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Trypanosoma cruzi, the causing agent of Chagas disease, leads to an activation of the immune system in congenitally infected infants. In this study, we measured a set of cytokines/chemokines and the levels of parasitemia by quantitative PCR in the circulation of neonates born to T. cruzi-infected mothers to evaluate the predictive value of these mediators as biomarkers of congenital transmission. We conducted a retrospective cohort study of 35 infants with congenital T. cruzi infection, of which 15 and 10 infants had been diagnosed by detection of parasites by microscopy in the first and sixth month after delivery, respectively, and the remaining 10 had been diagnosed by the presence of T. cruzi-specific Abs at 10-12 mo old. Uninfected infants born to either T. cruzi-infected or uninfected mothers were also evaluated as controls. The plasma levels of IL-17A, MCP-1, and monokine induced by IFN-γ were increased in infants congenitally infected with T. cruzi, even before they developed detectable parasitemia or seroconversion. Infants diagnosed between 6 and 12 mo old also showed increased levels of IL-6 and IL-17F at 1 mo of age. Conversely, infants who did not develop congenital T. cruzi infection had higher levels of IFN-γ than infected infants born to uninfected mothers. Monokine induced by IFN-γ, MCP-1, and IFN-γ production induced in T. cruzi-infected infants correlated with parasitemia, whereas the plasma levels of IL-17A, IL-17F, and IL-6 were less parasite load dependent. These findings support the existence of a distinct profile of cytokines and chemokines in the circulation of infants born to T. cruzi-infected mothers, which might predict congenital infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Chagas congenital infection is an important health problem in endemic and non-endemic areas in which Trypanosoma cruzi-infected women can transmit the parasite to their offspring. In this study, we evaluated the antibody levels against the T. cruzi Shed Acute Phase Antigen (SAPA) in 91 binomial samples of seropositive pregnant women and their infected and non-infected children by ELISA. In 70 children without congenital T. cruzi transmission, the titers of anti-SAPA antibodies were lower than those of their seropositive mothers. In contrast, 90.5% of 21 congenitally infected children, at around 1 month of age, showed higher anti-SAPA antibody levels than their mothers. Subtracting the SAPA-ELISA mother OD value to the SAPA-ELISA child OD allowed efficient detection of most T. cruzi congenitally infected children immediately after birth, when total anti-parasite antibodies transferred during pregnancy are still present in all children born to seropositive women. A positive correlation was observed between parasitemia levels in mothers and infants evaluated by quantitative DNA amplification and anti-SAPA antibody titers by ELISA. As SAPA serology has proved to be very efficient to detect T. cruzi infection in mother-child binomial samples, it could be of extreme help for early diagnosis of newborns, in maternities and hospitals where DNA amplification is not available. This prompt diagnosis may prevent drop out of the long-term follow-up for future diagnosis and may ensure early trypanocidal treatment, which has proved to be efficient to cure infants with congenital Chagas disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10-12 months of age., Methodology and Principal Findings: Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI., Significance: This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.

The relationship between parasite burden and vertical transmission of Trypanosoma cruzi was studied in pairs of chronically infected women and their children in a non-endemic area. Parasitemia was quantified by quantitative polymerase chain reaction (qPCR) in the peripheral blood amplifying a nuclear T. cruzi DNA and expressed as equivalent amounts of CL Brener parasites DNA per ml (eP/ml). Similar levels of parasitemia were found in non-transmitting pregnant women and in non-pregnant women: 1.8 ± 0.5 and 1.5 ± 0.7 eP/ml, respectively. In women pregnant with infected children parasitemia was 11.0 ± 2.7 eP/ml (n=20). In 12 of their neonates the infection was detected by microscopic observation of the parasites in peripheral blood in the 1(st) month of age. These children had variable levels of parasitemia (13,000 ± 7000 eP/ml), that were about 600-fold higher than that found in their mothers. To our knowledge, this is the first quantitative evaluation of parasitemia in these three groups of women and in their congenitally infected children. These parasite quantifications could be a basis to plan the control of mother-to-child transmission of T. cruzi., (Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Objective: The presence of autoantibodies with adrenergic and cholinergic activity, capable of triggering neurotransmitter receptor-mediated effects, has been associated with pathogenesis in T. cruzi-infected hosts. The goal of this study was to investigate the production of anti-M2 muscarinic receptor autoantibodies (Anti-M2R AAbs) as well as the IFN-γ profile in children at the early stage of Chagas disease, and to examine whether trypanocidal chemotherapy with benzonidazole (BZ) could modify both response patterns., Methods: This study comprised 30 T. cruzi-infected children (mean age: 13.8 years) and 19 uninfected controls (mean age: 12.7 years). Infected patients were treated with BZ and followed-up. Blood samples collected at diagnosis-T0, end of treatment-T1, and six months later-T2 were analysed by ELISA for detection of Anti-M2R AAbs and circulating levels of IFN-γ., Results: At T0, anti-M2R AAbs were demonstrated in 56.7% of T. cruzi-infected patients, whereas uninfected controls were 100% negative. The average age of Anti-M2R AAbs(+) patients was higher than that from negative population. Infected children also displayed significantly stronger serum IFN-γ responses than controls. Upon BZ treatment, a significant linear decreasing trend in Anti-M2R AAb reactivity was recorded throughout the follow-up, with 29.7-88.1% decrease at T2. IFN-γ circulating levels also declined by T2., Conclusion: Anti-M2R AAbs and IFN-γ raise early during chagasic infection in children and are downmodulated by BZ therapy. These findings reinforce the usefulness of early BZ treatment not only to eliminate the parasite but also to reduce potentially pathogenic immune responses.

EC-MPS was designed to improve MPA-related GII because of MMF, by delaying the release of MPA until reaching the small intestine. At present, its immunosuppressive activity in pediatric renal transplant recipients with GII has not been clarified. We studied eight renal transplant recipients before and after three months of the conversion from MMF to equimolar doses of EC-MPS. After three months of treatment with EC-MPA, GII decreased between 100% and 12.5%. The predose levels of MPA were about 60% higher on EC-MPS (6.9 +/- 1.1 microg/mL) compared with MMF administration (4.2 +/- 0.9 microg/mL). Hemoglobin decreased significantly post-conversion (12.0 +/- 0.4 to 11.0 +/- 0.5 g/dL). Serum creatinine, creatinine clearance, and urinary protein excretion did not change. Also, proliferative response and cytotoxic antibodies showed no significant change. The release of interleukin-10 was strikingly augmented with MMF or EC-MPS therapy; meanwhile, gamma-interferon and TNF were low under both treatments. Our data indicate that conversion from MMF to EC-MPS leads to an improvement in GII without altering key elements of immunosuppression.

In the neonates born to T. cruzi infected mothers, the diagnosis of the congenital transmission relays on the detection of the parasites and/or the specific antibodies non-transferred by their mothers, in the absence of blood transfusion and vectorial transmission. In the early stage, approximately until the 7th month of life, when maternal immunoglobulins could be present, the diagnosis depends on the detection of the parasite. Then, in the late stage, from the 8th month, the detection of specific antibodies by at least 2 of 3 serological tests confirms the infection in the neonates. The diagnostic follow up of the children born to a group of sero-reactive pregnant women was carried out in the INP. The 11% of the mothers (29 out of 267) transmitted the infection to their children. The neonates of 20 of these mothers were diagnosed in the early stage, 14 and 6 in one or two controls, respectively. In the 9 remaining mothers the children were diagnosed in the late stage of the infection, mainly serologicaly. Our analisis of previously published reports stressed that the maternal-fetal transmission rate depends on the time of diagnostic follow up of the child. In this reports, mean values of mother to child transmission reported was 2% and 9% when the diagnosis of the neonates born to sero-reactive mothers was carried out only in the early stage or in the early and also the late stage, respectively.

Most congenital transmissions of Trypanosoma cruzi are not detected. As the levels of mediators regulating the immune response might be different in the absence or in the presence of transmission, we explored the levels of tumor necrosis factor (TNF) and soluble TNF receptors TNF-R1 and -R2 in T. cruzi-infected pregnant women and the neonates. We previously found that the circulating levels of TNF were higher in non-transmitting than in transmitting pregnant women. This observation has now been extended to the spontaneous release of TNF by peripheral blood leukocytes (PBLs) that was also higher in non-transmitting than in transmitting pregnant women. As their mothers, non-infected neonates had higher circulating levels of TNF than congenitally infected children. The circulating levels of sTNF-R1 increased in non-transmitting and transmitting mothers and in infected and non-infected neonates. The circulating levels of sTNF-R2 were approximately 60% higher in infected than in non-infected neonates (1,635 +/- 101 and 1,027 +/- 100 pg/mL, respectively) and remained higher at 1 year of age. This important increase, only observed in infected neonates, could be useful to orientate to the presence of vertical transmission of T. cruzi infection.

The outcome of the kidney allograft mainly depends on the immune response and on its complex regulation, where the cytokine network and other mediators play an important role. At present, kidney biopsy is the most useful tool for monitoring the transplant rejection and the diagnosis of the associated nephropathies, in spite of the invasiveness of the procedure. Thus, it is of great interest to find alternative tools for diagnosis. The evaluation of regulatory cytokines is a simple procedure of low cost that could be useful to increase the sensitivity of the detection of polymorphic differences, to predict the graft acceptance and for the early detection of rejection. Recent studies suggest that the high production of pro-inflammatory mediators, such as Th1 cytokines, could be detrimental, whereas the production of anti-inflammatory regulatory cytokines, such as interleukin (IL)-10 and tumor necrosis factor (TGF)-beta, could be beneficial for graft survival. In the early stages, the cellular cytotoxicity is activated by the Th1 response and the detection of cytotoxic molecules is associated to the acute rejection. Later, the balance between pro and anti-inflammatory mediators and the regulation of their levels could be more important. In this regard, TGF-beta is also fibrogenic and a high local production can contribute to kidney damage. On the other hand, the increased production of IL-10 in response to the allogeneic stimuli could be, in most cases, an important marker of long-term acceptance.

The aim of this study was to study the incidence of chronic renal dysfunction in patients with more than 5 yr of follow-up following liver transplantation and to evaluate the benefit of decreasing cyclosporine A (CsA) dose combined with mycophenolate mofetil (MMF) on renal function and immune response in these patients. Between 1988 and 1994, 60 children were transplanted, and 86% survived >5 yr post-liver transplantation. Fourteen patients developed chronic renal dysfunction secondary to CsA toxicity as evaluated by renal biopsy. In 11 patients CsA dose was decreased to 40-90 mg/ml target levels and MMF 600 mg/m(2) twice daily was added to the immunosuppressive regimen. Plasma creatinine decreased (from 1.0 +/- 0.03 to 0.8 +/- 0.03 ng/dl, p < 0.007), creatinine clearance increased (from 66.8 +/- 3.0 to 99.2 +/- 6.3 ml/min/1.73 m(2), p < 0.002) and microalbuminuria decreased (from 21.0 +/- 8.6 to 3.6 +/- 1.1 mg/24 h, p < 0.05) after 12 months of CsA combined with MMF therapy. During combined therapy the proliferative, cytolytic response and cytotoxic antibodies showed no significant changes, whereas CD4/CD8 ratio increased (from 1.2 +/- 0.2 to 1.4 +/- 0.1, p < 0.05). Tumor necrosis factor-alpha secretion increased (p < 0.005) during MMF therapy. The release of interleukin-10 was strikingly augmented under both immunosuppressive regimens, but the release of transforming growth factor-beta and interferon-gamma did not change. Our findings indicate that initiation of MMF combined with reduced doses of CsA allowed the recovery of renal function with minor changes in the immune response., (Copyright 2004 Blackwell Munksgaard)

Trypanosoma cruzi induces inflammatory reactions in several tissues. The production of prostaglandin F2alpha, 6-keto-prostaglandin F1alpha and thromboxane B2, known to regulate the immune response and to participate in inflammatory reactions, was studied in mice experimentally infected with T. cruzi. The generation of nitric oxide (NO), which could be regulated by cyclooxygenase metabolites, was also evaluated. In the acute infection the extension of inflammatory infiltrates in skeletal muscle as well as the circulating levels of cyclooxygenase metabolites and NO were higher in resistant C3H mice than in susceptible BALB/c mice. In addition, the spontaneous release of NO by spleen cells increased earlier in the C3H mouse strain. In the chronic infections, the tissue inflammatory reaction was still prominent in both groups of mice, but a moderate increase of thromboxane B2 concentration and in NO released by spleen cells was observed only in C3H mice. This comparative study shows that these mediators could be mainly related to protective mechanisms in the acute phase, but seem not to be involved in its maintenance in the chronic T. cruzi infections.

Mother-to-child transmission of intracellular parasites could be related to the production of immunoregulatory cytokines. The levels of gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and lnterleukin (IL)-10 were evaluated during pregnancy in sera of women chronically infected with Trypanosoma cruzi that delivered infected or non-infected children. The levels of IL-10 increased in both, women only pregnant and only infected, compared to non-infected non-pregnant women. However, in pregnant women chronically infected with T. cruzi, IL-10 did not increase significantly, neither in the mothers of infected nor in the mothers of non-infected children. The levels of the inflammatory cytokine TNF-alpha were not affected in normal pregnancy but increased in the infected mothers of non-infected children. The levels of IFN-gamma did not increase in the groups studied, indicating that the production of this pro-inflammatory cytokine was controlled, even when the levels of IL-10 did not increase, as in pregnant women chronically infected with T. cruzi.

T and NK cells play a key role in resistance to Trypanosoma cruzi infections, mainly through IFN-gamma production. The expression of T and NK cells surface markers was studied in NWNA spleen cells of resistant C3H and susceptible BALB/c mice that release IFN-gamma in the early and late acute infection, respectively. In the progressively enlarged spleens, we found: (a) an increased percentage and number of NK blast cells as early as at 2 days post-infection (pi), (b) an enrichment of T and NK cells, in both the total and blast populations, during the late acute phase. At 17 days pi, there was also an accumulation of TCR- alphabeta+DX5+, NKT cells, mainly in resistant mice. At 21 days pi, the enrichment of NK cells ceased, while spleen cells and the T cell compartment continued their expansion. In the chronic stage, TCR-alphabeta+ blasts were expanded in both mouse strains, but NK blasts increased only in BALB/c that, unlike C3H mice, release IFN-gamma. As T and NK cell proliferation is not always associated to IFN-gamma release the experimental downregulation of their expansion to avoid tissue damage could be explored.

Background: Tacrolimus (Tac) has immunosuppressant properties similar to those of cyclosporine A (CsA), but it is more potent. At present, however, its immunosuppressive activity in renal transplant recipients with ongoing chronic rejection has not been clarified., Methods: We studied changes in kidney function, mixed lymphocyte culture, cell-mediated lympholysis, cytotoxic antibodies, lymphocyte population, and cytokine response before and after the conversion from CsA to Tac in 14 pediatric renal transplant recipients with chronic rejection. CsA (5.9+/-0.2 mg/kg/d) was replaced by Tac (0.1+/-0.004 mg/kg/d)., Results: Serum creatinine decreased (2.3+/-0.2-1.9+/-0.2 mg/dL, P <0.005), creatinine clearance increased (36.8+/-2.5-46.1+/-4.4 mL/min/1.73 m, P <0.005), and urinary protein excretion decreased (0.4+/-0.01-0.2+/-0.04 g/24 hr, P <0.03) after 6 months, and these values were maintained after 2 years with Tac treatment. During Tac therapy, anti-donor and anti-control mixed lymphocyte culture decreased 38% and 31% (P <0.05), respectively. Cell-mediated lympholysis did not change. CD3 decreased from 87%+/-2% to 80%+/-2% (P <0.005), and CD8 decreased from 34%+/-3% to 27%+/-2% (P <0.005). The switch to Tac decreased the interferon-gamma production in vitro (P <0.05) and increased tumor necrosis factor-alpha levels (P <0.05). The release of interleukin-10 was strikingly augmented with CsA or Tac therapy (P <0.01), but transforming growth factor-beta secretion was similar., Conclusions: Our data indicate that conversion from CsA to Tac therapy leads to an improvement in renal function without altering key elements of the immunosuppression in children with ongoing chronic rejection.

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.

Resistance to acute Trypanosoma cruzi infection is mainly associated with a Th1 immune response, characterized by gamma-interferon (IFN-gamma) production and activation of macrophages. The outcome of the Th1 response in the spleen and serum of BALB/c and C3H mice infected with T. cruzi, Tulahuén strain was studied. The levels of interleukin-12 p40 (IL-12 p40) and IFN-gamma, as well as natural killer (NK) cell cytotoxicity were determined at different time-points during the acute phase, and the production of cytokines was also studied in the chronic infection. At 2 days post-infection (pi), spleen cells from C3H mice increased their NK cell activity and the ex vivo spontaneous release of both IL-12 p40 and IFN-gamma. On the other hand, BALB/c mice reached low levels of NK cell cytotoxicity and no IFN-gamma production was detected at this time pi, but the cytokine was released at high amounts in the second week of the infection. Seric IL-12 p40 concentrations showed a 3-fold increase in both mouse strains on the second day pi and remained high throughout the acute phase. However, seric IFN-gamma levels increased during the late acute infection and were higher in BALB/c than in C3H mice. In chronically infected mice IL-12 p40 was as high as in the acute phase in the serum of both strains, but only BALB/c mice still produced IFN-gamma. To the authors' knowledge this is the first report showing the protein levels of IL-12 p40 determined in vivo in acute and chronic T. cruzi infections. The results reveal differences between both mouse strains in the mechanisms controlling the onset and fate of the Th1 response triggered by the parasite and a long lasting pro-inflammatory stimuli.

Specific antibodies and the activation of phagocytic cells by IFN-gamma are the key elements of the immune response involved in protection of the T. cruzi infected host. The central role of the IFN-gamma in vivo seems to be the activation of the inducible nitric oxide synthetase of macrophages (iNOS) and the production of nitric oxide (NO degree) for the intracellular destruction of the parasite. Interleukin 12 (IL-12), the cytokine that stimulates NK cells for IFN-gamma production, seems to trigger the TH1 response in the acute phase. Other cell types, such as lymphocytes Thy-1+CD4-CD8-, CD4+ and CD8+, are also involved in IFN-gamma production. The down regulation of the TH1 response could in part depend on the decrease in the macrophage activation, as a result of the controlled parasite burden, and on the production of IL-10 and transforming growth factor beta (TGF-beta). The protective TH1 immune response seems to be also related to both the tissue damage and the alterations of the immune response observed during the infection. We studied the kinetics of both NK cell activity, and the production of IL-12 and/IFN-gamma by spleen cells, as well as the seric levels of these cytokines, in BALB/c and C3H mice infected with T. cruzi, Tulahuén strain. In the spleen, we found that the production of IL-12 and the NK cell activity increased in the very early acute infection, and that in C3H the effect was higher than in BALB/c mice. IFN-gamma increased in C3H at the same time, but in the BALB/c strain it increased later in the acute phase. The infection induced a very early increase in the seric levels of IL-12, that remained high throughout the acute phase, in both mouse strains. However, the levels of IFN-gamma in the serum increased a few days before the peak of parasitemia, reaching higher values, and earlier, in BALB/c than in C3H mice. Surprisingly, in the chronic infection IL-12 production remained high in both mouse strains, but IFN-gamma production was only observed in BALB/c mice. The immune response was predominantly TH1 in both mouse strains, in spite of the higher susceptibility of BALB/c compared to C3H. The early control of the parasite burden could be evaluated as the expression of the TH1 response in spleen cells, while the seric levels of IL-12 and IFN-gamma would be related to the induction of tissue damage. Our data indicate that the protective TH1 immune response has a different expression according to the host-parasite relationship, and that the factors controlling the response are of primary importance to determine the quali- and quantitative expression of IL-12/NK/IFN-gamma as well as their involvement in resistance and tissue damage.

The changes in the T cell subsets of the Peyer's patches and the thymus were analyzed in BALB/c mice infected with Trypanosoma cruzi, Tulahuén strain. During the acute stage of the infection both lymphoid organs drastically reduced their cellularity. This was mainly due to the decrease in the immature CD4+CD8+ T cell population in the thymus and in both T and B cells in the Peyer's patches. In the acute infection, few Peyer's patches were found and the histological studies revealed a depletion of the thymic-dependent areas, paralleling the decreased number of cells expressing CD4 and alpha beta T cell receptor. After 14 weeks, in the late stage of the infection, the cellularity and the levels of the T cell subsets studied returned to values similar to those of noninfected mice.

The release of reactive oxygen intermediates (ROI), mediators of inflammatory reactions, was evaluated in murine Trypanosoma cruzi infection. In acutely infected BALB/c mice, spleen cells were stimulated, either with epimastigote or trypomastigote forms of the parasite, and the effect was enhanced by serum from infected mice. Only opsonized parasites triggered the release of ROI by normal mouse cells and this response was several times lower than in infected mice. This seems to indicate that cells from acutely infected mice reacted to T. cruzi and that neither parasites nor serum factors blocked the release of ROI. During the acute stage of the infection, both the parasitemia and the release of ROI by spleen cells were higher in BALB/c than in C3H mice (ROI generated in response to a phagocytic stimulation was 12 and 3 times the normal levels, respectively). In addition, in BALB/c mice infected with different numbers of parasites, the production of ROI was related to parasitemia. On the other hand, during the chronic stage of the infection, the inflammatory reaction in myocardium was greater in C3H than in BALB/c mice, and the increase in ROI production was 30% and 100% above the normal levels in BALB/c and C3H mice, respectively. This suggests that the increased ROI production paralleled the parasite burden in the acute phase, and could be related to inflammatory processes after the control of the parasitemia.

The acute stage of Trypanosoma cruzi infections related to high parasitemia is characterized by the presence of inflammatory infiltrates in several tissues, including the heart and squeletal muscle, as well as by an increased production of inflammatory mediators, such as gamma-interferon (IFN-gamma), tumor necrosis factor (TNF), interleukin-1 (IL-1) and oxygen and nitrogen reactive intermediates. The activation of phagocytic cells seems to be closely related to both the inflammatory process and host resistance to the infection. Herein, the inflammatory mediators produced in vivo and their relationship with the tissue damage and TH1 immune response are reviewed.

The discovery of the superantigens (SAgs) offered new insights on the interaction between microorganisms and the host immune system. Associated to Major Histocompatibility Complex (MHC) class II molecules, SAgs bind to the variable domain of the beta chain (V beta) of the TCR alpha beta engaged in the family specificity of lymphocytes. Therefore, these molecules are able to activate a high number of T lymphocytes as well as surface MHC class II bearing cells, leading to an overriding release of cytokines and inflammatory mediators, which have been related to their toxic effects. Endogenous SAgs are encoded by murine tumor proviruses (Mtv) which are integrated in the genome of mice. Bacteria and viruses produce exogenous SAgs and those related to food poisoning have been widely studied. The presence of parasite SAgs is still unclear and further studies are required to establish their existence and effects on the corresponding infections.

The T cell receptor (TCR) V beta repertoire was studied in BALB/c, CBA/HJ, and CBA/J mice experimentally infected with Trypanosoma cruzi. The percentage of expression of 14 V beta chains of the variable domain of the TCR in the thymus and spleen was evaluated. In the thymus of acutely infected with BALB/c and CBA/HJ mice there was an increase in the expression of positively selected V beta families. These changes in the V beta chains usage in the thymus paralleled the enrichment of CD4+ and CD8+ single-positive T cells. During the acute infection, several changes were observed in the peripheral expression of V beta families, such as of V beta 6 in BALB/c (a 36% increase in CD8+ T cells of the corresponding levels of V beta), of V beta 8 in CBA/HJ (a 37% decrease in CD8+ cells), and of V beta chains 8 and 14 in CBA/J mice (V beta 14+CD4+ cells increased 19%, and V beta 8 expression decreased 19 and 33% in CD4+ and CD8+ cells, respectively). In chronically infected BALB/c and CBA/HJ mice, no change in the V beta families was observed, neither in the thymus nor in the spleen. In acutely infected mice, the alterations of the peripheral expression of positively selected V beta families could be due to the stimulation by T. cruzi antigens and/or cytokines; the homeostatic mechanism/s that maintains the selection of the TCR V beta repertoire did not seem to be severely affected during the infections.

Effector mechanisms of resistance exerted by T cells from BALB/c mice chronically infected with Trypanosoma cruzi, Tulahuén strain, were studied. Spleen cells from chronically infected mice (Chro-SC) prestimulated with heat-killed trypomastigotes (HKT) and/or IL-2 destroyed PHA-labeled p-815 mastocytoma cells, HKT-pulsed macrophages, and normal peritoneal macrophages. However, HKT-stimulated Chro-SC did not affect the infectivity of free bloodstream forms of the parasite. Upon HKT stimulation, Chro-SC or their culture supernatant activated peritoneal macrophages for the destruction of intracellular amastigotes. The effect was abolished after Thy 1.2+ cell depletion. The addition of Cyclosporin A (CyA), which blocks T-cell activation, during HKT-stimulation of Chro-SC, diminished their ability to activate the trypanocidal activity of macrophages. CyA also inhibited the production of both macrophage-activating factors and interferon-gamma by HKT-stimulated Chro-SC. CyA administration to recipients of nylon-wool nonadherent spleen cells from chronically infected mice inhibited their adoptively acquired resistance against T. cruzi, suggesting that the conferred resistance depended on the effect of specifically activated cells. When administered during the chronic stage of the infection, CyA abrogated the antigen-specific delayed type hypersensitivity response but increased the levels of anti-T. cruzi IgG antibodies. Neither parasitemia, tissular parasitism in myocardium or skeletal muscle, nor mortality were detected after CyA treatment, suggesting the presence of a CyA nonsensitive mechanism(s) in the control of T. cruzi during the chronic phase of the infection.

This study has been designed to find an easy method to evaluate the motor unit alterations induced during experimental T. cruzi infections. Different mouse strains infected with three strains of T. cruzi were used to perform conventional needle electromyography, in one of the lower limb hamstring muscles; amplitude, duration and number of phases of single motor unit potentials were measured. The following parasite strain to mouse strain relationship was investigated, in mice inoculated intraperitoneally with bloodstream forms of T. cruzi: Tulahuen and C3H/HeN, C57Bl, Balb/c, Swiss; CA-I and C3H/HeN, Rockland, NIH; RA and C3H/HeN, Rockland. T. cruzi-induced denervating alterations were found in both C3H/HeN and C57Bl mice infected with the Tulahuen strain, as well as in C3H/HeN mice inoculated with the CA-I strain. Moreover, CA-I trypomastigotes could produce primary muscle changes in C3H/HeN and NIH mice. The technique employed in this investigation proved to be an easy and adequate way to detect changes within the motor unit during T. cruzi infection in mice.

Release of reactive oxygen species (ROS) by cells from BALB/c mice was studied during the acute stage of the infection with 50 bloodstream forms of Trypanosoma cruzi, Tulahuén strain. Production of ROS by spleen and peritoneal cells was evaluated by chemiluminescence using luminol as enhancer (CL-Lum). Three to four weeks after infection, CL-Lum response after the addition of opsonized zymosan to spleen and peritoneal cells from infected mice was 13 and 98 times, respectively, above the levels obtained with cells from noninfected mice. The kinetics of this hyperactivity was similar to that of the parasitemia. Both reached maximal values on the third to fourth weeks and decreased at 7 weeks postinfection. During this hyperactivation stage, spleen and peritoneal cells from infected mice showed a "spontaneous" CL-Lum response (without any stimulus added in vitro) absent in noninfected mice. Both, "spontaneous" and zymosan stimulated CL-Lum responses were inhibited by 100 microM azide and by 0.8 microM superoxide dismutase, suggesting the involvement of hemoproteins and superoxide anion in the measured responses. Moreover, spleen cells from acutely infected mice displayed a hyperactivity in the CL-Lum response when recombinant interferon-gamma was added in vitro. Supernatants of spleen cells from both normal or infected mice, stimulated in vitro with concanavalin A, contained similar levels of interferon and were equally able to stimulate the trypanocidal activity of normal macrophages. These results suggest that mediators of activation of phagocytic cells can be produced during acute T. cruzi infection. In addition, phagocytic cells from acutely infected mice were activated in vivo and were hyperactive to the in vitro stimulation.

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.

In this work, we describe skeletal muscle, neuromuscular junction, nerve and spinal cord lesions in the mouse model system of Chagas' disease. Myositis was a common finding and Trypanosoma cruzi amastigote nests were frequently found in the muscle fibers. Angular atrophy, targetoid fibers, groups of atrophic fibers, fibrosis, myofiber necrosis and phagocytosis of cellular debris were also observed. The neuromuscular junction studies showed degeneration of intramuscular nerve fibers, swelling and distortion of nerve endings and multiple ramifications on the same muscle fiber. Collateral, terminal and ultraterminal axonal sprouts were also present. Inflammatory neuropathy was seen in all of the infected mice. Demyelination, axonal degeneration, remyelination and axonal regeneration were observed in the transverse sections. There was an average reduction of 29% in the total number of myelin fibers. The teasing of single myelin fibers showed segmental and paranodal demyelination and remyelination more frequently than axonal degeneration and regeneration. The lumbar spinal cords presented inflammatory cell infiltration associated with tissue destruction. Amastigote nests were found in 3 out of the 8 infected mice studied. There was a mean loss of 21% of the large cytoneurons of the anterior horn of the lumbar spinal cord.

The temperature-dependence of some processes involved in the killing of sensitized T. cruzi epimastigotes by human polymorphonuclear leukocytes (PMN) was determined. The rate of the reactions was related to the temperature of incubation according to the Arrhenius equation and the apparent energies of activation (Ea) were calculated. The Ea values separated these complex reactions into two groups: one with Ea of about 10 kcal/mol for the phagocytosis of the parasites and the release of lysosomal enzymes by PMN, and the other with Ea of about 22 kcal/mol for the cytotoxicity against sensitized T. cruzi, the rate of oxygen consumption by PMN, and the lysis of the parasites with added hydrogen peroxide.

The humoral and cellular immune responses were studied in mice immunized with flagellar fraction (F), F plus Bordetella pertussis as adjuvant (F-Bp), and microsomal (Mc) subcellular fractions from the epimastigote forms of Trypanosoma cruzi. The immune response was studied before and after the challenge with 50 bloodstream forms of T. cruzi, Tulahuén strain. The immunization with F-Bp, but not with Mc or F and Bp separately, protected mice, in terms of parasitemia and mortality, from the challenge with the parasite. Before the challenge, levels of specific antibodies in mice immunized with F-Bp were higher than in mice immunized with F or Mc. Antibody levels 17 days after the infection were similar in the three groups of mice while nonimmunized mice reached lower levels. Early during the infection nonimmunized infected mice lacked delayed-type hypersensitivity (DTH) responses to parasite antigens and to concanavalin A (Con A). Mice immunized with F-Bp, however, presented positive DTH responses to parasite antigens and Con A both, before and after the challenge with T. cruzi. DTH reaction was transferred with spleen cells. Mice immunized with Mc behaved similarly to infected nonimmunized animals in their reactivity to parasite antigens. These results indicated striking differences between protected and nonprotected mice in humoral and cellular immune responses during experimental T. cruzi infection.

This study provides biochemical and electron spin resonance spectroscopic evidence that contract of human polymorphonuclear leukocytes with antibody-coated Trypanosoma cruzi triggers the respiratory burst. Oxygen consumption, superoxide anion and hydrogen peroxide release were stimulated under conditions of polymorphonuclear leukocyte-mediated killing. This stimulation did not occur under non-killing conditions when antibody was omitted. A common mechanism of cytotoxicity of human polymorphonuclear leukocytes against different T. cruzi forms is suggested by the triggering of the respiratory burst by antibody-coated epimastigotes and trypomastigotes.

T lymphocytes provide a major line of defence against many protozoan parasites. The aim of this work was to determine the role of T-cell helper/inducer subset (T h/i) in the resistance to Trypanosoma cruzi in a murine model. The importance of natural killer (NK) cells in the resistance to the parasite was also evaluated. BALB/c and C57BL/6 mice were injected with either monoclonal antibodies against L3T4, Thy 1.2, NK1.1, or with a polyclonal rabbit antiserum against NK cells (anti-asialo GM-1). The effect of in vivo administration of these antibodies was tested in separate functional assays. After antibody treatment, mice were infected with a low dose of T. cruzi in the bloodstream form. Mice depleted of, or reduced in T, T h/i, or NK cell activity all developed higher parasitaemia and had higher mortality than their control counterparts. Mice injected with anti-L3T4 monoclonal antibodies were unable to generate a specific antibody response to the parasite. Treatment of mice with alpha/beta interferon, which is known to boost NK cell activity, resulted in an enhanced resistance to the parasite. Our data indicate that T h/i cells as well as NK cells are of vital importance in controlling parasitaemia and reducing mortality in T. cruzi-infected mice. Finally, we also demonstrate that the production of antibodies specific for T. cruzi is strictly T helper cell-dependent.

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.

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 degrees 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.

Infective forms of Trypanosoma cruzi were used to evaluate the complement-mediated lysis (CoML) of the parasites in the presence of anti-T. cruzi sera. Parasites released to the supernatant from infected Vero cell monolayers were used. Cultures of 1-3 x 10(6) parasites/ml were incubated for 24 h in the presence of 10 microCi/ml of 3H-uridine. Under these conditions 10(5) parasites used for each determination incorporated about 9600 dpm of the radioactive material. The release of tritium from labelled parasites after incubation with antiserum and complement correlated with the percentage of lysed parasites evaluated by optical microscopy. Normal sera from humans, a guinea pig, a rabbit, and mice were tested as complement sources. Only human sera were suitable for the evaluation of CoML in the presence of antisera, and the levels of lysis attained depended on the serum donor.

Positive skin reactions to PPD in guinea-pigs immunized with Freund's complete adjuvant (FCA) were reversed after treatment with 10 mg/kg/day nifurtimox for 12 days. The in vitro migration of peripheral blood leucocytes from FCA-immunized guinea-pigs was inhibited with PPD, but it returned to normal values after nifurtimox treatment. Furthermore, the cell-free supernatant from PPD-stimulated lymphocytes from FCA-immunized nifurtimox-treated guinea-pigs did not inhibit the migration of normal cells. Thus the administration of nifurtimox impaired the specific cell-mediated immune response to PPD both in vivo and in vitro.

Normal human polymorphonuclear leukocytes (PMN) are cytotoxic to T. cruzi epimastigotes sensitized with specific antiserum (T. cruzi + Ab). The damage follows an early phagocytic event, suggesting the intracellular destruction of the parasites. We have studied the characteristics of the killing using metabolic inhibitors of the effector cells. Oxygen consumption by PMN with unsensitized parasites was similar to the uptake by resting cells, but increased two- to fourfold when T. cruzi + Ab was used. This increase in O2 consumption, associated with phagocytosis of T. cruzi + Ab was not sensitive to 2 mM cyanide nor 100 microM azide. Addition of T. cruzi + Ab to human PMN also stimulated H2O2 production. When PMN were incubated with phenylbutazone, cyanide or azide, an inhibition of cytotoxicity against sensitized T. cruzi was observed. Under the same experimental conditions phagocytosis was unaffected. These results indicate that active oxygen reduction products and myeloperoxidase are involved in the destruction of sensitized, T. cruzi epimastigotes by normal PMN.

Peripheral leucocyte migration inhibition (LMI) with Trypanosoma cruzi-specific antigens, measured as a migration index (MI), was studied in chronic Chagas' disease patients. The MI of untreated patients with polymerized antigens from culture forms (epimastigotes) of T. cruzi was significantly lower than that of controls. In contrast, when chronic Chagas' patients were treated with nifurtimox, 10 mg/kg/day for 2 months, the MI was not different from control values. Treated and untreated patients had normal T- and B-lymphocyte markers, measured by the ability to form rosettes either with sheep erythrocytes (E-RFC) or with sheep erythrocytes--antibody--complement (EAC-RFC). In addition, the number of lymphocytes bearing surface membrane Ig (SMIg) was the same as that of controls. Non-specific functional assays, such as PHA-induced blastogenesis and antibody-dependent cell-mediated cytotoxicity (ADCC) to sensitized chicken erythrocytes were also normal, both in treated and untreated patients. Thus, nifurtimox produced a particularly effect on cell-mediated immunity, specially detectable using LMI.

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the relationship between phagocytosis and cytotoxicity of human polymorphonuclear leucocytes (PMN) to sensitized Trypanosoma cruzi. Assays were done simultaneously using [3H]-uridine labelled epimastigotes as target cells. Phagocytosis was evaluated by the uptake and cytotoxicity by the release of parasite associated [3H]-uridine. Both reactions reached maximum levels at the same effector- to target-cell ratio and antibody concentration. Uptake of epimastigotes by PMN was highest at 30 min and intracellular disruption and release of parasite debris took place later. In conditions that precluded repeated uptake of sensitized radiolabelled T. cruzi, the release profile of [3H]-uridine from PMN that contained intracellular parasites was similar to that of the standard cytotoxic assay. However, as the ingestion phase was separated from the release step, no lag in the onset of the reaction was observed. Although we cannot rule out extracellular killing, the results of this study demonstrate that the bulk of damaged T. cruzi epimastigotes had been previously internalized by the PMN.