The involvement of CD4+CD25+ T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.     

Trypanosoma cruzi: effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-γ were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-γ when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas’ disease, enhancing pathogenesis through disturbance of the host’s immune system.     

Trypanosoma cruzi: plasma corticosterone after repetitive stress during the acute phase of infection

An increased level of plasma corticosterone is one manifestation of severe environmental or physiologic stress. The stress response mediated by the hypothalamic-pituitary-adrenal axis is already known to suppress immunoglobulin production and to impair immune function, but there are few studies relating stress and plasma corticosterone to the outcome of Trypanosoma cruzi infection. In this study, male Wistar rats were infected with the Y strain of T. cruzi and then subjected to repetitive stress by exposure to ether vapor for 1min twice a day during the acute phase of infection. Stressed animals showed decreased lytic antibody activity and lowered levels of peritoneal macrophages. Despite an increase in the weight of the spleen, histological analyses demonstrated tissue alterations, the presence of amastigote nests, and a complete absence of activated lymphoid follicles. These results suggest that stress-induced increases in plasma corticosterone can suppress the immune response and worsen tissue injury during the acute phase of T. cruzi infection.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Trypanosoma cruzi: correlation of muscle lesions with contractile properties in the acute phase of experimental infection in mice (Mus musculus)

Parasitism in skeletal muscles and myositis are commonly observed during experimental Trypanosoma cruzi infection. The effect of T. cruzi infection on contractile properties of skeletal muscles in consecutive periods of the acute infection in BALB/c mice was studied. Albarrada strain (clone 4) which was isolated in Mexico and has demonstrated a high level of blood parasitemia and parasitism in skeletal muscles was used. Isolated strips of rectus abdominis muscle were subjected to direct electrical field in vitro. Alternatively, plantaris muscles were stimulated in situ through the sciatic nerve. The peak amplitudes of a single twitch and tetanus contractions were considered to estimate the mechanical properties of muscles. Histopathological analysis was performed to correlate functional changes with the evolution of tissue parasitism and tissue injury. Contractile properties of muscles were significantly attenuated during acute T. cruzi infection. The percentage of damaged muscles rather than the character of tissue pathology affected their contractile properties significantly.     

Trypanosoma cruzi: effect of the infection on the 20S proteasome in non-immune cells

Human infection with the protozoan Trypanosoma cruzi leads to Chagas disease. After 10-20 years of the normal acute phase, this disease develops to a chronic phase characterized mainly by dilated congestive cardiomyopathy. The mechanisms involved in the chronic phase are poorly understood, and it has been suggested that the parasite evades immune surveillance by down regulating the MHC class I antigen processing pathway. Here we analyzed whether composition or expression of the 20S proteasome, the major proteinase responsible for the generation of MHC class I ligands, were altered upon infection of HeLa cells by T. cruzi. Two-dimensional gel electrophoresis and RT-PCR experiments comparing non-infected and infected cells did not show differences between the composition of 20S proteasome or expression of its subunits. However, the proteasome’s trypsin- and chymotrypsin-like activities were 2.5 and 3.6 times higher in infected cells than in non-infected cells. Our results suggest that in vitroT. cruzi infection of human or rat cells do not alter the expression of 20S proteasomal subunits or particle composition, and fails to induce the formation of immunoproteasome. However, a significant increase in the trypsin- and chymotrypsin-like activities of the host proteasome was observed.     

Heart FoF1-ATPase changes during the acute phase of Trypanosoma cruzi infection in rats

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K_0.5 values, and a decreased total V_max. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.     

Active substances against trypomastigote forms of Trypanosoma cruzi and microorganisms are produced in sequence by Talaromyces flavus

The conditions for the sequential production of antibiotic activity by Talaromyces flavus were determined. The highest level of activity against Trypanosoma cruzi was obtained from the aqueous extract of the Czapeck's fermentative culture after 48 hours, with lysis of 97.58% of the trypomastigote forms of Trypanosoma cruzi (red blood cells remained normal). The antimicrobial activity was detected in the extracts of fermentative cultures from different media just after 144 hours of incubation. Maximum activities against Micrococcus luteus, Staphylococcus aureus and Candida albicans were present in chloroform, butanolic and water extracts, in this order, when Talaromyces flavus was cultivated at pH 5.0. The minimal inhibitory concentration (MIC) of extracts of Takeuchi's cultures were determined.     

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.     

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with (3)H-androstenedione, T. cruzi trypomastigotes synthesized (3)H-testosterone (T), (3)H-17beta-estradiol (E(2)) and (3)H-estrone (E(1)). Metabolism of (3)H-DHEA by the parasites yielded (3)H-androstendione and (3)H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the (3)H-androstendiol and (3)H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Structural variation in the glycoinositolphospholipids of different strains ofTrypanosoma cruzi

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasiteTrypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man₄(AEP)GlcN-Ins-PO₄ core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors ofT. cruzi. The core can be further substituted by terminal (1–3)-linked -galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of -galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection byT. cruzi.Abbreviations GIPL glycoinositolphospholipid - LPPG lipopeptidophosphoglycan - GPI glycosylphosphatidylinositol - AEP 2-aminoethylphosphonic acid - PI phosphoinositol - GC gas-liquid chromatography - MS mass spectrometry - FAB fast atom bombardment - NMR nuclear magnetic resonance - DQF-COSY double quantum-filtered correlation spectroscopy - TOCSY total correlation spectroscopy - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - EtNP ethanolaminephosphate - HMQC heteronuclear multiple quantum coherence - Man mannose - Galf galactofuranose - GlcN glucosamine - Ins inositol - InsP inositolphosphate - Man 3 third mannose distal to inositol - NOE nuclear Overhauser effect - [M+H]⁺ protonated molecule - [M–H]^– deprotonated molecule - RMM relative molecular mass (monoisotopic)     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.     

Pivotal role for TGF-beta in infectious heart disease: The case of Trypanosoma cruzi infection and consequent Chagasic myocardiopathy

This paper summarizes recent data from the literature suggesting that transforming growth factor-β (TGF-β) participates at least in four different processes influencing development of myocardiopathy in Chagas disease, a major parasitic illness caused by Trypanosoma cruzi infection: (a) invasion of cardiac fibroblasts and myocytes; (b) intracellular parasite cycle; (c) regulation of inflammation and immune response; (d) fibrosis and heart remodeling during acute and chronic disease. All these effects point to an important role of TGF-β in Chagas disease myocardiopathy and suggest that monitoring the circulating levels of this cytokine could be of help in clinical prognosis and management of patients. Moreover, TGF-β-interfering therapies appear as interesting adjuvant interventions during acute and chronic phases of T. cruzi infection.     

Trypanosoma cruzi: effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced.     

Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes

The polymerase chain reaction showed high sensitivity for detecting Trypanosoma cruzi in the blood of mice, independent of clonal genotype (19, 20-T. cruzi I; 32, 39-T. cruzi II) or phase of the infection (acute or chronic).     

Energetics of heart mitochondria during acute phase of Trypanosoma cruzi infection in rats

The energetics of heart mitochondria was studied in the acute phase of Trypanosoma cruzi infection in rats. Wistar rats were infected with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi, and heart mitochondria and submitochondrial particles isolated after 7 and 25 days of infection. Ultrastructure of mitochondria seemed to be preserved, but cytochrome c levels were significantly depressed. Respiratory control ratios (RCR) were decreased for glutamate and succinate oxidations, as a consequence of inhibition of respiration in state 3 and/or of stimulation of respiration in state 4. Stimulation of hydrolytic activity of FoF1-ATPase by energization of mitochondria was approx. 2-fold higher in relation to controls. Mitochondrial ATP concentration remained constant. In conclusion, during the acute phase of T. cruzi infection in rats there is an energy impairment at the level of heart mitochondria, but their ultrastructure and ATP concentration seem to be preserved; the maintenance of ATP may be due to an adaptative mechanism of the cell which includes inhibition of the hydrolytic activity of FoF1-ATPase.     

Trypanosoma cruzi: effects of repetitive stress during the development of experimental infection

Activation of the hypothalamus-pituitary-adrenal axis plays a major role in the suppression of the immune system. We have investigated the effects of repetitive stress on Wistar rats infected with the Y strain of Trypanosoma cruzi and a control group that underwent stressor stimuli by exposure to ether vapor for one minute twice a day. Repetitive stress resulted in an elevated number of circulating parasites accompanies by deep tissue disorganization, and cardiac histopathological alterations. The infected and stressed group displayed a decrease in body weight, and an increased parasite burden in heart tissue, and adrenal glands. Histological analysis of the heart also showed a moderate to severe diffused mononuclear inflammatory process. These results suggest that repetitive stress could be considered an important factor during development of experimental Chagas' disease, enhancing pathogenesis through disturbance of the host's immune system.