Antibody-dependent cytotoxicity of human and mouse mononuclear cells against Trypanosoma cruzi epimastigotes

Human peripheral mononuclear cells were cytotoxic to antibody-sensitized Trypanosoma cruzi epimastigotes. The cytotoxic effect depended on the concentration of effector cells and antiserum, and was progressive until 17 hr of incubation at 28 °C. After 3 hr of incubation the highest specific activity was achieved at a 50:1 effector to target cell ratio. A nonspecific cytotoxic effect in the absence of antiserum was observed at a 100:1 parasite to cell ratio or after 17 hr of incubation. When the human mononuclear cell population was depleted of adherent cells by Sephadex G-10 filtration or adsorption to glass, the cytotoxic effect was greatly reduced. Similar results were obtained using mouse spleen cells, indicating that only the adherent cells were cytotoxic to sensitized T. cruzi in both systems. When human mononuclear cells were incubated with amobarbital, cyanide, azide, or aminotriazole, an inhibition of cytotoxicity against sensitized T. cruzi was observed, suggesting that oxygen reduction products and myeloperoxidase were involved in the destruction of sensitized T. cruzi epimastigotes by normal human mononuclear cells.     

Phagocytosis of Trypanosoma cruzi by Human Polymorphonuclear Leukocytes1

Phagocytosis of culture forms of Trypanosoma cruzi was assayed by a radioisotopic method. Purified polymorphonuclear leukocytes (PMN) were mixed with ³H-uridine-labeled T. cruzi epimastigotes in the presence or absence of anti-T. cruzi antibodies. The reaction was stopped by adding N-ethyl-maleimide, and noningested parasites were lysed by complement. The percentage of radioactivity incorporated into the PMN pellet was recorded. The phagocytosis reaction was rapid, yielding maximum incorporation at 30 min at which point the radioactivity associated with the PMN cells decreased through release of the isotope to the supernatant. The degree of incorporation of radio-labeled parasites was a function of the effector/target cell ratio and the antibody concentration. The method is suitable for the quantitative determination of phagocytosis of T. cruzi by normal PMN.     

Determination of chemical structure and anti-Trypanosoma cruzi activity of extracts from the roots of Lonchocarpus cultratus (Vell.) A.M.G. Azevedo & H.C. Lima

Trypanosoma cruzi is the agent of Chagas disease, an infection that affects around 8 million people worldwide. The search for new anti-T. cruzi drugs are relevant, mainly because the treatment of this disease is limited to two drugs. The objective of this study was to investigate the trypanocidal and cytotoxic activity and elucidate the chemical profile of extracts from the roots of the Lonchocarpus cultratus. Roots from L. cultratus were submitted to successive extractions with hexane, dichloromethane, and methanol, resulting in LCH, LCD, and LCM extracts, respectively. Characterization of extracts was done using ¹H-RMN, ¹³C-RMN, CC and TLC. Treatment of T. cruzi forms (epimastigotes, trypomastigotes, and amastigotes) with crescent concentrations of LCH, LCD, and LCM was done for 72, 48, and 48 h, respectively. After this, the percentage of inhibition and IC₅₀/LC₅₀ were calculated. Benznidazole was used as a positive control. Murine macrophages were treated with different concentrations of both extracts for 48 h, and after, the cellular viability was determined by the MTT method and CC₅₀ was calculated. The chalcones derricin and lonchocarpine were identified in the hexane extract, and for the first time in the genus Lonchocarpus, the presence of a dihydrolonchocarpine derivative was observed. Other chalcones such as isocordoin and erioschalcone B were detected in the dichloromethane extract. The dichloromethane extract showed higher activity against all tested forms of T. cruzi than the other two extracts, with IC₅₀ values of 10.98, 2.42, and 0.83 µg/mL, respectively; these values are very close to those of benznidazole. Although the dichloromethane extract presented a cytotoxic effect against mammalian cells, it showed selectivity against amastigotes. The methanolic extract showed the lowest anti-T. cruzi activity but was non-toxic to peritoneal murine macrophages. Thus, the genus Lonchocarpus had demonstrated in the past action against epimastigotes forms of T. cruzi but is the first time that the activity against infective forms is showed, which leading to further studies with in vivo tests.     

Trypanosoma cruzi: Nucleotide and vitamin requirements of growing epimastigotes

Using a defined culture medium it was shown that Trypanosoma cruzi epimastigotes (strains Y, Ma, and F1) do not require exogeneous nucleotides for continuous cultivation. Biochemical determinations carried out on parasites grown in the presence or absence of exogenous nucleotides revealed no differences in intracellular nucleotide concentrations. This suggests that T. cruzi epimastigotes have the capacity for de novo nucleotide synthesis. Choline and folic acid were necessary only for high yields of T. cruzi, suggesting that epimastigotes can partially satisfy their vitamin requirements.     

Inhibition of mammalian host cell infection by insect-derived, metacyclic forms of Trypanosoma cruzi in the presence of human or rabbit anti-T. cruzi antibodies

The presence of serum from chronic chagasic patients or rabbits immunized with killed epimastigote forms of Trypanosoma cruzi inhibited infection of rat heart myoblasts by insect-vector (Triatoma infestans)-derived, metacyclic forms of Trypanosoma cruzi. The effect was produced even after diluting the chagasic serum to non-agglutinating levels and was evidenced by marked reductions in both the percentage of infected myoblasts and the number of parasites per 100 cells. Human IgG or IgM purified from chronic chagasic serum and serum from rabbits immunized with killed T. cruzi epimastigotes also reduced both parameters. While previous work has shown that immunological destruction of invasive forms of T. cruzi may underlie the protective effects of the humoral immune response against this parasite, the present in vitro results suggest that specific anti- T. cruzi antibodies could also contribute to protection via inhibition of host cell infection by the vectortransmissible form of the parasite.     

Humoral antibody response and Ig characterization of the specific agglutinins in rabbits during experimental American trypanosomiasis

A comparative follow up study of the specific agglutinins detected by direct agglutination (DA) test and the immune response detected by specific lysis (SL), indirect immunofluorescence (IFA), indirect hemagglutination (IHA) and complement fixation (CF) tests in rabbits inoculated with trypomastigotes of T. cruzi is reported here.The specific antibody response was detected first by DA test. Reductive cleavage of sera with 2-mercaptoethanol produced a drop in the agglutinin titer of the sera during the first 30 days of infection.The next test to become positive was SL and later on the IFA, IHA and CF tests became positive simultaneously.When fractions obtained by column chromatography in Sephadex G-200 were tested serologically it was demonstrated that specific antibodies were detected mainly in fraction I (IgM) of the pooled rabbit sera obtained 15 days after inoculation (acute stage), and in fraction II (IgG) of the pooled sera obtained from rabbits 90 days after inoculation (chronic stage).Antigens prepared with trypsinized and formolized epimastigotes of three T. cruzi strains, belonging to each one of the different immunological groups described, worked similarly in the detection of specific agglutinin antibodies.Trypanosoma cruzi agglutinins were highly specific in their reaction with their homologous T. cruzi antigens as was proved by the low agglutinin titer obtained in sera from infected rabbits when, instead of T. cruzi epimastigotes, promastigotes of L. donovani were used as antigen, and by the incapacity of this parasite to absorb the T. cruzi agglutinins.     

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.     

MDL28170, a calpain inhibitor, affects Trypanosoma cruzi metacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut

Background

Trypanosoma cruzi is the etiological agent of Chagas'' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host.

Methodology/Principal Findings

In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption.

Conclusions/Significance

The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi.     

Involvement of STI1 protein in the differentiation process of Trypanosoma cruzi

The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

CD8low T cells expanded following acute Trypanosoma cruzi infection and benznidazole treatment are a relevant subset of IFN-γ producers

CD8 T cells are regarded as pivotal players in both immunoprotection and immunopathology following Trypanosoma cruzi infection. Previously, we demonstrated the expansion of CD8⁺ T lymphocytes in the spleen of T. cruzi-infected mice under treatment with benznidazole (N-benzyl-2-nitroimidazole acetamide; Bz), a drug available for clinical therapy. This finding underlies the concept that the beneficial effects of Bz on controlling acute T. cruzi infection are related to a synergistic process between intrinsic trypanocidal effect and indirect triggering of the active immune response. In the present study, we particularly investigated the effect of Bz treatment on the CD8⁺ T cell subset following T. cruzi infection. Herein we demonstrated that, during acute T. cruzi infection, Bz treatment reduces and abbreviates the parasitemia, but maintains elevated expansion of CD8⁺ T cells. Within this subset, a remarkable group of CD8^low cells was found in both Bz-treated and non-treated infected mice. In Bz-treated mice, early pathogen control paralleled the lower frequency of recently activated CD8^low cells, as ascertained by CD69 expression. However, the CD8^low subset sustains significant levels of CD44^highCD62L^low and CD62L^lowT-bet^high effector memory T cells, in both Bz-treated and non-treated infected mice. These CD8^low cells also comprise the main group of spontaneous interferon (IFN)-γ-producing CD8⁺ T cells. Interestingly, following in vitro anti-CD3/CD28 stimulation, CD8⁺ T cells from Bz-treated T. cruzi-infected mice exhibited higher frequency of IFN-γ⁺ cells, which bear mostly a CD8^low phenotype. Altogether, our results point to the marked presence of CD8^low T cells that arise during acute T. cruzi infection, with Bz treatment promoting their significant expansion along with a potential effector program for high IFN-γ production.     

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.     

Trypanosoma cruzi: effect of olivacine on macromolecular synthesis, ultrastructure, and respiration of epimastigotes.

Olivacine (a pyridocarbazole derivative) causes ultrastructural and metabolic alterations in Trypanosoma cruzi epimastigotes. The cytoplasm of cells grown in the presence of olivacine appears vacuolated, and swelling of the mitochondria is observed. Concomitant with the ultrastructural alterations, there was a reduction of the respiratory rate as well as inhibition of pyruvate oxidation. A marked inhibition of protein synthesis and a slower although significant inhibition of DNA, RNA, and lipid synthesis were also observed. The in vivo activity of olivacine does not parallel its in vitro effects suggesting inactivation of the drug by the host.     

Cloning of a Partial Length cDNA Encoding the C-Terminal Portion of the 75-77-kDa Antigen of Trypanosoma cruzi

ABSTRACT It has been suggested that several Trypanosoma cruzi antigens have possible protective epitopes which may be suitable vaccine candidates. We found previously that animals resistant to T. cruzi infection produced antibodies against the 75-77-kDa parasite antigen. To test the ability of the recombinant form of this antigen to protect animals from T. cruzi infection, the cDNA which encodes a portion of the 75-77-kDa antigen was cloned using a cDNA library constructed in an orientation-specific bacteriophage expression vector (λgt11) from poly (A)⁺ RNA of Brazil strain epimastigotes. One clone, named SFS-40, was selected by screening the library using affinity purified antibodies specific for the 75-77-kDa parasite antigen as probe. The cDNA corresponding to the 1.7-kilobase insert of SFS-40 was subcloned into plasmid vectors and characterized. The cDNA sequence encodes a polypeptide of about 40 kDa. The putative product of the cDNA was homologous to members of the 70-kDa stress protein family. When epimastigotes were shifted from 29° C to 37° C, there was no change in the level of SFS-40 mRNA. In contrast, the 70-kDa heat shock protein mRNA of the parasite was increased about four fold by this treatment.     

Trypanosoma cruzi: ability of T-cell-enriched and -depleted lymphocyte populations to passively protect mice

T-Cells and a T-cell-depleted population were prepared from the spleens of C3H mice immunized with epimastigotes of the Brazil strain of Trypanosoma cruzi. Both populations of cells, as well as unfractionated spleen cells, were capable of reducing parasitemias and protecting against death when transferred to susceptible C3H mice 24 hr before challenge with 10⁴ Brazil strain trypomastigotes. The immune T-cell-depleted subpopulation was, on an equal cell basis, more effective in engendering resistance than the immune T-cell subpopulation. Protection could also be transferred with unfractionated immune spleen cells if the cells were given within 8 days following challenge of recipient mice. Transfer after 8 days led to significantly reduced parasitemias but all mice died.     

Cultivation of Trypanosoma cruzi epimastigotes in low glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes

This study offers an insight into why Trypanosoma cruzi epimastigotes lose their capacity to differentiate into metacyclic forms, if maintained in culture media long-term through serial passages. The biological and metabolic behaviour of two T. cruzi strains isolated from various origins (human, opossum), and maintained under two schedules (alternate triatomine/mouse passages and serial culture media) were compared. To determine the effect of the environment on the parasites, the epimastigotes were grown under extreme conditions (high and low glucose concentrations), and the glucose consumption, ammonia production and changes in pH, either in one compartment (along the growth curve) or two compartments (induced metacyclogenesis) were compared. The glucose effect on the stages involved in metacyclogenesis at antigenic level was also evaluated. The results indicate that T. cruzi adapts to various environmental conditions and also that the ability of epimastigotes to undergo metacyclogenesis are influenced by the maintenance schedule. Antigenic profile analysis supports the idea that epimastigotes adapted to culture media do not complete their molecular differentiation into the trypomastigote metacyclic stage. These transition forms conserve some degree of gene expression of the epimastigote stage.