Efficacy of voriconazole in a murine model of acute Trypanosoma cruzi infection.

OBJECTIVES: Antifungal triazole derivatives have been studied as possible alternatives for the treatment of Chagas' disease. Voriconazole has demonstrated in vitro activity against Trypanosoma cruzi, but its efficacy in vivo has not yet been tested. We aimed to determine the effect of voriconazole in a murine model of acute T. cruzi infection. METHODS: Treatment efficacy was evaluated by comparing parasitaemia, mortality and organ involvement (by histological examination) of infected mice. RESULTS: Treatment with voriconazole significantly lowered parasitaemia and mortality compared with controls, reduced the percentage of mice with amastigote nests in heart and skeletal muscle and moderately decreased myocardial inflammation. CONCLUSIONS: Our findings support the potential of voriconazole for the treatment of acute Chagas' disease and motivate future animal studies using varying doses and treatment schemes. Further evaluation of voriconazole for clinical use in human Chagas' patients is warranted.

Reduction of parasite levels in blood improves pregnancy outcome during experimental Trypanosoma cruzi infection.

Infection with a myotropic Trypanosoma cruzi clone induces maternal fertility failure. In the current work, we evaluated whether reduction of maternal parasitaemia before mating has beneficial effects on pregnancy outcome. Female mice were subjected to benznidazole (Bz) treatment after infection. On day 30 of therapy, mating was assessed and pregnancy outcome was determined on day 14 of gestation. Fetal resorptions diminished in T. cruzi-infected Bz-treated mice compared with T. cruzi-infected untreated mice. This was in agreement with the reduction in the blood/solid tissue parasite load and with the percentage of necrotic foci in placental samples from T. cruzi-infected Bz-treated females. To study eventual changes in the immune homeostasis of T. cruzi-infected Bz-treated mice, activation of the immune system was evaluated at the end of Bz therapy and before mating. We found specific IgG1 reduction resulting in a predominance of specific IgG2a, reduced numbers of CD69+ CD4+ cells and diminished frequency and numbers of CD44+ T cells. Concanavalin A-stimulated splenocytes from T. cruzi-infected Bz-treated mice produced higher amounts of IFN-gamma than T. cruzi-infected untreated mice. These results indicate that reduction of maternal parasite load improves pregnancy outcome. These findings correlate with a favourable modulation of the immune response.

Behavior of atrial natriuretic factor in an experimental model of Trypanosoma cruzi infection in rats.

Enhanced atrial natriuretic factor (ANF) production by the heart is related to hemodynamic overload, cardiac growth, and hypertrophy. The heart is one of the most affected organs during Trypanosoma cruzi infection. We tested the hypothesis that myocarditis produced by parasite infection alters the natriuretic peptide system by investigating the behavior of plasma ANF during the acute and chronic stages of T. cruzi infection in rats. Sprague-Dawley rats were infected with T. cruzi clone Sylvio-X10/7. Cardiac morphology showed damage to myocardial cells and lymphocyte infiltration in the acute phase; and fibrosis and cell atrophy in the chronic period. Plasma ANF levels (radioimmunoassay) were significantly higher in acute (348 +/- 40 vs. 195 +/- 36 pg/ml, P < 0.05, n = 17) and chronic T. cruzi myocarditis (545 +/- 81 vs. 229 +/- 38 pg/ml, P < 0.001, n = 11) than in their respective controls (n = 10 and 14). Rats in the chronic phase also showed higher levels than rats in the acute phase (P < 0.01). The damage of myocardial cells produced by the parasite and the subsequent inflammatory response could be responsible for the elevation of plasma ANF during the acute period of T. cruzi infection. The highest plasma ANF levels found in chronically infected rats could be derived from the progressive failure of cardiac function.

Alterations in cardiac beta-adrenergic receptors in chagasic mice and their association with circulating beta-adrenoceptor-related autoantibodies.

OBJECTIVE: Cardiac tissue from chagasic mice was studied to evaluate the expression and biological activity of beta-adrenoceptors in association with circulating beta-adrenoceptor-related autoantibodies. METHODS: BALB/c inbred mice that were either treated or not treated with atenolol (2.5 mg/kg) and infected or not infected with 1 x 10(4) trypomastigotes (CA-1 strain) were sacrificed weekly up to week nine. Morphological, binding and contractility studies were performed on the four different groups of animals. The effect of their serum antibodies was also assayed in binding and contractility studies on normal heart preparations. RESULTS: Hearts from chagasic myocarditis mice showed a beta-adrenoceptor-related dysfunction, with a decrease in heart contractility, impaired response to exogenous beta-adrenoceptor agonist and a significant reduction in beta-adrenergic binding sites. Those effects were maximum at eight-nine weeks post-infection and were improved by treating infected mice with atenolol. In addition, serum or IgG from chagasic myocarditis mice was capable of interacting with cardiac beta-adrenoceptors, reducing the number of binding sites and inhibiting the contractile response to exogenous norepinephrine. IgG effects that were observed in normal myocardium, were highest in sera from mice eight-nine weeks post-infection and correlate with the degree of myocarditis. Moreover, chagasic autoantibodies from infected mice recognized a peptide corresponding to the sequence of the second extracellular loop of the human beta 1-adrenoceptor. CONCLUSIONS: (1) The development of alterations in beta-adrenergic receptors, related to cardiac dysfunction, may be associated with the presence of circulating antibodies against these receptors and (2) it is possible that the chronic deposits of these autoantibodies in cardiac beta-adrenoceptors could lead to a progressive blockade with sympathetic denervation, a phenomenon that has been described in the course of chagasic myocarditis.

[Myocardial cell response to Trypanosoma cruzi infection]. / Respuesta de las células musculares cardíacas a la infeccion con Trypanosoma cruzi.

The aim of this study is to establish the response of cardiac myocytes to the infection with Trypanosoma cruzi. The role of myocardial cell proliferation on heart remodelation and the ability of these cells to produce nitric oxide and control intracellular parasite growth during T. cruzi infection were evaluated. The presence of proliferating cell nuclear antigen (PCNA) was determined in myocardial cells of Wistar rats infected with T. cruzi, resulting in a significant increase of PCNA+ labelling in all stages of disease. The ability of myocardial cells to control growth of intracellular parasites and the production of nitric oxide were evaluated in cultures of cardiac myocytes obtained from neonatal rats. Different combinations of cytokines were added to culture media. The number of cardiac cells displaying intracellular amastigotes was lower in cultures supplemented with IL-1b, TNF-a and IFN-g than with other cytokine combinations and controls. The addition of cytokines resulted also in an increase of nitric oxide production in both infected and non-infected controls. These results demonstrate that myocardial cells participate actively in the response of the heart to the infection with T. cruzi.

Glycosylphosphatidylinositols are required for the development of Trypanosoma cruzi amastigotes.

Induction of a glycosylphosphatidylinositol (GPI) deficiency in Trypanosoma cruzi by the heterologous expression of Trypanosoma brucei GPI-phospholipase C (GPI-PLC) results in decreased expression of major surface proteins (N. Garg, R. L. Tarleton, and K. Mensa-Wilmot, J. Biol. Chem. 272:12482-12491, 1997). To further explore the consequences of a GPI deficiency on replication and differentiation of T. cruzi, the in vitro and in vivo behaviors of GPI-PLC-expressing T. cruzi were studied. In comparison to wild-type controls, GPI-deficient T. cruzi epimastigotes exhibited a slight decrease in overall growth potential in culture. In the stationary phase of in vitro growth, GPI-deficient epimastigotes readily converted to metacyclic trypomastigotes and efficiently infected mammalian cells. However, upon conversion to amastigote forms within these host cells, the GPI-deficient parasites exhibited a limited capacity to replicate and subsequently failed to differentiate into trypomastigotes. Mice infected with GPI-deficient parasites showed a substantially lower rate of mortality, decreased tissue parasite burden, and a moderate tissue inflammatory response in comparison to those of mice infected with wild-type parasites. The decreased virulence exhibited by GPI-deficient parasites suggests that inhibition of GPI biosynthesis is a feasible strategy for chemotherapy of infections by T. cruzi and possibly other intracellular protozoan parasites.

Trypanosoma cruzi infection in MHC-deficient mice: further evidence for the role of both class I- and class II-restricted T cells in immune resistance and disease.

The role of T cell population in immune control of Trypanosoma cruzi infection and subsequent development of disease has been examined using gene knockout mice deficient in the expression of either or both class I and Class II MHC. Mice deficient in either class I- or class II-restricted T cell populations show a striking similarity in their mortality rate, parasite load and tissue inflammatory response following infection with the Brazil strain of T. cruzi. In both cases all animals died during the acute phase of the infection with high parasitemias and high parasite loads in the heart and skeletal muscle, but with reduced tissue inflammatory response. Mice deficient in both class I and class II MHC expression demonstrated even higher numbers of circulating and tissue parasites, essentially non-existent tissue inflammatory responses, and succumbed to infection earlier than single-deficient mice. MHC class I-deficient mice which survive into the chronic phase following infection with the M/78 or M/80 clones of T. cruzi have both relatively higher tissue parasite loads and more extensive and severe inflammatory responses than control immunocompetent mice. Immunologically, the acute infection in the double-deficient mice was accompanied by a marked increase in CD4(-)CD8(-)alphabetaTCR+ cells in the spleen. Surprisingly, both class I- and class II-deficient mice produce detectable but sub-normal levels of anti-parasite antibodies while double-deficient mice produced little to no detectable anti-parasite antibody. These results establish the importance of both class I- and class II-restricted T cells in immune control of circulating blood stages and intracellular states of T. cruzi. In addition, this work reinforces the relationship between tissue parasite load and the severity of the inflammatory lesions in chronically infected animals.

In vitro culture of cardiac mast cells from mice experimentally infected with Trypanosoma cruzi.

In this study we describe a mast cell population obtained by in vitro culture of hearts of Trypanosoma-cruzi-infected mice. Heart tissue was placed in culture and observed daily for the efflux of cells. Mast cells appeared in the medium during the 1st week of culture in the area immediately surrounding the tissues and increased in numbers over time. The cells were identified as mast cells by electron microscopy and by positive staining of granules with Giemsa, toluidine blue, and berberine sulfate techniques. Histopathological analysis of the cultured heart fragments from infected mice showed numerous mast cells, located mostly in areas where the histologic structure was abnormal and surrounded by fibrous connective tissue. This is the first report on in situ proliferation and migration of mast cells form inflamed heart tissues. In situ grown mast cells might be useful in developing in vitro models to study the role of these cells in T. cruzi-induced and other myocardiopathies.

Depletion of T-cell subpopulations results in exacerbation of myocarditis and parasitism in experimental Chagas' disease.

The contribution of T-cell subpopulations to immunopathology in murine Trypanosoma cruzi infection was studied by using in situ localization of lymphocytes and in vivo depletion of T-lymphocyte populations. CD8+ T cells were the major lymphocyte population in the inflamed hearts of C3H/HeSnJ mice infected with the Sylvio X10/4 clone of T. cruzi at all time points of the acute and chronic phases of the infection examined. Depletion of CD8+ and/or CD4+ T cells beginning on day 20 of the infection resulted in a moderate decrease in the inflammation and an increase in parasite burden in the hearts of mice at day 30 of infection. Longer-term depletion, beginning at day 20 and extending as long as 200 days of infection, resulted in an increased inflammatory response in the heart. A large proportion of the inflammatory cells in the hearts of anti-CD8- or anti-CD4- and anti-CD8-treated mice were Thy1+ and CD4- CD8-. At 200 days of infection, the increased inflammation was accompanied by an increase in the parasite load in the heart. These results show that T-cell subset depletion does not prevent the inflammatory response associated with acute and chronic T. cruzi infection. The increased parasite load in T-cell-depleted mice also demonstrates the participation of these T-cell subsets in regulation of parasite load throughout the course of the infection. The increased inflammatory response despite T-cell depletion and in association with increased numbers of tissue parasites suggests that intracellular parasites are a driving force behind the inflammatory response in chronic murine T. cruzi infection.

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.

T lymphocytes from T. cruzi-infected mice alter heart contractility: participation of arachidonic acid metabolites.

T lymphocytes from T. cruzi infected mice susceptible to the development of myocarditis altered the contractility of normal mouse atria in vitro. While lymphocytes obtained from normal mice had no effect, lymphocytes from T. cruzi-infected mice cultured with normal atria induced negative or positive inotropic effects depending upon the post-infection period; negative inotropism was induced by lymphocytes obtained from animals at 1 to 4 weeks post-infection, and positive inotropism was induced by lymphocytes taken at 7 to 14 weeks post-infection. These effects were mediated by soluble factors as evidenced by the ability of lymphocyte culture supernatants to alter contractility. Cell enrichment experiments indicated that T lymphocytes rather than B lymphocytes were responsible for these inotropic effects. Lyt(2+)-enriched T lymphocytes were found to be responsible for triggering the negative inotropic effect at 3 weeks post-infection when myocarditis was less intense, whereas Lyt1(+)-enriched T lymphocytes induced the positive inotropic effect at 8 weeks after T. cruzi infection when myocarditis was severe. Furthermore, inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism blunted the negative inotropic effect while inhibitors of lipoxygenase pathway inhibited the positive inotropic effect. PGE2 was found to be spontaneously released by Lyt(2+)-enriched T cells obtained at 3 weeks post-infection while LTC4 was released by atria cultured in the presence of Lyt 1+ T cells obtained at 8 weeks post-infection. In conclusion, these findings suggest that infiltrating T lymphocytes may contribute to myocardial dysfunction during T. cruzi infection by releasing or inducing the release of harmful arachidonic acid metabolites such as PGE2 and LTC4 which alter normal cardiac function.

Studies of Trypanosoma cruzi clones in inbred mice. III. Histopathological and electrocardiographical responses to chronic infection.

Histopathological and electrocardiographical (ECG) changes occur in the heart of C3H/HeN and C57BL/6 mice infected for 1 year with Trypanosoma cruzi clones Sylvio-X10/4 (X10/4), Miranda/78 (M/78), or Miranda/80 (M/80). Heart parasitism and a variable degree of inflammation occurred following infection with clones X10/4 or M/78 but not with M/80. Clone X10/4 caused more extensive myocardial inflammation and fibrosis than clone M/78. Myocardial fibrosis was more extensive in C3H than in C57 mice infected with clone X10/4. The normal ECG pattern of C3H mice is distinctly different from C57 mice. The PR intervals of mice infected with clone X10/4 greater than M/78 greater than M/80 approximately equal to controls. ECG abnormalities occurred more frequently in mice infected with clone X10/4 than in controls or mice infected with either M/78 or M/80 regardless of strain or sex and were generally more severe in C57 than in C3H infected with X10/4. First degree atrioventricular block occurred more frequently in C3H mice infected with clone X10/4 or M/78 and C57 mice infected with X10/4 than in all other groups. Complete atrioventricular dissociation occurred frequently in C57 mice infected with X10/4 and rarely in other mice. These results demonstrate that the myocardial response of mice to T. cruzi infection, both histological and electrophysiological, is modulated by both the mouse strain and the parasite isolate used.

Comparative studies of the infection of Lewis rats with four Trypanosoma cruzi clones.

Quantitative and qualitative differences in parasitaemia levels and histopathological characteristics occurred in Lewis rats infected with four Trypanosoma cruzi clones. Rats infected with clone Sylvio-X10/7 sustained high parasitaemias and died during the acute phase whereas rats infected with clones Sylvio-X10/4, Miranda/78 or Miranda/80 did not. Myocardial fibrosis was more extensive in rats chronically infected with clone X10/4 than in rats infected with the 2 Miranda clones. Myocardial fibrosis developed more rapidly in rats than has been reported in mice. These characteristics are advantageous in developing animal models of human chagasic myocardiopathy.

A histopathological analysis of the course of myocarditis in C3H/He mice infected with Trypanosoma cruzi clone Sylvio-X10/4.

The histopathology of hearts of C3H/He (MTV-) mice infected for 2 to 440 days with Trypanosoma cruzi clone Sylvio-X10/4 was examined. Inflammation was present in most hearts at 14 days post-infection (d.p.i.) and persisted thereafter. Myocardial fibrosis, which was first seen 45 d.p.i. and was more marked after 200 d.p.i., developed at a time when few parasites were present. Fibrosis predominated in subepicardial and subendocardial myocardium, areas of most severe inflammation. Parasites were restricted to myocardial fibres and were found in all mice infected for 14 to 30 days and, in fewer numbers, in 54% of mice infected for more than 30 days. T. cruzi-induced myocarditis in mice was not a self-resolving disease but a dynamic, active process occurring continuously over a long time following infection. This clone-derived T. cruzi stock produced a myocarditis in inbred mice similar to that seen in human chronic Chagas' disease.

Trypanosoma cruzi: constancy of clone pathogenicity for inbred mice during long-term in vitro maintenance.

The reproducibility of infection of C3H/He mice with T. cruzi clones Sylvio-X10/4 and Sylvio-X10/7 maintained in the laboratory for 946 and 496 days respectively was assayed. Clone X10/7 from 15 different in vitro passages consistently induced an acute lethal infection (94.3% mortality) and constant survival time (Mean = 24.6 d.p.i.). Female mice survived significantly longer than males and mice older than 30 days at the time of infection survived significantly longer than younger mice. The mortality of mice infected with clone X10/4 from 23 different in vitro passages was lower (5.1%) and their survival longer (Mean = 42.7 d.p.i.) than mice infected with clone X10/7. High anti-T. cruzi IgG titres were detected in the plasma of all mice killed after 30 d.p.i. Histologically, the heart, skeletal muscles and/or large intestine contained intracellular parasites in 59% of the mice; parasites were found in mice of all groups tested. The hearts of all mice were comparably inflammed regardless of parasite passage number or duration of infection. These data demonstrate that the presentation and course of infection of inbred mice with two T. cruzi clones is not changed by either the duration or protocol used for in vitro maintenance; the reported loss or change in virulence of T. cruzi strains during long-term in vitro maintenance did not occur. Consequently, T. cruzi clones and inbred mice provide a dependable and reproducible means to study host-parasite interactions.

Studies of Trypanosoma cruzi clones in inbred mice. II. Course of infection of C57BL/6 mice with single-cell-isolated stocks.

A study of the course of parasitemia and mortality of C57BL/6 mice infected with the Trypanosoma cruzi Sylvio-X10 strain and two single-cell-isolate clones of the strain confirms our previous report on the existence of intra-strain subpopulations differing in their pathogenicity for inbred mice. In contrast to the generally accepted pattern of mouse strain susceptibility, C57BL/6 mice are more susceptible than C3H/HeN mice when infected with the Sylvio-X10 isolates. In addition, sex-related differences in susceptibility occurred depending upon the strain of the mouse and parasite isolate used. These data infer an interplay of host and parasite genetic factors influencing the outcome of a mouse infection with T. cruzi.

Studies of Trypanosoma cruzi clones in inbred mice. I. A comparison of the course of infection of C3H/HEN- mice with two clones isolated from a common source.

Two single-cell-isolate cloned stocks of the Sylvio-X10 strain, recovered from an acute human Trypanosoma cruzi infection, were used to infect C3H/HEN mice. The Sylvio-X10/4 clone produced a chronic infection in mice; clone Sylvio-X10/7 produced an acute lethal infection under identical experimental conditions. The course of infection of mice with the Sylvio-X10/7 clone was characterized by higher peripheral blood parasitemia and greater tissue involvement, an earlier appearance of specific anti-T. cruzi plasma IgG and shorter survival times than in mice infected with the Sylvio-X10/4 clone. The course of infection in mice with the Sylvio-X10 strain was intermediate between that of the two clones. This is the first demonstration of the pluripotential pathogenetic nature of a T. cruzi strain due to genetic heterogeneity of the population of parasites that constitute the strain. This experimental system is highly stable and reproducible. Consequently, the use of inbred mice and T. cruzi clones appears to provide an excellent model to study factors which influence the course of Chagas' disease.