CD4+ CD25+ FOXP3+ Treg cells induced by rSSP4 derived from T. cruzi amastigotes increase parasitemia in an experimental Chagas disease model.

Currently, there is a considerable controversy over the participation of Treg cells during Trypanosoma cruzi infection, the main point being whether these cells play a negative or a positive role. In this work, we found that the adoptive transfer of CD4(+)CD25(+)FOXP3(+) T cells from rSSP4- (a recombinant Trypanosoma cruzi amastigote derived protein, previously shown to have immunomodulatory properties on macrophages) immunized BALB/c donors into syngenic recipients simultaneously with T. cruzi challenge reduces cardiac inflammation and prolongs hosts' survival but increases blood parasitemia and parasite loads in the heart. These CD4(+)CD25(+)FOXP3(+) Treg cells from immunized mice have a relatively TGF-ß-dependent suppressive activity on CD4(+) T cells. Therefore, regulatory CD4(+)CD25(+) T cells play a positive role in the development of acute T. cruzi infection by inducing immunosuppressive activity that controls early cardiac inflammation during acute Chagas disease, prolonging survival, but at the same time promoting parasite growth.

Differential immune response in mice immunized with the A, R or C domain from TcSP protein of Trypanosoma cruzi or with the coding DNAs.

In a murine model of experimental Trypanosoma cruzi (H8 strain) infection, we investigated the induction of protective immunity against the domains [amino (A), repeats (R) and carboxyl (C)] of the surface protein (SP), a member of the trans-sialidase (TS) superfamily. Recombinant proteins and plasmid DNA coding for the respective proteins were used to immunize BALB/c mice, and the humoral response and cytokine levels were analysed. Immunization with the recombinant proteins induced higher levels of anti-TcSP antibodies than immunization with the corresponding DNAs, and analysis of serum cytokines showed that immunization with both recombinant proteins and naked DNA resulted in a Th1-Th2 mixed T-cell response. Mice immunized with either recombinant proteins or plasmid DNA were infected with blood trypomastigotes. The recombinant protein-immunized mice showed a variable reduction in peak parasitemia, and most died by day 60. Only the pBKTcSPR-immunized mice exhibited a significant reduction in peak parasitemia and survived the lethal challenge. DNA-based immunization with DNA coding for the repeats domain of TcSP is a good candidate for the development of a vaccine against experimental T. cruzi infection.

Subcellular distribution of the Entamoeba histolytica 140 kDa FN-binding molecule during host-parasite interaction.

Entamoeba histolytica trophozoites recovered from the host-parasite interface during abscess development obtain different stimuli compared with long-term cultured cells. In order to have a better understanding about the mechanisms in which the 140 kDa fibronectin (FN)-binding molecule (EhFNR) is involved during the invasive process, we decided to compare the regulation process of this molecule among long-term cultured trophozoites, FN-stimulated trophozoites, and trophozoites recently recovered from a liver abscess. A cDNA clone (5A) containing a fragment of the EhFNR that shows identity to the C-terminal region of the intermediate galactose lectin subunit Igl, was selected with a mAb (3C10). Identity of EhFNR with Igl was confirmed by immunoprecipitation with 3C10 and EH3015 (against the Gal/GalNAc intermediate subunit) mAbs. The 3C10 mAb was used as a tool to explore the modulation of the amoebic receptor (EhFNR). Our results showed specific regulation of the EhFNR in FN-interacted amoebas, as well as in trophozoites recovered at different stages of abscess development. This regulation involved mobilization of the receptor molecule from internal vesicles to the plasma membrane. Therefore, we suggest that in the host-parasite interface, the EhFNR (Igl) plays an important role in the adhesion process during abscess development.

Recombinant SSP4 protein from Trypanosoma cruzi amastigotes regulates nitric oxide production by macrophages.

Acute infection with Trypanosoma cruzi is characterized by immunosuppression mediated by T cells and macrophages (Mphis). Nitric oxide (NO) production during the initial phase of acute infection might participate in the clearance of parasites by Mphis, whereas its overproduction during the late phase of acute infection would account for the immunosuppression observed. Trypanosoma cruzi molecules that might regulate the host responses have not been fully identified. Here, we demonstrate that active immunization with MBP::SSP4, a recombinant protein derived from a surface antigen specific of T. cruzi amastigotes (TcSSP4), was able to stimulate Ab production (IgG1, IgG2a, and IgG2b). On the other hand, MBP::SSP4 was able to stimulate NO production by peritoneal Mphis from BALB/c mice and Mphis from the J774 cell line. This effect was also observed at the level of inducible nitric oxide synthase (iNOS) detected by Western Blot. Furthermore, MBP::SSP4 was also shown to induce the expression of IL-1alpha, IL-6, IL-12, IFN-gamma, and TNF-alpha in normal animals, and IL-10 in immunized animals. In addition the protein MBP::SSP4 was able to bind to the surface of PMphis and J774 Mphis. These results suggest that TcSSP4 could modulate Mphi NO production and this may represent a mechanism participating in the immunoregulatory processes during Chagas' disease.

Stage specific kinetoplast DNA-binding proteins in Trypanosoma cruzi.

Knowledge regarding kinetoplast DNA organization in all members of the Trypanosomatid family is incomplete. Recently, the presence of kinetoplast-associated proteins in condensing kDNA networks in Crithidia fasciculata has been described and a role for these proteins in the maintenance of these complex structures was suggested. To investigate the presence of protein components in Trypanosoma cruzi kinetoplast, we previously described seven epimastigote kinetoplast-associated proteins. We report here the existence of kinetoplast binding proteins in amastigote and trypomastigote stages of T. cruzi, which could bind both mini and maxicircles components with a stage specific elements for every infective form of the parasite. We propose three major classes of kinetoplast-associated proteins related to the basic processes of this intricate disc structure and suggest a possible function of these binding proteins in the T. cruzi mitochondrial DNA organization.

American trypanosomiasis (Chagas' disease) and blood banking in Mexico City: seroprevalence and its potential transfusional transmission risk.

BACKGROUND: American trypanosomiasis (Chagas' disease), an anthropozoonosis fairly common in rural Latin America, has become an urban disease due to continuous migration, intra- and internationally. Blood transfusion, the second important pathway for transmission, increases its impact. Recognition of seropositive subjects among blood donors is now recommended, and clinical and serological screening enforced. Maneuvers to inactivate or remove Trypanosoma cruzi present in collected blood are recommended. METHODS: We surveyed voluntary donors at the National Institute of Cardiology in Mexico City in search of anti-T. cruzi by indirect immunofluorescence, ELISA, and Western blot analysis. Seropositive donors were identified and tested for immunoglobulin. We used types and fractions of donated blood to extract DNA and perform the PCR technique using kinetoplast primers seeking parasite DNA in blood. RESULTS: After 3,300 donors were screened, we identified 10 seropositive subjects (0.3%). These subjects were considered as indeterminate chagasic patients, came mainly from rural areas, and had IgG (100%) and IgA (30%) antibodies against a crude extract as well as a recombinant T. cruzi antigen. Identification of parasite DNA in red cell and platelet fraction was achieved from eight blood units. CONCLUSIONS: The present data provide evidence that blood donors at an urban hospital are seropositive for T. cruzi and at least 50% of donors carry the parasite potentially able to transmit T. cruzi in their cellular blood products. Serological screening should be included in routine blood-making. It is also necessary to adopt measures to inactivate or eliminate organisms in donated blood.

Humoral nitric oxide levels and antibody immune response of symptomatic and indeterminate Chagas' disease patients to commercial and autochthonous Trypanosoma cruzi antigen.

We report here the evaluation of chagasic patients for the presence and/or severity of the disease, antibody to Trypanosoma cruzi, and nitric oxide (NO) serum levels. Serum samples tested by ELISA with autochthonous and commercial antigen revealed that 10% and 7.5% of the individuals were anti-T. cruzi antibody-positive, respectively. Ten of 21 seropositive individuals had no clinical signs, the other 11 cases presented cardiomyopathy and/or mega-gastrointestinal syndromes, and three patients presented a combined form. A statistical difference (P < 0.001) in antibody titer between asymptomatic and symptomatic patients with autochthonous antigen was detected, and serum NO levels was found to be three times higher in cases than in controls. These results suggest that it is recommended to use a sole source of antigen (autochthonous) for the serodiagnosis of Chagas' disease, and that the pathogenic role of NO in this disease should be evaluated.

Serodiagnosis of American trypanosomosis by using nonpathogenic trypanosomatid antigen.

Crithidia luciliae, a nonpathogenic trypanosomatid, could provide a good alternative source of antigen for serodiagnosis of Chagas' disease. An enzyme-linked immunosorbent assay showed 100% sensitivity and 83% specificity when 91 human serum samples from Chagas' disease patients and 127 human serum samples from people suffering from toxoplasmosis (21 samples), leishmaniasis (32 samples), systemic rheumatic diseases (33 samples), and heart diseases (41 samples) were tested simultaneously with Trypanosoma cruzi and C. luciliae crude extracts. By Western blotting, an immunodominant band (30 kDa) was recognized by chagasic sera on the C. luciliae crude extract; specificity reached 97% with respect to this protein band. The carbohydrate moieties were not antigenic.

Role of prostaglandin E2 on amoebic liver abscess formation in hamsters.

Entamoeba histolytica can modulate macrophage functions and cytokine production by a prostaglandin E2 (PGE2) mechanism. To study the participation of PGE2 on amoebic liver abscess formation, we tested the effect of the PG synthesis inhibitor indomethacin (INDO) on abscess development in hamsters infected intrahepatically with E. histolytica trophozoites. Male infected animals had higher levels of plasma PGE2 (5.7 +/- 0.7 pg/ml pre-infection; 26.0 +/- 2.0 pg/ml 7 days postinfection; p < 0.001). INDO prevented this increase, so that infected-treated and control non-infected animals had similar levels of plasma PGE2. INDO reduced liver and abscess weight by 18% and 30% respectively (p < 0.05). Cyclooxygenase (COX) activity determination by thin layer chromatography using (1-14C) arachidonic acid (AA) showed that liver microsomes from infected animals produced more PGE2 than controls. COX activity was considerably inhibited in infected INDO-treated animals. Our data suggest that E. histolytica can stimulate the hepatic production of PGE2 which contributes to pathogenesis of amoebic abscesses through generation and support of the inflammation. The partial effect of INDO treatment suggests that additional factors are involved.

Leishmania mexicana: binding of promastigotes to type I collagen.

During leishmania infection, parasites are inoculated to the human host through the bite of a sandfly vector into the dermis, where they first interact with tissue components, cells and extracellular matrix molecules. Since collagen is the most abundant component of the skin matrix, we investigated whether there is a specific interaction of Leishmania mexicana promastigotes with this host component. Promastigotes were able to attach to collagen fibrils and move through the matrix of mouse skin sections and to penetrate easily into a type I collagen gel. Denatured type I collagen coated beads (Cytodex 3) readily bound to the parasite surface. The interaction of promastigotes with type I collagen was dose dependent and saturable and was competitively and specifically inhibited with increasing concentrations of gelatin. Biotin-labeled parasite surface molecules were able to associate with both denatured collagen from microcarriers and native type I collagen from bovine kidney. It is suggested that the presence of parasite cell membrane receptors to collagen may confer a specific tropism for the skin, where collagen is the most abundant component of the matrix.

American trypanosomosis: in situ and generalized features of parasitism and inflammation kinetics in a murine model.

American trypanosomiasis: In situ and generalized features of parasitism and inflammation kinetics in a murine model. Experimental Parasitology 83, 267-274. Mimicking the natural conditions of mammalian infection, metacyclic trypomastigote forms of a Mexican isolate (Ninoa) of Trypanosoma cruzi were inoculated into mice in order to study inflammation kinetics and parasite clearance at the inoculation site, parasite tropism to different organs, and local inflammatory cell infiltrates. Polymorphonuclear cells were detected at the inoculation site as early as 1 hr after inoculation. Peak cell infiltrate was observed at 24 hr; at 96 hr polymorphonuclear cells had disappeared. Mononuclear cell infiltrates began at 24 hr, peaking at Day 15, and then stared disappearing by Day 30. Antigens and parasites were detected by conventional techniques up to 15 min and thereafter became undetectable. Amplification of the hypervariable region of kinetoplast minicircle DNA by polymerase chain reaction was positive from 24 hr to Day 15, and the reaction became negative on Day 30. Myositis was observed in skeletal muscle from Days 7 to 180, it progressed from slight to severe, with an inflammation process which included macrophages, plasmatic cells, and a few eosinophils, the phenotype of the infiltrating cells included LyT2+ and LyT1+ on Day 30, and both cell populations decreased in parallel on Day 180. Antigen and parasite nests were present from Day 15 to 180; in muscle the earliest time at which minicircle DNA was detected was Day 7 and it was present until Day 180. Myocarditis was also observed; it developed from slight to severe in between Days 7 and 30, then gradually decreased, and cleared up. Mononuclear cell infiltrates in the myocardium were present from Days 7 to 180. Antigen and parasite nests were detected at Days 15 and 30 and disappeared at Day 180, although minicircle DNA was detected until the last day of observation. Both skeletal and heart muscles showed inflammatory reaction foci containing T. cruzi antigen. There was twice the number of foci in heart as in skeletal muscle. This ratio was maintained by Day 30; later skeletal muscle showed persistent inflammation which was practically cleared up in the heart. Parasites or antigen were not detected by Day 180 in both skeletal and cardiac muscle; however, minicircle DNA was amplified, indicating that an small proportion of parasites evaded immune response. According to these data, Mexican Ninoa Strain has a classification as biodeme 3.

T-cell suppression and selective in vivo activation of TH2 subpopulation by the Entamoeba histolytica 220-kilodalton lectin.

A 220-kDa surface protein (L220) with lectin activity from Entamoeba histolytica trophozoites has been characterized previously (J. L. Rosales-Encina, I. Meza, A. López-de-León, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:790-797, 1987). This molecule is involved in the adhesion process (I. Meza, F. Cázares, J. L. Rosales-Encina, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:798-805, 1987) and is very immunogenic. In this work, we studied both the humoral and the cellular immune responses to L220. We compared L220 with L220-derived components, such as a fusion peptide (M-11) and chemically obtained peptides (by treating the 220-kDa molecule with N-chlorosuccinimide-urea). Spleen cells from L220-immunized mice were unable to proliferate in vitro when stimulated with the protein. However, a proliferative response was obtained when mice were immunized with the L220-derived fusion peptide or the cleaved lectin. To find out if there was a correlation between the observed responses and TH1 or TH2 activation, we analyzed patterns of cytokine secretion (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon). Cells from mice immunized with peptides that induced cell proliferation (100, 80, and 47 kDa) with the peptides (P < 0.01) and with the intact molecule secreted IL-2 and gamma interferon, showing a TH1-subset pattern. Conversely, cells from mice immunized with the intact 220-kDa molecule secreted IL-4 and IL-10, typical of a TH2 subpopulation; however, antibodies from each group recognized the 220-kDa molecule as determined by Western blotting (immunoblotting). These results suggest that various epitopes in the 220-kDa molecule generate different response patterns, suppressing or activating T-cell responses.

A beta 1 integrin-like molecule in Entamoeba histolytica.

Human invasive amoebiasis is highly destructive, causing rapid necrosis and liquefaction of all tissues reached by the trophozoites. Degradation of extracellular matrix components (EMC) has been demonstrated during invasion of the basal lamina. Pursuing the idea that trophozoites might behave similarly to other invasive cells with respect to their interaction with EMC, plasma membrane proteins biochemically or functionally related to integrins were looked for. A 140 kDa molecular mass membrane protein from Entamoeba histolytica trophozoites with the characteristics of a beta 1 integrin-like fibronectin receptor was identified.