Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y, CL and Gilmar). In all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas’disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curve of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phase with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Unusual Structures in the Flagellar Apparatus of a Strain of Trypanosoma cruzi*

Certain structures, associated with the flagellum, and which had hitherto been described as appearing occasionally in some species of trypanosomes, were found very frequently in epimastigote forms of strain F of Trypanosoma cruzi: (a) a group of tubular elements in an electron-dense mass enclosed within a swelling of the flagellar membrane as the flagellum emerges from its reservoir; (b) an expansion of the flagellar membrane at the point of the above swelling, which in cross-sections appears as a ring; and (c) an electron dense band in the body of the organism alongside the border of the flagellar pocket. The possible significance of these structures and the fact that so far they have been found only in one strain of T. cruzi are discussed.     

Binding of Lectins to the Cell Surface of Trypanosoma cruzi*

SYNOPSIS. Differences in the composition and distribution of cell membrane carbohydrates were demonstrated in the 3 life cycle forms of 3 Trypanosoma cruzi strains by using lectins with different specificities. The results suggest that lectin binding may be useful in characterization of the parasite strains.     

Strain-Dependent Thermosensitivity Influencing Intracellular Differentiation of Trypanosoma cruzi in Cell Culture*

SYNOPSIS. Temperature strongly influenced morphogenesis of intracellular trypomastigotes in cell culture infected with 2 different strains of T. cruzi. With the Gilmar strain the amastigote-to-trypomastigote differentiation readily occurred at 33 and 37 C, whereas with the CL strain differentiation took place at 33 C but was inhibited at 37 C. The possibility of this selective thermosensitivity resulting from mutational adaptation of the parasite is discussed.     

Growth and Differentiation of Trypanosoma cruzi Cultivated with a Triatoma infestans Embryo Cell Line*

SYNOPSIS. Trypanosoma cruzi strain Peru was cultivated in the presence of an established cell line of Triatoma infestans embryo cells (TI-32). Bloodstream trypomastigotes differentiated into amastigote-like cells (first differentiation phase) which multiplied to form large clusters of cells. Because of their clustering nature, a new term, “staphylomastigotes,” has been proposed for this stage. After 10 days of cultivation, 90% of the staphylomastigotes underwent differentiation (2nd differentiation phase) to trypomastigotes (?98%) or epimastigotes (?2%). Bloodstream trypomastigotes cultivated without TI-32 cells underwent the first, but not the 2nd differentiation phase, although occasional epimastigotes were seen (< 1%). The evidence presented suggests that TI-32 cells produce a labile factor(s) important not only for initiation of the 2nd differentiation phase but also for maintaining the parasites in the trypomastigote stage. The pH of the culture medium was not the initiating factor for the 2nd differentiation phase. Infectivity studies indicated that staphylomastigotes were as infective as bloodstream trypomastigotes, but that metacyclic trypomastigotes isolated from culture after the 2nd differentiation phase were slightly more infective than bloodstream forms. Electromicrographs of styphylomastigotes do not provide any evidence of exchange of genetic material between cells.     

Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi

Abstract Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH₂-OCO-(2,4,6-Me₃)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi . The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 μM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi , and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.     

Density of Parasites in Various Organs and the Relation to Numbers of Trypomastigotes in the Blood during Acute Infections of Trypanosoma cruzi in Mice*

Quantitative methods were used to (a) determine the density of Trypanosoma cruzi in organs of CF₁ mice following intraperitoneal inoculation of 50,000 trypomastigotes of a Brazil strain of T. cruzi and (b) study the relation of the numbers of these intracellular stages to the numbers of trypomastigotes in the blood. Tissue stages (predominantly amastigotes) in heart, skeletal muscle (triceps), diaphragm, cerebrum, cerebellum, and musculature of stomach, duodenum, esophagus, jejunum, cecum, and rectum increased in numbers during the 1st 3 weeks of infection, reached maximum density 21–28 days after inoculation and subsequently declined in numbers until mice were histologically negative for intracellular parasites by 30–40 days. The density of tissue stages in the urinary bladder, uterine body, and ileum was similar with the exception that maximum numbers of parasites were observed slightly earlier at 15 days. The greatest density of intracellular stages was seen in heart, urinary bladder, diaphragm, and triceps muscle where mean counts of 44.6–60.0 × 10⁶ parasites/cc of muscle were recorded while maximum density of parasites in the uterine body, cerebrum, stomach, cerebellum, duodenum, esophagus, jejunum, ileum, cecum, and rectum was 13.0 × 10⁶/cc of muscle or less. Amastigotes were not observed in sections of lymph node, thymus, salivary glands, liver, spleen, or kidney and only a single pseudocyst containing 5 amastigotes was seen in lung. With the exception of the brain and lung, intracellular parasites were located exclusively in the musculature. Trypomastigotes in the blood increased during the 1st 3 weeks of infection, reached maximum numbers 21–28 days after initiation of infection, and subsequently decreased until by 30–40 days parasites were observed only rarely in the blood of a few animals. Thus generally close correlation was noted between the numbers of intracellular stages of T. cruzi in the organs and trypomastigotes in the blood throughout acute Chagas’ disease in mice as evidenced by the concomitant increase in numbers of both stages, the coincidence of days of maximum parasite levels, and the simultaneous decline in numbers of both stages. The mean number of parasites/pseudocyst section varied in the organs studied. Of the 15 positive organs studied, the pseudocyst sections in skeletal muscle contained the highest mean number of parasites (64.3 parasites/pseudocyst section) and those pseudocyst sections seen in the musculature of the small intestine contained the lowest mean number (5.5–6.8 parasites/pseudocyst section respectively in ileum and jejunum). Serial sections of skeletal muscle, heart, urinary bladder, and stomach revealed the largest pseudocysts in skeletal muscle while those in the musculature of the urinary bladder were the smallest.     

Antigens of Subcellular Fractions of Trypanosoma cruzi. III. Humoral immune response and histopathology of immunized mice*

SYNOPSIS. The relation of humoral antibody response to protection was evaluated in mice immunized with whole homogenates of Trypanosoma cruzi or with its flagellar fraction by direct agglutination and indirect fluorescent antibody test as well as by lytic and neutralizing activity against blood trypomastigotes. The results indicated that lytic antibodies were not implicated directly in protection against these trypanosomes. It was evident from histopathologic examination that the higher the degree of protection achieved, the lower the tissue damage observed in the challenged mice. Serum-neutralizing activity was highest in the groups protected most effectively.     

The in vivo distribution of 51Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Comparative Kinetics of Arginine and Lysine Transport by Epimastigotes and Trypomastigotes from Two Strains of Trypanosoma cruzi*

SYNOPSIS. Three-day-old cultures of Y and MR strains of Trypanosoma cruzi had a higher rate of lysine and arginine uptake than 10-day cultures. Amino acid uptake by cells of the MR strain was consistently higher than that of the Y strain. Flagellates separated on DEAE-cellulose columns have normal structure, motility, and infectivity; they have higher rates of lysine and arginine uptake than the original 3- and 10-day cultures. In addition, passage through DEAE-cellulose columns modified the kinetic behavior of amino acid transport systems in the flagellate membranes. Methionine inhibited uncompetitively uptake of lysine and arginine by MR and Y strains. Lysine inhibited arginine uptake by both strains by an uncompetitive mechanism. Lysine, however, inhibited the uptake of arginine by 10-day culture cells of the Y strain by a mixed-type of inhibition. Arginine also inhibited the lysine uptake of both strains by an uncompetitive mechanism. In all experiments, beyond a certain level, a further increase in inhibitor concentration resulted in a decreased inhibition, which eventually disappeared altogether. Inhibition of amino-acid uptake by any of the substances tested was not observed after passage of flagellates through a DEAE-cellulose column. A model for amino acid transport was formulated which includes a recognition site amenable to modulation by effectors.     

Mapping of B cell epitopes in an immunodominant antigen of Trypanosoma cruzi using fusions to the Escherichia coli LamB protein

The JL8 protein antigen from Trypanosoma cruzi, a dominant immunogen in man, has been characterized as containing tandem amino acid repeats. Here, we describe the use of the LamB protein of Escherichia coli as a carrier of JL8 derived sequences in order to map the immunodominant B cell epitopes in this antigen. Five different sequences of JL8 were inserted in the LamB protein and the JL8-LamB fusion proteins were tested by ELISA with human chronic chagasic sera. The fusion carrying the sequence AEKQKAAEATKVAE was recognized by most sera. This protein was also capable of inhibiting the binding of human chagasic antibodies to GST-JL8 in competitive ELISA suggesting that it contains an immunodominant B cell epitope of JL8.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.     

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.     

Curative Effects of the Antipiroplasms Amicarbalide and Imidocarb on Trypanosoma brucei Infection in Mice*

SYNOPSIS The babesicides imidocarb and amicarbalide, which have structural similarities to the antitrypanosomatid diamidines, proved active against Trypanosoma brucei mouse infections: both cured infections when doses were administered daily for 3 days 24 h post-inoculation (curative dose imidocarb, 10 mg/kg; amicarbalide, 25 mg/kg). Mice were considered cured after survival 30 days longer than untreated infected controls, with no trypanosomes present in blood or cerebrospinal fluid smears. Both agents also cured when administered 48 and 72 h after challenge with T. brucei and prolonged the lives of animals 94 h after challenge. The results are discussed in respect to the potential of these carbanilides and their precursors, the antitumor phthalanilides, as lead compounds in chemotherapy of mammalian trypanosomiases.     

Trypanosoma cruzi: Correlation of Growth Kinetics to Zymodeme Type in Clones Derived from Various Sources*†

SYNOPSIS. Trypanosoma ( Schizotrypanum ) cruzi clones were derived from isolates of an acute human case of Chagas' disease (strain Esmereldo), a human case of T. cruzi infection (strain CAN-III) and from a naturally infected opossum (strain WA-250). The isoenzyme patterns and growth rates of the clones were stable during long-term cultivation, by serial passages, of the parasites in liquid medium. Both clones of strain Esmereldo were zymodeme II; the 2 clones of strain CAN-III, zymodeme III; and the 5 clones of strain WA-250, zymodeme I. The range in doubling times of the parasite populations in liquid medium were 36–49. 7 h (strain WA-250), 117.2–133.7 h (Esmereldo clones) and 169–208 h (CAN-III clones).     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

On the Fine Structure of the Nucleus in Trypanosoma cruzi in Tissue Culture Forms. Spindle Fibers in the Dividing Nucleus*

The fine structure of the dividing nucleus in the intracellular amastigote forms of Trypanosoma cruzi from tissue cultures has been described. In the first phase of division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metozoan cells then appear, running from one pole to the other. They disappear when the division of the nucleus is complete and the chromatin and the nucleolar material reorganize into their former positions.     

Inhibition of xenograft rejection reaction in the bug Triatoma infestans during infection with a protozoan, Trypanosoma cruzi

Autografts and allografts implanted into the body cavity of the bug Triatoma infestans provoked a poor hemocyte encapsulation, while xenografts stimulated significantly stronger reaction. This reaction of xenograft rejection, however, appeared distinctly inhibited in Trypanosoma cruzi-infected bugs. Similar effect was produced in triatomas by injecting them with the fluid from culture of the parasites. The results of these studies suggest that under natural conditions some parasites may avoid destruction and develop within the insect's body cavity following their suppression of host's defense reactions.     

Mechanism of Acquired Immunity Induced by “Leptomonas pessoai” against Trypanosoma cruzi in Mice*

Living culture forms of “Leptomonas pessoai” cross protected mice against T. cruzi challenge infection. Circulating antibodies have been detected in the immunized mice by immunodiffusion analysis, passive hemagglutination, complement fixation test and antibody binding assay; these antibodies cross reacted with T. cruzi extracts. A cellular immune response was indicated by leucocyte migration inhibition using L. pessoai and T. cruzi antigens, strongly suggesting a role for cell-mediated immunity in the mechanism of protection induced by L. pessoai.     

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas' disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas' disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.