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina, 2Laboratory of Parasitology, Microbiology Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina and 3Endocrinology Service, Clinic Biochemistry Department, Jose de San Martin Hospital, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina * Corresponding author

Unique pharmacological properties of serotonergic G-protein coupled receptors from cestodes

Background Cestodes are a diverse group of parasites, some of them being agents of neglected diseases. In cestodes, little is known about the functional properties of G protein coupled receptors (GPCRs) which have proved to be highly druggable targets in other organisms. Notably, serotoninergic G-protein coupled receptors (5-HT GPCRs) play major roles in key functions like movement, development and reproduction in parasites. Methodology/Principal findings Three 5-HT GPCRs from Echinococcus granulosus and Mesocestoides corti were cloned, sequenced, bioinformatically analyzed and functionally characterized. Multiple sequence alignment with other GPCRs showed the presence of seven transmembrane segments and conserved motifs but interesting differences were also observed. Phylogenetic analysis grouped these new sequences within the 5-HT7 clade of GPCRs. Molecular modeling showed a striking resemblance in the spatial localization of key residues with their mammalian counterparts. Expression analysis using available RNAseq data showed that both E. granulosus sequences are expressed in larval and adult stages. Localization studies performed in E. granulosus larvae with a fluorescent probe produced a punctiform pattern concentrated in suckers. E. granulosus and M. corti larvae showed an increase in motility in response to serotonin. Heterologous expression revealed elevated levels of cAMP production in response to 5-HT and two of the GPCRs showed extremely high sensitivity to 5-HT (picomolar range). While each of these GPCRs was activated by 5-HT, they exhibit distinct pharmacological properties (5-HT sensitivity, differential responsiveness to ligands). Conclusions/Significance These data provide the first functional report of GPCRs in parasitic cestodes. The serotoninergic GPCRs characterized here may represent novel druggable targets for antiparasitic intervention.

The Circadian System of Trypanosoma cruzi-Infected Mice

The effects of Chagas disease on the mammalian circadian system were studied in Trypanosoma cruzi-infected C57-Bl6J mice. Animals were inoculated with CAI or RA strains of T. cruzi or vehicle, parasitism confirmed by blood specimen visualization and locomotor activity rhythms analyzed by wheel-running recording. RA-strain infected mice exhibited significantly decreased amplitude of circadian rhythms, both under light–dark and constant dark conditions, probably due to motor deficiencies. CAI-treated animals showed normal locomotor activity rhythms. However, in these mice, reentrainment to a 6h phase shift of the LD cycle took significantly longer than controls, and application of 15min light pulses in DD produced smaller phase delays of the rhythms. All groups exhibited light-induced Fos expression in the suprachiasmatic nuclei. We conclude that the main effect of T. cruzi infection on the circadian system is an impairment of the motor output from the clock toward controlled rhythms, together with an effect on circadian visual sensitivity.