B-Cell Responses in Chronic Chagas Disease: Waning of Trypanosoma cruzi-Specific Antibody-Secreting Cells Following Successful Etiological Treatment.

BACKGROUND: A drawback in the treatment of chronic Chagas disease (American trypanosomiasis) is the long time required to achieve complete loss of serological reactivity, the standard for determining treatment efficacy. METHODS: Antibody-secreting cells and memory B cells specific for Trypanosoma cruzi and their degree of differentiation were evaluated in adult and pediatric study participants with chronic Chagas disease before and after etiological treatment. RESULTS: T. cruzi-specific antibody-secreting cells disappeared from the circulation in benznidazole or nifurtimox-treated participants with declining parasite-specific antibody levels after treatment, whereas B cells in most participants with unaltered antibody levels were low before treatment and did not change after treatment. The timing of the decay in parasite-specific antibody-secreting B cells was similar to that in parasite-specific antibodies, as measured by a Luminex-based assay, but preceded the decay in antibody levels detected by conventional serology. The phenotype of total B cells returned to a noninfection profile after successful treatment. CONCLUSIONS: T. cruzi-specific antibodies in the circulation of chronically T. cruzi-infected study participants likely derive from both antigen-driven plasmablasts, which disappear after successful treatment, and long-lived plasma cells, which persist and account for the low frequency and long course to complete seronegative conversion in successfully treated participants.

Efficacy of continuous versus intermittent administration of nanoformulated benznidazole during the chronic phase of Trypanosoma cruzi Nicaragua infection in mice.

BACKGROUND: Benznidazole and nifurtimox are effective drugs used to treat Chagas' disease; however, their administration in patients in the chronic phase of the disease is still limited, mainly due to their limited efficacy in the later chronic stage of the disease and to the adverse effects related to these drugs. OBJECTIVES: To evaluate the effect of low doses of nanoformulated benznidazole using a chronic model of Trypanosoma cruzi Nicaragua infection in C57BL/6J mice. METHODS: Nanoformulations were administered in two different schemes: one daily dose for 30 days or one dose every 7 days, 13 times. RESULTS: Both treatment schemes showed promising outcomes, such as the elimination of parasitaemia, a reduction in the levels of T. cruzi-specific antibodies and a reduction in T. cruzi-specific IFN-γ-producing cells, as well as an improvement in electrocardiographic alterations and a reduction in inflammation and fibrosis in the heart compared with untreated T. cruzi-infected animals. These results were also compared with those from our previous work on benznidazole administration, which was shown to be effective in the same chronic model. CONCLUSIONS: In this experimental model, intermittently administered benznidazole nanoformulations were as effective as those administered continuously; however, the total dose administered in the intermittent scheme was lower, indicating a promising therapeutic approach to Chagas' disease.

Sequential combined treatment with allopurinol and benznidazole in the chronic phase of Trypanosoma cruzi infection: a pilot study.

OBJECTIVES: Even though the use of combined drugs has been proved to be effective in other chronic infections, assessment of combined treatment of antiparasitic drugs in human Chagas' disease has not been performed. Herein, a pilot study was conducted to evaluate the tolerance and side effects of a sequential combined treatment of two antiparasitic drugs, allopurinol and benznidazole, in the chronic phase of Trypanosoma cruzi infection. PATIENTS AND METHODS: Changes in total and T. cruzi-specific T and B cells were monitored during a median follow-up of 36 months. Allopurinol was administered for 3 months (600 mg/day) followed by 30 days of benznidazole (5 mg/kg/day) in 11 T. cruzi-infected subjects. RESULTS: The combined sequential treatment of allopurinol and benznidazole was well tolerated. The levels of T. cruzi-specific antibodies significantly decreased after sequential combined treatment, as determined by conventional serology and by a multiplex assay using recombinant proteins. The frequency of T. cruzi-specific interferon-γ-producing T cells significantly increased after allopurinol treatment and decreased to background levels following benznidazole administration in a substantial proportion of subjects evaluated. The levels of total naive (CD45RA + CCR7 + CD62L+) CD4 + and CD8 + T cells were restored after allopurinol administration and maintained after completion of the combined drug protocol, along with a decrease in T cell activation in total peripheral CD4 + and CD8 + T cells. CONCLUSIONS: This pilot study shows that the combination of allopurinol and benznidazole induces significant modifications in T and B cell responses indicative of a reduction in parasite burden, and sustains the feasibility of administration of two antiparasitic drugs in the chronic phase of Chagas' disease.

Altered frequency and phenotype of CD4+ forkhead box protein 3+ T cells and its association with autoantibody production in human immunodeficiency virus-infected paediatric patients.

The association between immune dysfunction and the development of autoimmune pathology in patients with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is not clear. The frequency and phenotype of regulatory T cells, as well as the presence of autoantibodies, were evaluated in a paediatric cohort of HIV-infected patients without clinical evidence of autoimmune disease. Lower absolute counts but higher percentages of total CD4(+) forkhead box protein 3 (FoxP3)(+) T cells were recorded in children with severe immunosuppression than in those without evidence of immunosuppression. The frequencies of classical CD4(+) CD25(+) FoxP3(+) regulatory T cells were not altered, whereas CD4(+) FoxP3(+) CD25(-) T cells were found increased significantly in patients with severe immunosuppression. Like classical regulatory T cells, CD4(+) FoxP3(+) CD25(-) T cells display higher cytotoxic T-lymphocyte antigen 4 (CTLA-4) but lower CD127 expression compared with CD4(+) FoxP3(-) CD25(+) T cells. An improvement in CD4(+) T cell counts, along with a decrease in viral load, was associated with a decrease in CD4(+) FoxP3(+) CD25(-) T cells. The majority of the patients with severe immunosuppression were positive for at least one out of seven autoantibodies tested and displayed hypergammaglobulinaemia. Conversely, HIV-infected children without evidence of immunosuppression had lower levels of autoantibodies and total immunoglobulins. A decline in CD4(+) FoxP3(+) T cell numbers or a variation in their phenotype may induce a raise in antigen exposure with polyclonal B cell activation, probably contributing to the generation of autoantibodies in the absence of clinical autoimmune disease.

Evaluation of immune responses raised against Tc13 antigens of Trypanosoma cruzi in the outcome of murine experimental infection.

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-gamma secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-gamma production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.

Immunological and pathological responses in BALB/c mice induced by genetic administration of Tc 13 Tul antigen of Trypanosoma cruzi.

Tc13 is a trans-sialidase family protein of Trypanosoma cruzi, the aetiological agent of Chagas' disease. Recently, in vitro studies had suggested that Tc13 might participate in the pathogenesis of the disease. In order to study the role of Tc13 antigens in an in vivo model, we administered plasmid DNA encoding a Tc13 antigen from the Tulahuén strain (Tc13 Tul) to BALB/c mice and evaluated the immunological and pathological manifestations as well as the capacity of this antigen to confer protection against T. cruzi infection. Tc13 Tul immunization did not elicit a detectable humoral immune response but induced specific memory T-cells with no capacity to produce IFN-gamma. Five months after DNA-immunization with Tc13 Tul, signs of hepatotoxicity and reactive changes in the heart, liver and spleen were observed in 40-80% of mice. When Tc13 Tul DNA-immunized animals were challenged with trypomastigotes, a significant decrease in parasitaemia in early and late acute phase was observed without modification in the survival rate. Surprisingly, Tc13 Tul-immunized mice chronically infected with T. cruzi showed a decrease in the severity of heart damage. We conclude that, in BALB/c mice, genetic immunization with Tc13 Tul mainly induces immune responses associated with pathology.

Value of echocardiography for diagnosis and prognosis of chronic Chagas disease cardiomyopathy without heart failure.

OBJECTIVES: To establish the usefulness of echocardiography for the clinical classification of patients with Chagas disease and to determine the predictors of mortality and clinical events. METHODS: 849 patients with chronic Chagas disease with a mean follow up of 9.9 years were studied. On admission, ECG, chest radiograph, and two dimensional echocardiogram were obtained from all patients. Clinical events were defined as new ECG abnormalities, change in clinical status resulting in transfer to another group, and death. Morphologically characterised segmental lesions were also seen in 12 patients on a second harmonic echocardiogram with intravenous contrast agent. Univariate and multivariate analysis for clinical events and mortality were performed. SETTING: Community of San Martín, Buenos Aires, Argentina. RESULTS: Change in clinical group (68 of 833 survivors v 15 of 16 who died, p < 0.001), left ventricular systolic dimension (mean (SD) 3.06 (0.72) cm v 4.71 (0.90) cm, p < 0.0001), and ejection fraction (mean (SD) 0.67 (0.11)% v 0.42 (0.17)%, p < 0.0001) were found to be the only predictors of mortality. ECG abnormalities related to the disease (in 220 of 699 patients with no clinical event v 98 of 150 patients with a clinical event, p < 0.0001), left ventricular diastolic dimension (mean (SD) 4.88 (0.54) cm v 5.44 (0.83) cm, p < 0.0001), left ventricular systolic dimension (mean (SD) 2.98 (0.62) cm v 3.64 (1.03) cm, p < 0.0001), and ejection fraction (mean (SD) 0.68 (0.10)% v 0.60 (0.16)%, p < 0.0001) were predictors of clinical events. Segmental lesions were observed in 211 of 849 patients (25%). Segmental lesions were seen in 66 (13%) and systolic dysfunction was seen in four of 505 (0.8%) patients with normal ECG. Significant differences were found between the groups of patients (group 0: reactive serology and normal ECG and chest radiography without cardiac enlargement and no signs of heart failure; group 1: reactive serology and abnormal ECG and chest radiography without cardiac enlargement; group 2: reactive serology and abnormal ECG and chest radiography with cardiac enlargement and no signs of heart failure). CONCLUSION: Echocardiography was useful both to characterise and to determine the prognosis of patients with chronic Chagas disease without heart failure.

Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease.

The humoral immune response to epitopes expressed on cruzipain was evaluated in 31 Chagas disease patients (CDP) with different degrees of cardiac dysfunction. We took advantage of the availability of anti-Trypanosoma cruzi microsomal fraction monoclonal antibodies (MoAbs) reactive with epitopes that are recognized (5A9B11) or not recognized (1A10C11) by CDP sera. The 5A9B11- and 1A10C11-like epitopes are expressed on cruzipain. The reactivity of 5A9B11 against cruzipain was completely inhibited by sera of severe cardiopathy patients while a partial inhibition was found with sera from chagasic patients with mild disease. CDP sera did not block cruzipain recognition by 1A10C11. The antigenic determinants recognized by CDP sera appeared to be linear and carbohydrate free. When the overall anti-cruzipain immune response was evaluated, 70% of CDP with severe disease showed cruzipain titers higher than 1/800 while none of them displayed titers lower that 1/400. This report shows for the first time that the humoral immune response against epitopes expressed on cruzipain appeared to be related with the severity of chronic Chagas disease.

alpha 4 Integrins and sialyl Lewis x modulation in chronic Chagas disease: further evidence of persistent immune activation.

We have previously shown that titers of soluble platelet selectin (s-P-selectin) and soluble vascular cell adhesion molecule-1 (s-VCAM-1) were increased in sera of patients with chronic Trypanosoma cruzi infection. In this study, we analyzed the expression of CD49d-integrins, that bind to VCAM-1, and sialyl Lewis x (SLe(x)), which binds selectins, in peripheral blood lymphocytes of 27 patients with Chagas' disease at different levels of disease severity. Patients with a mild form of Chagas' disease showed a lower number of CD49d(+) cells, in comparison with those with severe chronic cardiopathy. Decreased levels of CD49d(+) cells were detected in CD3(-) cell populations. Conversely, SLe(x) expression was found to be decreased in patients with severe Chagas' disease. Levels of soluble platelet endothelial cell adhesion molecule-1 (s-PECAM-1) were significantly increased in the plasma of patients with severe Chagas' disease while unaltered levels of MCP-1 were recorded. These data show that VCAM-1 and P-Selectin ligands are differentially expressed during the chronic phase of the Trypanosoma cruzi infection. These findings also reinforce a role of the P-selectin/SLe(x) adhesion pathway rather than very late antigen-4 (VLA-4)/VCAM-1, in the pathogenesis of Chagas' disease.

Prognostic value of soluble intercellular adhesion molecule-1 (s-ICAM-1) in HIV-infected children.

Central events in the host defence system and immune-mediated damage are tightly regulated by cell adhesion molecules. Sera from 28 human immunodeficiency virus (HIV)-1 infected children divided into groups according to disease severity, six seroreverting (SR) children and 25 healthy controls were studied to detect the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1). Soluble ICAM-1 levels were found to be significantly increased in HIV-infected children in comparison with SR children or healthy controls. Levels of soluble ICAM-1 were higher in patients with severe forms of HIV-infection than in those with a milder form of the disease. Significant differences in titers of s-ICAM-1 were recorded between SR children and HIV-infected children with mild disease or healthy controls. There was a significant correlation between s-ICAM-1 levels and the concentrations of beta 2 microglobulin (beta 2m) and, to a lesser extend, immunoglobulin A levels (IgA). Soluble ICAM-1 levels didn't change considerably in HIV-infected children in stable clinical conditions, independently of their clinical stage of the disease, during a follow-up period of 9-12 months. Conversely, s-ICAM-1 levels increased simultaneously with the appearance of new well-defined clinical disorders or decreased during the improvement of clinical conditions. A significant negative correlation was recorded between the titers of the s-ICAM-1 and the CD4(+) T-cell levels. These results suggest that the s-ICAM-1 might be another useful tool to evaluate disease progression.

Soluble platelet selectin (sP-selectin) and soluble vascular cell adhesion molecule-1 (sVCAM-1) decrease during therapy with benznidazole in children with indeterminate form of Chagas' disease.

The immune response against Trypanosoma cruzi infection has been associated with both protection and pathogenesis. Central events in host defence system- and immune-mediated damage are tightly regulated by cell adhesion molecules (CAM). Levels of sP-selectin and sVCAM-1 were measured in sera from 41 children with the indeterminate phase of Chagas' disease. Simultaneously, levels of soluble adhesion molecule were also quantified in Chagas' disease children undergoing specific chemotherapy with benznidazole. Levels of sP-selectin and sVCAM-1 were found to be elevated in children with indeterminate Chagas' disease before aetiologic therapy was started. However, a small group of patients showed sP-selectin and sVCAM-1 levels comparable to those of non-infected children. A positive correlation between levels of sVCAM-1 and sP-selectin in sera from Chagas' disease patients was found. There was a significantly greater decrease in the titres of sP-selectin and sVCAM-1 in those children receiving benznidazole therapy compared with those children receiving placebo. Measurement of soluble adhesion molecules revealed differences in the activation of the immune system in children with the indeterminate form of Chagas' disease. The early decrease of sP-selectin and sVCAM-1 levels after anti-parasitic treatment suggests that these molecules might be valuable indicators of effective parasitologic clearance.

Soluble intercellular adhesion molecule-1 in paediatric connective tissue diseases.

The intercellular adhesion molecule-1 (ICAM-1) is a cytokine-induced glycoprotein involved in the recruitment of cells into tissues undergoing inflammatory responses. The aim of this study was to compare the levels of soluble ICAM-1 (s-ICAM-1) in children with juvenile chronic arthritis (JCA) and systemic lupus erythematosus (SLE) and to evaluate the usefulness of this molecule as marker of disease activity. Levels of s-ICAM-1 were measured in sera using a monoclonal antibody sandwich enzyme-linked immunoassay. Serum levels (mean+/-SD) of s-ICAM-1 in 37 children with JCA, 18 patients suffering from SLE and 25 healthy controls were 609+/-184, 513+/-139 and 210+/-95 ng/ml, respectively. A significant difference could be demonstrated between the levels of s-ICAM-1 in sera from each disease, as a group, and those of healthy controls. Higher levels of s-ICAM-1 were recorded in JCA patients with systemic features and patients who had polyarthritis than in children who were pauciarticular. A positive correlation was observed between s-ICAM-1 levels and disease activity score in SLE patients. Moreover, s-ICAM-1 levels closely followed clinical conditions in five children with SLE during follow-up. The data show that s-ICAM-1 levels are increased in children suffering from connective tissue diseases and reflect disease status or activity, suggesting the usefulness of this molecule in the follow-up of these diseases.

Haptoglobina y a1-antitripsina: proteínas de valor predictivo en la progresión del SIDA pediátrico / Haptoglobin and A1-antitrypsin: prognosis value in pediatric AIDS

El propósito de este trabajo fue determinar la utilidad de la heptoglobina (HP), a1-antitripsina (a1-AT), transferrina (TRF) y pre-albúmina (PA) en el SIDA pediátrico como marcadores de progresión de enfermedad. En este estudio, se utilizó la clasificación del Centro de Control y Prevención de Enfermedades (CDC), 1994. Para evaluar el comportamiento de estas proteínas en relación a los principales parámetros usados en esta clasificación, es decir la sintomatología clínica y el grado de compromiso inmunológico, los pacientes se reagruparon de acuerdo a estos parámetros en dos categorías: considerando la sintomatología clínica (N-A-B-C), y de acuerdo al grado de compromiso inmunológico (EI 1- EI 2- EI 3). Hp, a1-AT, TRF y PA son proteínas sintetizadas en el hígado. La HP y la a1-AT, pertenecientes a las proteínas reactantes de fase aguda, aumentan significativamente sus niveles plasmáticos en la inflamación e infección mientras que la TRF y la PA, proteínas de vida media corta, disminuyen sus niveles en diversas enfermedades hepáticas. Se estudiaron 53 niños infectados por el virus HIV, 27 niños y 26 niñas con edades comprendidas entre 2 meses y 10 años y 30 niños sanos como controles (Co). Dentro de la categoría clínica, los niveles de todas las proteínas estudiadas fueron similares (p > 0,05) entre los distintos grupos de pacientes HIV y los controles. Sin embargo, de acuerdo al grado de compromiso inmunológico, se observó una disminución significativa (p < 0,05) en los niveles plasmáticos de HP en pacientes HIV con evidencias de inmunosupresión severa (EI 3) con respecto a pacientes HIV con evidencia de inmunosupresión moderada (EI 2) y a los controles (EI 3: n = 8 x ñ ES = 77 ñ 25; EI 2: n = 23, x ñ ES = 193 ñ 19; Co: n = 30, x ñ ES = 193 ñ 90 mg por ciento). Por otro lado, se observó un aumento significativo (p < 0,05) en los niveles de a1-AT en EI 2 con respecto a los pacientes HIV- sin compromiso inmunológico (EI 1) y a los controles, manteniéndose dichos niveles en EI 3 (EI 3: n = 8, x ñ ES = 404 + 34; EI 2: n = 23, x ñ ES = 353 ñ 24; EI 1: n = 22, x ñ ES = 277 ñ 33; Co: n = 30, x ñ ES = 260 ñ 80 mg por ciento). Contrariamente, los niveles plasmáticos de TRF y PA fueron similares entre los distintos grupos de la categoría inmunológica...

Haptoglobina y a1-antitripsina: proteínas de valor predictivo en la progresión del SIDA pediátrico / Haptoglobin and A1-antitrypsin: prognosis value in pediatric AIDS

El propósito de este trabajo fue determinar la utilidad de la heptoglobina (HP), a1-antitripsina (a1-AT), transferrina (TRF) y pre-albúmina (PA) en el SIDA pediátrico como marcadores de progresión de enfermedad. En este estudio, se utilizó la clasificación del Centro de Control y Prevención de Enfermedades (CDC), 1994. Para evaluar el comportamiento de estas proteínas en relación a los principales parámetros usados en esta clasificación, es decir la sintomatología clínica y el grado de compromiso inmunológico, los pacientes se reagruparon de acuerdo a estos parámetros en dos categorías: considerando la sintomatología clínica (N-A-B-C), y de acuerdo al grado de compromiso inmunológico (EI 1- EI 2- EI 3). Hp, a1-AT, TRF y PA son proteínas sintetizadas en el hígado. La HP y la a1-AT, pertenecientes a las proteínas reactantes de fase aguda, aumentan significativamente sus niveles plasmáticos en la inflamación e infección mientras que la TRF y la PA, proteínas de vida media corta, disminuyen sus niveles en diversas enfermedades hepáticas. Se estudiaron 53 niños infectados por el virus HIV, 27 niños y 26 niñas con edades comprendidas entre 2 meses y 10 años y 30 niños sanos como controles (Co). Dentro de la categoría clínica, los niveles de todas las proteínas estudiadas fueron similares (p > 0,05) entre los distintos grupos de pacientes HIV y los controles. Sin embargo, de acuerdo al grado de compromiso inmunológico, se observó una disminución significativa (p < 0,05) en los niveles plasmáticos de HP en pacientes HIV con evidencias de inmunosupresión severa (EI 3) con respecto a pacientes HIV con evidencia de inmunosupresión moderada (EI 2) y a los controles (EI 3: n = 8 x ñ ES = 77 ñ 25; EI 2: n = 23, x ñ ES = 193 ñ 19; Co: n = 30, x ñ ES = 193 ñ 90 mg por ciento). Por otro lado, se observó un aumento significativo (p < 0,05) en los niveles de a1-AT en EI 2 con respecto a los pacientes HIV- sin compromiso inmunológico (EI 1) y a los controles, manteniéndose dichos niveles en EI 3 (EI 3: n = 8, x ñ ES = 404 + 34; EI 2: n = 23, x ñ ES = 353 ñ 24; EI 1: n = 22, x ñ ES = 277 ñ 33; Co: n = 30, x ñ ES = 260 ñ 80 mg por ciento). Contrariamente, los niveles plasmáticos de TRF y PA fueron similares entre los distintos grupos de la categoría inmunológica... (AU)

Soluble cell adhesion molecules in human Chagas' disease: association with disease severity and stage of infection.

Formation of inflammatory lesions, one of the pathologic consequences of infection with Trypanosoma cruzi, involves intricate cell-cell interactions in which cell adhesion molecules (CAMs) are involved. Sera from 56 Chagas' disease patients grouped according to disease severity were studied for the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1), soluble endothelial selectin (s-E-selectin), soluble vascular cell adhesion molecule-1 (s-VCAM-1), soluble platelet selectin (s-P-selectin), and s-CD44 were studied to determine if they could be used alone or in different combinations as markers for specific diagnostic procedures. Comparisons were made between congenitally, acutely, and chronically infected patients and aged-matched, noninfected individuals, as well as between patients with chronic Chagas' disease grouped according to the severity of their heart-related pathology. No differences in levels of s-CAMs were detected between sera from children with congenital T. cruzi infection and sera from noninfected infants born from chagasic mothers. In contrast, titers of s-ICAM-1, s-VCAM-1, s-selectin, and s-CD44 but not s-P-selectin were significantly increased in sera from patients during the acute phase of infection with T. cruzi. Titers of s-VCAM-1 and s-P-selectin were increased in chronically infected patients. A positive association with disease severity in sera from patients with chronic disease was observed for the levels of s-P-selectin. In contrast, we found no association between clinical symptoms and levels of s-VCAM-1. Patients with chronic disease with severe cardiopathy also showed diminished levels of s-CD44 in comparison with healthy controls or patients with mild disease. The results are discussed in the context of pathology of Chagas' disease.

Epitopes common to Trypanosoma cruzi and mammalian tissues are recognized by sera from Chagas' disease patients: prognosis value in Chagas disease.

Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.

Papel de las citoquininas en la resistencia y patología durante la infección con Trypanosoma cruzi / Role of cytokines in the resistance and pathology during infection with Trypanosoma cruzi

La enfermedad de Chagas se asocia con diversos trastornos inmunológicos. La participación del sistema inmune del huésped infectado en la resistencia a Trypanosoma cruzi es bien conocida, no así su papel patogénico. En esta dinámica, las citoquinas juegan un papel fundamental ya que su actividad determina el inicio, duración y composición de la respuesta inmune. El interferón-gamma (IFN-t) es la citoquina más relacionada con la resistencia a T. cruzi dada su capacidad para activar a los macrófagos cuya actividad tripanocida se correlaciona con la producción de especies reactivas del oxígeno (ROS) y óxido nítrico (NO). Otras citoquinas como las interleuquinas 4 (IL-4) y 10 (IL-10) y el factor de crecimiento y transformación beta (TGF-ß) inhibirían los efectos del IFN-t durante la infección con T. cruzi, ya que desactivan la capacidad tripanocida de macrófagos e inhiben la liberación de NO. Durante la infección con T. cruzi, el factor de necrosis tumoral alfa (TNF-a) se ha asociado tanto a la resistencia como al desarrollo de patología mientras que las interleuquinas 1 (IL-1) y 6 (IL-6) se han relacionado con alteraciones del endotelio vascular responsables del espasmo microvascular observado en la miocardiopatía chagásica. Diversas citoquinas inducen la expresión de moléculas de adhesión que contribuirían al desarrollo de las procesos inflamatorios a través de las interacciones celulares del sistema inmune y de éste con las células blanco de los tejidos. En la infección aguda con distintas cepas de T. cruzi se demostró la expresión de la molécula de adhesión intercelular 1 (ICAM-1) en miocardiocitos y leucocitos inflamatorios en forma paralela a la producción de citoquinas proinflamatorias. Los resultados descriptos en modelos experimentales muestran que la infección con T. cruzi induce la producción de citoquinas cuyas actividades biológicas regulan la resistencia al parásito y posiblemente la patología de la forma crónica de la enfermedad de Chagas. Más aún, el delicado balance entre citoquinas determinaría el curso de la infección ya que los mismos mecanismo involucrados en la resistencia participarían en el desarrollo de patología y la enfermedad se apoyaría en una alteración del equilibrio regulatorio de la respuesta inmune

Papel de las citoquininas en la resistencia y patología durante la infección con Trypanosoma cruzi / Role of cytokines in the resistance and pathology during infection with Trypanosoma cruzi

La enfermedad de Chagas se asocia con diversos trastornos inmunológicos. La participación del sistema inmune del huésped infectado en la resistencia a Trypanosoma cruzi es bien conocida, no así su papel patogénico. En esta dinámica, las citoquinas juegan un papel fundamental ya que su actividad determina el inicio, duración y composición de la respuesta inmune. El interferón-gamma (IFN-t) es la citoquina más relacionada con la resistencia a T. cruzi dada su capacidad para activar a los macrófagos cuya actividad tripanocida se correlaciona con la producción de especies reactivas del oxígeno (ROS) y óxido nítrico (NO). Otras citoquinas como las interleuquinas 4 (IL-4) y 10 (IL-10) y el factor de crecimiento y transformación beta (TGF-ß) inhibirían los efectos del IFN-t durante la infección con T. cruzi, ya que desactivan la capacidad tripanocida de macrófagos e inhiben la liberación de NO. Durante la infección con T. cruzi, el factor de necrosis tumoral alfa (TNF-a) se ha asociado tanto a la resistencia como al desarrollo de patología mientras que las interleuquinas 1 (IL-1) y 6 (IL-6) se han relacionado con alteraciones del endotelio vascular responsables del espasmo microvascular observado en la miocardiopatía chagásica. Diversas citoquinas inducen la expresión de moléculas de adhesión que contribuirían al desarrollo de las procesos inflamatorios a través de las interacciones celulares del sistema inmune y de éste con las células blanco de los tejidos. En la infección aguda con distintas cepas de T. cruzi se demostró la expresión de la molécula de adhesión intercelular 1 (ICAM-1) en miocardiocitos y leucocitos inflamatorios en forma paralela a la producción de citoquinas proinflamatorias. Los resultados descriptos en modelos experimentales muestran que la infección con T. cruzi induce la producción de citoquinas cuyas actividades biológicas regulan la resistencia al parásito y posiblemente la patología de la forma crónica de la enfermedad de Chagas. Más aún, el delicado balance entre citoquinas determinaría el curso de la infección ya que los mismos mecanismo involucrados en la resistencia participarían en el desarrollo de patología y la enfermedad se apoyaría en una alteración del equilibrio regulatorio de la respuesta inmune (AU)

Trypanosoma cruzi and mammalian heart cross-reactive antigens.

Monoclonal antibodies produced against T. cruzi microsomal fraction (Mc) were used to investigate the presence of molecular mimicry between the parasite and mammalian tissues. A total of 42 cell lines secreting anti-Mc antibodies were characterized and selected by ELISA, dot blotting and Western blotting assays. Twenty seven supernatants reactive with Mc and/or parasite cytosol (CS) also reacted with human myocardial and/or skeletal muscle antigens by dot blotting assay. Twelve among those cross-reactive hybridomes, which happen to be all of the IgM isotype and to recognize structures on the surface and/or flagellum of the parasite, were selected for cell cloning. Western blotting analysis of these 12 monoclonal antibodies revealed that they mainly recognized bands of 65, 45, 34 and 27 kDa on myocardium and bands of 71, 59, 44 and 30-27 kDa on skeletal muscle. Moreover, seven among them, when assayed by immuno-histochemistry on human and hamster myocardium and skeletal muscle, recognized cytoplasmic antigens, although the monoclonal antibodies 5F2 and 5A9B11 did also bind to the vessel muscle layer. Competitive assays proved the specificity of tissue structures recognition by these monoclonal antibodies. Moreover, this reactivity resulted to be organ specific as they failed to react on lung, stomach and kidney samples. These results demonstrate the cross-reactivity of mammalian and parasite antigens, thus supporting the possibility that molecular mimicry plays a central role in the development of chagasic cardiomyopathy.