Gastric acid secretion response in the Cebus apella. A monkey model of chronic Chagas' disease.

The objective was to study the secretory pattern, both basal and stimulated either by histamine (0.1 mg/kg) or pentagastrin (64 micrograms/kg) in eighteen Cebus apella monkeys chronically infected with different T. cruzi strains (CA1, n = 10; Colombian, n = 4 and Tulahuen, n = 4) and to describe the morphological findings in the gastrointestinal tract in twelve infected (6 sacrificed and 6 spontaneously dead) and four healthy monkeys. All infected monkeys and 35 healthy ones were evaluated by contrast X-ray examination. No differences were observed in basal acid output between control and infected groups. Animals infected with the Tulahuen and Colombian strains showed significant lower values of peak acid output in response to histamine or pentagastrin (p < 0.01 and p < 0.05 respectively; "t" test) in comparison to the controls. Barium contrast studies showed enlargement and dilatation of the colon in three infected animals. Histopathological lesions were seen in 75% of the autopsied animals either in colon alone (33%) or both, in colon and esophagus (42%). The normal secretion observed in the CA1 infected group could be due to a lower virulence of the strain, a lower esophageal tropism or the necessity of a longer post-infection time to cause lesions.     

CTLA-4 regulates the murine immune response to Trypanosoma cruzi infection

Infection with Trypanosoma cruzi causes a profound suppression of T cell responsiveness to polyclonal or antigenic stimuli. In this study, we quantified expression of the negative T cell regulatory molecule CTLA-4 in T. cruzi infected mice and analysed its influence on the immune suppression. Levels of splenic CTLA-4 expression were highest around day 10 after infection, reaching 5% in resistant B6D2F1 mice, but exceeding 10% of CD4(+) T cells in C57BL/6 mice that were susceptible to mortal disease. The proliferative response of explanted splenocytes to CD3-mediated stimulation was strongly suppressed in both the susceptible and the resistant strains. Blockade of CTLA-4 in vitro with a monoclonal antibody affected neither proliferative response nor cytokine production (IFN-gamma, IL-4 and IL-2) by splenic T cells from infected C57BL/6 mice. Treatment of mice with anti-CTLA-4 antibody on the day of infection decreased IFN-gamma production and reduced mortality by about 50%. We conclude that high CTLA-4 expression is a hallmark of severe disease in murine T. cruzi infection, and that CTLA-4 has a regulative influence at the early stages during priming of the immune reaction to the parasite, augmenting a strong Th1-biased response.     

Cyclophosphamide adjuvant arthritis in Trypanosoma cruzi infected rats with inflammatory cytokine effects

OBJECTIVE: To analyze whether the cyclophosphamide (CYC) induced reestablishment of adjuvant arthritis (AA) in chronically Trypanosoma cruzi infected rats correlates with changes in the secretion of pro- and antiinflammatory cytokines by popliteal lymph node cells. METHODS: Inbred "l" rats infected with T. cruzi 90 days earlier and age matched controls were given CYC (25 mg/kg body weight) or physiologic saline 48 h before arthritis induction. Popliteal lymph node cells were collected at the time of AA induction (48 h after CYC treatment) or during the peak response, to study the concanavalin-A (ConA) or Mycobacterium tuberculosis-driven in vitro proliferation of several cytokines in their culture supernatants. Results. Infected rats given CYC were recovered from the otherwise decreased ConA induced proliferation seen at the time of peak AA. The CYC mediated reestablishment of AA in T. cruzi infected rats coexisted with an increased presence of tumor necrosis factor-a in supernatants from either antigen or ConA stimulated cultures as well as interleukin 12 (IL-12) in the latter case. CYC also lowered to normal the increased IL-10 levels from ConA stimulated cultures that the T. cruzi group displayed at the time of inducing AA. Conclusion. The process by which CYC restores the clinical expression of AA affects the balance between cytokines that influence the regulation of arthritis in favor of the inflammatory component.     

Activity of cholinesterases and adenosine deaminase in blood and serum of rats experimentally infected with Trypanosoma cruzi

This study aimed to evaluate the activity of cholinesterases and adenosine deaminase (ADA) in blood and serum of rats infected with Trypanosoma cruzi. Twelve adult rats were used in the experiment divided into two uniform groups. Rodents from group A (control group) were non-infected and animals from group B served as infected, receiving intraperitoneally 3·3×10(7) trypomastigotes/each. Blood collection was performed at days 60 and 120 post-infection (PI) in order to evaluate the hemogram, blood activity of acetylcholinesterase, and serum butyrylcholinesterase and ADA activities. Hematological parameters did not differ between groups. A significant increase (P<0·05) of acetylcholinesterase activity was observed in blood while butyrylcholinesterase had a significant reduction (P<0·01) in serum of infected rats at days 60 and 120 PI. ADA activity in serum showed an inhibition in infected animals when compared to non-infected at day 120 PI. Based on these results, it is possible to conclude that the activity of cholinesterases and ADA were changed in animals infected with T. cruzi. The possible causes of these alterations will be discussed in this paper.     

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.     

Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells

Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.     

The effects of Lampit (Bayer 2502) on the interaction of Trypanosoma cruzi with vertebrate cells in vitro.

The effects of 3-methyl-4-(5'-nitrofurfurylidene-amino)-tetrahydro-4H-1,4-thiazine-1,1-dioxide [Lampit, Bayer 2502] on the intracellular cycle of Trypanosoma cruzi were studied with an in vitro steady-state culture system that permitted the continuous analysis of individual host cell-parasite interactions. Lampit concentrations of 10(-4) and 10(-5) M significantly affected the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations above 10(-4) M were toxic to the host cell population and Lampit concentrations below 10(-5) M did not affect the ability of trypomastigotes to penetrate vertebrate cells. Lampit concentrations of 10(-5) M or greater completely inhibited the intracellular cycle of T. cruzi whereas Lampit concentrations of 10(-7) M or less had no effect on the intracellular cycle. Continuous perfusion with 10(-6) M Lampit resulted in a linear increase in intracellular parasite reproduction time. A stable strain of T. cruzi was produced in vitro that was resistant to a 100-fold greater concentration of Lampit that the parent strain.     

The course of trypanosoma cruzi infection in nonsplenectomized SPE rats with and without Haemobartonella muris infection (author's transl)]

Rats were infected with H. muris from the first to the second week of their life. Four weeks later 20 of the infected rats were inoculated with 1.5 x 10(5) T. cruzi. Control animals were given the same number of parasites. In both experimental groups no hemoflagellate could be seen in blood smears four weeks after infection. The comparison of the course of infection of T. cruzi in rats with and without H. muris was made using t- and Wilcoxon-Test for Pair Differences. Rats with H. muris infections showed a low development of T. cruzi up to the 16th day of infection with this flagellate (P less than 0.0005). This difference in the development of the trypanosomes is lost from the 17th to the 27th day after infection.     

Food restriction in female Wistar rats. VI. Effect of reduced glutathione on the proliferative response of splenic lymphocytes from ad libitum fed and food restricted animals

The effect of reduced glutathione (GSH) on the proliferative response of splenic lymphocytes from young, adult and old ad libitum (AL) fed as well as from old food-restricted rats was investigated. Food restriction was applied on an every-other-day schedule (EOD) starting from the age of 3.5 months. As was expected, the cells from EOD fed animals responded to concanavalin A (Con A) much better than those from age-matched ad libitum fed rats. The presence of the antioxidant GSH in the culture medium increased the response of lymphocytes in all the models taken into account; furthermore, it decreased the differences due to aging and application of food restriction. According to present knowledge, mitogenic stimulation induces free radical production, and GSH has, among others, a strong antioxidant activity. Thus, present data suggest that splenocytes from EOD animals tolerated the peroxidative stress resulting from mitogenic stimulation better than those from AL fed ones.     

Lymphoid abnormalities in rats with adjuvant-induced arthritis. I. Mitogen responsiveness and lymphokine synthesis.

Lewis rats injected in the hind paw with Mycobacterium butyricum develop a severe polyarthritis which shares certain features in common with rheumatoid arthritis in man. Spleen and peripheral blood mononuclear cells from rats with this form of arthritic disease proliferate poorly in vitro in response to concanavalin A (con A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM). The splenic hyporesponsiveness appears within four days of M. butyricum injection (three to five days prior to the development of detectable arthritis), reaches a peak 16-22 days following injection, and persists for at least 40 days. Buffalo strain rats injected with M. butyricum do not develop arthritis, and their spleen cells respond normally to con A, PHA, and PWM. In response to lipopolysaccharide (LPS) the synthesis of interleukin 1 (IL-1) by spleen or peritoneal macrophages from arthritic Lewis rats equalled or exceeded that of macrophages from normal rats. In contrast splenic T cells from arthritic rats produced reduced amounts of interleukin 2 (IL-2; T cell growth factor) in response to stimulation with PHA or con A. Moreover, con-A-activated spleen cells from arthritic rats failed to bind IL-2 and to respond to this growth factor with increased 3H-TdR uptake as did normal spleen cells. In-vitro treatment of 'arthritic' cells with 10(-5) M indomethacin did not restore to normal their reduced mitogen responsiveness, and spleen cells from normal and arthritic rats were equally sensitive to the inhibitory effects of prostaglandin E2 on con-A-induced proliferative responses. These results indicate that peripheral lymphoid function is compromised in rats with adjuvant-induced arthritis and that this functional deficit is mediated by aberrant synthesis of and response to IL-2 by T cells of arthritic animals.     

A complement-fixing antigen from Trypanosoma cruzi grown in cell cultures.

A complement-fixing (CF) antigen was prepared from amastigotes and trypomastigotes of Trypanosoma cruzi (Ernestina strain) grown in beef embryo cell cultures. Multiple lots of the antigen, which consisted of a supernate of washed and disrupted organisms, required material from 10(6) to 10(7) total organism per ml for optimum CF activity. Antibody at dilutions up to 1:256 was demonstrable in various sera from infected animals or patients. Contaminating beef cells from infected cultures were shown to be partly responsible for crossreactions of the antigen by CF with sera from cases of cutaneous leishmaniasis in whom concomitant infection with T. cruzi could be excluded. There were no cross-reactions with syphilitic sera and the frequency of positive reactions with normal sera was very low. Some characterisitics of the antigen included stability to storage at -20 degrees C and -70 degrees C for months, inactivation at 60 degrees C and by lyophilization, and an estimated molecular size of between 50,000 and 100,000 on the basis of membrane filtration.     

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.     

Congenital transmission of Trypanosoma cruzi is associated with maternal enhanced parasitemia and decreased production of interferon- gamma in response to parasite antigens

The conditions and mechanisms of congenital transmission of Trypanosoma cruzi remain largely unknown. In the present study, we compared the parasitic loads and the immune responses of pregnant T. cruzi-infected women who transmitted parasites to their fetus ("M+B+ mothers") with those of such women who did not transmit parasites to their fetus ("M+B- mothers"). M+B+ mothers had a higher frequency of positive results of hemoculture for T. cruzi than did M+B- mothers, in association with depressed production of parasite-specific interferon- gamma by blood cells that persisted after delivery. In contrast, the production of interleukin (IL)-2, IL-4, and IL-10 and transforming growth factor- beta 1 was similar between both groups of infected mothers, after stimulation with T. cruzi lysate. Flow cytometric analysis showed that T cells and monocytes of M+B+ mothers were less activated than were those of M+B- mothers. Altogether, these results indicate that congenital transmission of T. cruzi is associated with high parasitic loads and peripheral deficient immunological responses in mothers.     

Passive immunization of mice against Trypanosoma cruzi using convalescent mouse serum.

Groups of 10 mice were infected by the s.c. injection of blood trypomastigotes of Trypanosoma cruzi, strains "Y" and "Tulahuen". They were injected i.p. with convalescent mouse-anti-T. cruzi serum (CMATS) at various times. A single dose of CMATS (Y) was most effective when injected one day after homologous infection -31 of 40 mice survived the infection, whereas all controls, both untreated and injected with normal serum, died. CMATS was significantly less effective in treating mice infected with the Tulahuen strain. Sera from animals immunized with killed epimastigotes or trypomastigotes showed only very slight passive immunizing properties.     

In-vivo treatment with benznidazole enhances phagocytosis, parasite destruction and cytokine release by macrophages during infection with a drug-susceptible but not with a derived drug-resistant Trypansoma cruzi population

To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y strain (drug-susceptible) of Trypanosoma cruzi and a drug-resistant parasite population derived from the same strain. Trypomastigotes isolated from untreated infected mice, as well as, 3 h after treatment with BZ were incubated with inflammatory macrophages and used to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RN!) synthesis. Phagocytosis and destruction of the drug-susceptible parasites were significant/v enhanced by drug treatment. These enhancements were accompanied by an increase in cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)alpha] and RNI release by murine inflammatory macrophages primed with IFN-gamma. In contrast, BZ treatment of mice infected with drug-resistant T. cruzi population showed no effect whatsoever. The synthesis of IFN-gamma and RNI by splenocytes of mice infected with either susceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. On/v the splenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFN-gamma and RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosis and the production of cytokines and RN!, thus, favouring the destruction of the intracellular parasites by the cellular compartment of the immune system.     

Pasteurization of human milk to prevent transmission of Chagas disease.

Although admittedly transmission of Trypanosoma cruzi infection through breastfeeding is a rare event, it involves serious risks. To test the effectiveness of pasteurization in preventing this mode of infection, three sets of samples of human milk were tested: a - contaminated with T. cruzi and pasteurized; b - contaminated with T. cruzi and non-pasteurized; c - non-contaminated and pasteurized. Samples from all sets were orally and intraperitoneally administered to 90 BALB/c mice. The animals inoculated with contaminated, non-pasteurized samples, got the infection. Controls and the animals inoculated with contaminated and pasteurized milk were not infected. The hypothesis was accepted that pasteurization inactivates T. cruzi trypomastigotes.     

Trypanosoma cruzi infection in dogs and cats and household seroreactivity to T. cruzi in a rural community in northeast Brazil.

The prevalence of Trypanosoma cruzi parasitemia as determined by xenodiagnosis on domestic dogs and cats was correlated with household rates of seroreactivity to T. cruzi and household Panstrongylus megistus infestation in a rural area in northeast Brazil where P. megistus was the only domiciliary triatomine vector. T. cruzi infection was present in about 18% of domestic dogs and cats. Two-thirds of seroreactive children below age 10 resided in houses with T. cruzi-infected animals. In houses with a T. cruzi-infected dog or cat, as well as at least one infected P. megistus, the household rate of seroreactivity to T. cruzi was five times greater than in houses with non-infected domestic animals and no infected triatomine vectors. Domestic dogs and cats are important reservoirs of T. cruzi in an endemic area where P. megistus is the only domiciliary triatomine vector.     

Thymocyte depletion in Trypanosoma cruzi infection is mediated by trans-sialidase-induced apoptosis on nurse cells complex

Trypanosoma cruzi, the causative agent of Chagas' disease, induces transient thymic aplasia early after infection-a phenomenon that still lacks a molecular explanation. The parasite sheds an enzyme known as trans-sialidase (TS), which is able to direct transfer-sialyl residues among macromolecules. Because cell-surface sialylation is known to play a central role in the immune system, we tested whether the bloodstream-borne TS is responsible for the thymic alterations recorded during infection. We found that recombinant TS administered to naive mice was able to induce cell-count reduction mediated by apoptosis, mimicking cell subsets distribution and histologic findings observed during the acute phase of the infection. Thymocytes taken after TS treatment showed low response to Con A, although full ability to respond to IL-2 or IL-2 plus Con A was conserved, which resembles findings from infected animals. Alterations were found to revert several days after TS treatment. The administration of TS-neutralizing Abs to T. cruzi-infected mice prevented thymus alterations. Results indicate that the primary target for the TS-induced apoptosis is the so-called "nurse cell complex". Therefore, we report here supporting evidence that TS is the virulence factor from T. cruzi responsible for the thymic alterations via apoptosis induction on the nurse cell complex, and that TS-neutralizing Abs elicitation during infection is associated with the reversion to thymic normal parameters.     

Depressed adjuvant arthritis in chronically Trypanosoma cruzi infected rats: reversal by cyclophosphamide.

Chronically Trypanosoma cruzi infected "I" rats and syngeneic naive recipients, transferred with a T cell enriched spleen cell population from infected donors, develop an attenuated arthritis when challenged with complete Freund's adjuvant. We report that cyclophosphamide, 40 mg/kg body weight, given 48 h before induction, was able to reestablish or exacerbate adjuvant arthritis in infected and control rats, respectively. Although the T cell enriched spleen cells from infected donors continued to down regulate adjuvant arthritis in syngeneic recipients given cyclophosphamide 48 h before cell transfer, treatment of infected donors with cyclophosphamide, 48 h before cell collection, prevented these cells from exerting such effect when transferred to healthy recipients receiving no cyclophosphamide. It is suggested that cyclophosphamide may primarily affect a suppressor cell population, present in the infected host, with regulatory activity on adjuvant arthritis.