Activation by synergism between endotoxin and lymphokines of the mouse macrophage cell line J774 against infection by Trypanosoma cruzi

The mouse macrophage cell line J774 was easily infected by T. cruzi epimastigotes which were transformed to amastigotes that multiplied inside the cells. Spleen-T-cells from T. cruzi immune mice stimulated with Concanavalin A or T. cruzi, but not with unrelated antigens, released lymphokines into the supernatants that when added to J774 cells were unable to induce complete trypanocidal activity, although they were able to delay the rate of infection by protecting the cells from being infected. Addition of bacterial lipopolysaccharide (LPS), although inactive by itself, acted synergistically with the supernatants in inducing complete trypanocidal activity without affecting the susceptibility of J774 cells to infection. Gamma-interferon (gamma-IFN) activity was detected in the supernatants, however, but was not solely responsible for the trypanocidal inducing activities, since: there was no correlation between the levels of gamma-IFN and macrophage activation; gamma-IFN alone was less effective than the supernatants alone; and two active fractions of 100,000-150,000 mol. wt and 30,000 mol. wt were separated by gel filtration chromatography of the lymphokine preparations. The latter, which showed the characteristics of gamma-IFN with respect to size, pH 2 sensitivity and antiviral activity, had some trypanocidal activity alone. However, the 100,000-150,000 mol. wt fraction was active only in the presence of LPS. Finally, this trypanocidal inducing activity of the supernatants was not due to the induction of synthesis of gamma-IFN by the J774 cells.     

Antibody-dependent cell cytotoxicity against Trypanosoma cruzi is only mediated by protective antibodies

The antibody-dependent cell cytotoxicity (ADCC) to Trypanosoma cruzi blood forms (Btry) using non-adherent spleen cells is only mediated by sera from chronic chagasic patients or mice. Both display 'lytic antibodies' (LA), which are immunoglobulins directed against epitopes only present in living BTry, and 'conventional serology antibodies' (CSA), which are responsible for the positive diagnostic tests in Chagas disease. Sera from mice immunized with different T. cruzi antigens or from treated patients (displaying only CSA but not LA) are unable to mediate ADCC. These data confirm the central role played by LA in the host resistance against T. cruzi. Moreover, they probably explain why most immunizing agents used as vaccines in Chagas' disease and which elicit CSA but not LA, do not display significant protection against T. cruzi. We also demonstrate that trypsinization of BTry increases significantly the rate of parasite destruction by ADCC, suggesting that enzyme sensitive membrane components may help BTry to evade from this immune effector mechanism.     

Susceptible mice present higher macrophage activation than resistant mice during infections with myotropic strains of Trypanosoma cruzi

The kinetics of macrophage activation were compared among inbred strains of mice (C3H, BALB, B6 and B10.A) that are known to differ in their relative resistance to infections with the myotropic strains (Colombian and CL) of Trypanosoma cruzi. The parameters utilized to measure macrophage activation were rapid spreading on glass surfaces, hydrogen peroxide release and tumour necrosis factor/cachectin production. Macrophages obtained from C3H (susceptible), BALB (intermediate) and B6 or B10.A (resistant) mice infected with both strains of T. cruzi began to spread rapidly at the onset of parasitaemia. Surprisingly, the amount of hydrogen peroxide released by peritoneal cells obtained from the more susceptible mouse strain (C3H) was significantly higher than in the other mouse strains. Also, only in the serum of C3H mice was tumour necrosis factor/cachectin detected. These results suggest that resistance against infections with myotropic strains of T. cruzi does not correlate with enhanced macrophage activation. It is also shown that the acquired macrophage activation is largely dependent on T-lymphocytes bearing the phenotypic marker CD4 (helper/inducer), since all parameters of macrophage activation were significantly inhibited in athymic mice or in C3H mice treated in vivo with monoclonal antibody anti-CD4+ T-cells.     

Direct lysis of Trypanosoma cruzi: a novel effector mechanism of protection mediated by human anti-gal antibodies

Anti-gal antibodies directed against a carbohydrate epitope present in mouse laminin (galactosyl alpha 1-3 galactose) and detected in high levels in sera from patients in the acute phase of Chagas disease are responsible for the direct lysis (DL) of Trypanosoma cruzi blood forms independent of either the classic or alternative complement pathways. Furthermore, the lectins Euonymus europaeus (EE) specific for the carbohydrates gal alpha 1-3 gal present a similar lytic activity against T. cruzi at the same concentrations of purified anti-gal antibodies. The DL activity was tested with several other lectins but Concanavalin A (Con A) specific for alpha-D-mannose and alpha-D-glucose was the only one also presenting lytic activity. The lectins and anti-gal antibodies lytic activity can be inhibited by specific carbohydrates suggesting that this phenomenon is related to the capability of these lectins or anti-gal antibodies to bind to a crucial surface component of T. cruzi. Moreover, the infectivity of T. cruzi blood forms to mice was clearly inactivated by incubation with acute chagasic sera (ACS) but not by ACS absorbed by immunoaffinity chromatography with mouse laminin, a strong evidence that high levels of anti-gal antibodies participate in the decline of the parasitaemia from the acute to the chronic phase in Chagas disease.     

Susceptibility to Trypanosoma cruzi is modified by a previous non-related infection

Helminth infections are frequently massive, chronic and strong inductors of Th2-type cytokines. This implies that infection by such parasites could alter the susceptibility to subsequent infections by other pathogens, particularly intracellular parasites. We therefore explored whether a persistent infection, caused by Taenia crassiceps cysticerci, in BALB/c mice could affect susceptibility to a later infection by Trypanosoma cruzi. We found that the presence of the cysticerci indeed modified the immune response and the susceptibility to T. cruzi, and that these modifications depended on the time-course evolution of the initial infection. Coinfection with the protozoan in the early stages of the helminth infection, induced a delay on the onset of parasitaemia, early specific production of IFN-gamma and high specific production of IL-4. A significant increase in susceptibility to T. cruzi was observed only when mice were coinfected in late stages when the helminth load is greater and a Th2 type response against it is predominant. The in vitro specific response to T. cruzi antigens was then characterized by low levels of both IFN-gamma and IL-4. These findings suggest that chronic helminth infections could potentially have a significant influence over the immune response and hence susceptibility to other pathogens.     

Trypanolytic activity and antibodies to metacyclic trypomastigotes of Trypanosoma cruzi in non-Chagasic human sera

Metacyclic trypomastigotes of Trypanosoma cruzi, derived either from triatomid vectors or axenic cultures, were found to be extensively lysed by sera of some non-Chagasic healthy individuals through activation of the alternative complement pathway. Antibodies to T. cruzi metacyclics were detected by direct agglutination test in normal human sera (NHS) containing trypanolytic activity. Absorption of lytic NHS with metacyclic trypomastigotes, but not with non pathogenic Herpetomonas samuelpessoai promastigotes, abolished the trypanolytic effect. Natural antibodies to trypomastigotes were not found in NHS devoid of trypanolytic activity. Precipitation of 131I-labelled metacyclic surface proteins with lytic NHS revealed as the major band a polypeptide with an apparent molecular weight of 75,000.     

Effect of protective and non-protective antibodies in the phagocytosis rate of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

The phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages is significantly enhanced by sera from chronic chagasic patients, rabbits and mice presenting 'lytic antibodies' (LA) which are associated with resistance and active infections as well as 'conventional serology antibodies' (CSA) which are immunoglobulins involved in the positivity of serological diagnostic tests. The phagocytosis rate, however, is not influenced by sera from mice immunized with T. cruzi antigen or chagasic patients submitted to specific treatment, both displaying only CSA but not LA. The efficacy of LA in increasing phagocytosis is related to their ability to bind to epitopes of living trypomastigotes, a property lacking in CSA that bind only to fixed parasites. This phenomenon is apparently the reason for the low effectiveness of antigens used for vaccination in Chagas' disease which only induce CSA, immunoglobulins apparently unable to mediate a number of regular effector immune mechanisms such as complement-mediated lysis, antibody-dependent cell cytotoxicity and phagocytosis.     

The cachexia associated with Trypanosoma cruzi acute infection in mice is attenuated by anti-TNF-a, but not by anti-IL-6 or anti-IFN-7 antibodies

BALB/c male mice acutely infected with Trypanosoma cruzi underwent a severe weight loss (around 20%, from day 18 to 31 post-infection), when compared to age-matched uninfected animals. Though mice regained weight later, when blood parasites were hardly detectable, wasting extended over the chronic phase of infection. The onset and the magnitude of weight loss were related to the mouse susceptibility to infection, since they were respectively earlier and higher in male mice which will die than in surviving ones, in males than in females, and in BALB/c than in B6D2 [(C57B1/6 × DBA/2)F1], a mouse strain more resistant to infection. Fat weight of infected mice (male BALB/c) was reduced by 60 to 80%, whereas lean mass was unaffected and water content rose by 6 to 10% in acute and chronic infection. Haematocrit was also decreased by 15–16% in acute infection. Animals failed to compensate their energetic loss since their food intake remained similar to that of uninfected animals. Injections of neutralizing anti-TNF-α monoclonal antibody into infected male mice, during the first two weeks but not later in infection, significantly attenuated the weight loss. Early administration of anti-IL-6 or anti-IFN-γ MoAbs did not improve the mouse wasting. Taken together, these data show that TNFis a key agent of cachexia occurring in the acute T. cruzi infection in mice.     

Protective effect of Trypanosoma rangeli against infections with a highly virulent strain of Trypanosoma cruzi

Summary We investigated the protective effect of Trypanosoma rangeli against infection with Trypanosoma cruzi in animal models of various ages and with different doses of inoculum. The age of the mice and the dose of parasites determined the course of the infection. When T. cruzi was inoculated into mice after challenge with T. rangeli , parasitaemia was more controlled, mortality decreased and histopathology showed lower inflammatory infiltration and pseudocysts. This study proposes a new murine model of the protective effect of recombinant proteins of T. rangeli for possible application in the vaccines field.     

Trypanosoma cruz/-induced decrease in the level of interferon-7 receptor expression by resting and activated human blood lymphocytes

A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-γ receptors (IFN-γR) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-γR. Decreased IFN-γR expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion ofB cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-γR+ and IFN-R +^bnght (cells expressing above-normal levels of surface IFN-γR) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-γR expression by B cells. When T. cruzi was present, the intracellular levels of IFN-γR molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-γR synthesis. Among T (CD3+) cells, 10–8% to 39–6% (7 donors) expressed surface IFN-γR and did so at a very low level. These percentages were also reduced by T. cruzi. If occurring in the host, downregulated expression of IFN-γR could curtail the utilization of IFN-γ, known to play a critical role in host defence against T. cruzi infection.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal oulbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B- lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., ³H-thymidine incorporation) and cell membrane levels of interleukin-2 receptors (H.-2R; determined by flow cytomet.y) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

The nature of immunity against Trypanosoma cruzi in mice recovered from acute infection

Protective immunity against a lethal, Y strain, T. cruzi infection could be transferred to normal mice by either serum or spleen cells from mice which had recovered from the acute phase of infection. The ability of spleen cells to transfer immunity was abolished by B lymphocyte removal (anti-Ig column fractionation), but was relatively insensitive to T lymphocyte depletion (anti-Thy 1.2 plus complement) or macrophage removal (Sephadex G-10 fractionation). Immune spleen cells gave an anamnestic antibody response when injected together with T. cruzi antigen into lethally irradiated recipients and these antibodies conferred protection in a passive transfer system. T cell depletion reduced, but did not abolish, this antibody response. These data imply that the protective immunity of T. cruzi-convalescent mice is predominantly B cell-mediated with T cell involvement being restricted to a helper role.     

Trypanosoma cruzi infection in mice induces a polyisotypic hypergammaglobulinaemia and parasite-specific response involving high lgG2a concentrations and highly avid lgG1 antibodies

Trypanosoma cruzi infection in BALB/c mice induced a reversible polyisotypic hypergammaglobulinaemia, with particularly high levels of IgG2a, IgM and IgE. Hypergammaglobulinaemia started during the acute phase of infection and persisted during chronic disease until 11–13 weeks post-infection (w.p.i.), when immunoglobulin levels, with the exception of IgE, returned near normal values. Parasite-specific antibodies counted for 14 to 23% of gammaglobulinaemia, in acute and chronic infection respectively. The titres of IgM antibodies rose from two w.p.i. IgA, IgE and IgG subclass antibodies built up gradually over the time of parasite clearance (i.e., between three and six w.p.i.). All antibody isotypes, including IgM reached significant and stable titres throughout chronic infection. IgG2a, IgG1 and IgM antibodies had constantly higher titres than the other antibody isotypes. The dominance of IgG2a antibodies was due to their high plasma concentrations, around 70% of all antibodies available in the chronic infection. IgG1 had the highest functional avidity, whereas its concentration corresponded to only 10% of the whole antibody fraction. These results indicate that T. cruzi infection in mice induces a polyisotypic humoral immune response, dominated by some antibody isotypes, with major differences in concentrations and functional avidities. This could be of crucial importance in determining the outcome of infection.     

Short communication: Trypanosoma cruzi lineage I in endomyocardial biopsy from a north-eastern Brazilian patient at end-stage chronic Chagasic cardiomyopathy

Trypanosoma cruzi, the agent of Chagas disease, is genetically classified into two major evolutionary lineages, T. cruzi I and T. cruzi II. In Southern American Cone countries it is T cruzi II which causes most cases of severe chronic Chagas disease. Contrary to this, we isolated T. cruzi I nested in endomyocardial biopsies of a chronic chagasic patient with end-stage heart failure. Our finding should alert clinicians to the possibility of severe Chagas disease in all regions where T. cruzi circulates, regardless of its lineage.     

Trypanosoma cruzi: susceptibility in mice carrying mutant gene lpr (lymphoproliferation)

Summary There is evidence that autoimmune aberrations may contribute to the immunopathological consequences of Chagas' disease and because of this we sought to determine whether four inbred strains of mice bearing the single autosomal recessive gene, lpr (lymphoproliferation), which controls certain autoimmune manifestations, are particularly susceptible to acute infection with the Y strain of Trypanosoma cruzi. MRL/MpJ-lpr/lpr, C57B1/6J-lpr/lpr, AKR/J-lpr/lpr, C3H/HeJ-lpr/lpr showed parasitaemias 2–10 times higher when compared to their congenic partners. Mortality was significantly higher in three of the four lpr strains. The results indicate that a single autosomal recessive gene which is associated with autoimmunity can influence susceptibility to acute T. cruzi infection in mice.     

Antibody-dependent cytotoxicity of Trypanosoma cruzi antigen-coated mouse cell lines by eosinophils and neutrophils

Eosinophils and neutrophils are shown to be cytotoxic against two syngeneic mouse cell lines cells when these are coated with T. cruzi antigen and anti-T. cruzi antibody. Activity is detected within 5 h of incubation. Highest levels of cytotoxicity are obtained at antibody dilutions of 1:100 and 1:1000, while antiserum at 1:10 is shown to be inhibitory. Eosinophils show significant activity at an effector to target ratio of 5:1. No cytotoxicity occurs in the absence of either antigen, antibody or effector cells. This phenomenon may be a model for the tissue destruction in acute T. cruzi infection, where the lysis of trypanosomes may lead to antigen coating of host cells, followed by antibody-dependent granulocyte-mediated cytotoxicity of the host cells.     

Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures – composed mainly of sphingosine as the long chain base acylated with stearic acid – have been determined for the first time in Trypanosoma cruzi epimastigotes by UV‐MALDI‐TOF‐MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C‐terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross‐reactivity maps to the C‐terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate‐containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.     

Peanut agglutinin affinity chromatography of Trypanosoma cruzi glycoproteins

Glycoproteins from Trypanosoma cruzi epimastigotes have been extracted by diiodosalycilic acid and lithium salts, and phenol-water biphasic partition. Peanut agglutinin has been used in a one step preparative method for fractionating the total extract in order to separate the so-called galactose-terminal glycoproteins. The different fractions have been studied by SDS electrophoresis, ultracentrifugation and immunoelectrophoresis techniques. The experimental immunogenicity, antigenicity and specificity of the PNA affinity fractions has been evaluated.