Overexpression of neural cell adhesion molecule in Chagas' myocarditis.

The expression of the neural cell adhesion molecule (NCAM) was studied in normal human myocardium and in Chagas' disease myocarditis. We found that NCAM is expressed in the conduction system as well as the myocardium in the fetal heart, but its expression is restricted to the conduction system and absent in the adult myocardium. Chagas' disease is an American endemic disease caused by the Trypanosoma cruzi parasite, which produces myocarditis and a blockade of the conduction system, resulting in cardiac dysfunction. We studied the expression of NCAM in paraffin-embedded human heart tissues from 34 autopsies of patients with Chagas' myocarditis and from murine and canine experimental acute Chagas' myocarditis, using a polyclonal anti-NCAM antibody and immunohistochemistry. Our results show a dramatic upregulation of NCAM expression in the intercalated discs of cardiomyocytes in acute and chronic Chagas' myocarditis. Surprisingly, the NCAM signal was detected in intracellular nests of amastigote forms of the parasite, within infected cardiomyocytes of human and experimental Chagas' myocarditis. In contrast, cardiac cell-cell adhesion proteins, N-cadherin and beta-catenin, were found in intercalated discs distorted by the infection but absent from the amastigote nests. Proteins reactive to several antibodies against NCAM were detected by Western immunoblotting in cultured T cruzi parasites and in trypomastigote forms of T cruzi extracted from the blood of infected mice. The upregulation of NCAM in Chagas' myocarditis and the expression of NCAM or a NCAM-like protein by T cruzi suggest that NCAM may act as a receptor for tissue targeting and cellular invasion by T cruzi in Chagas' disease.

Immunoblot analysis of Leishmania panamensis antigens in sera of patients with American cutaneous leishmaniasis.

Sera from 86 Colombian patients with parasitologically confirmed cutaneous leishmaniasis were studied by immunoblot analysis in order to identify a specific pattern for Leishmania infection. A soluble extract of Leishmania panamensis was used as the antigen. Sera from patients with Chagas' disease and sera from patients with no record of infection with trypanosomatids were also studied. The sera from patients with cutaneous leishmaniasis specifically recognized fractions of 120 kDa (76.7%), 123 and 129 kDa (69.7%), 138 kDa (61.6%), 141 kDa (53.4%), and 78 kDa (44.1%). No band common to all patients infected with Leishmania parasites was found at the time of diagnosis. Likewise, the pattern of immunoblot change after the patients were treated and apparently cured with meglumine antimoniate (Glucantime) was studied by evaluating sera pretreatment and 1 year posttreatment. Only minor changes in the color intensity at the same serum dilution between pre- and posttreatment sera were found, although the antibody titers by indirect immunofluorescence were negative for the posttreatment sample. This study shows that Western blot analysis is a more sensitive test for the detection of anti-Leishmania antibodies. However, the importance of whether the presence of antibodies correlates with the presence of Leishmania antigens could not be resolved by the data obtained from this study.

Defective production of interleukin 2 in patients with Chagas' disease. Purified IL-2 augments in vitro response in patients with chagasic cardiomyopathy.

The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas' disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No differences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL-2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentrations of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

Cell mediated immune response in patients with Chagas. Correlation with the presence of Chagasic cardiomyopathy.

We are reporting data from a series of studies undertaken to evaluate if there was any correlation between the cell mediated immune response and the presence of Chagasic cardiomyopathy. Infected patients without evidence of heart pathology (INF), infected patients with Chagasic cardiomyopathy (CDM) and healthy controls, were studied. We evaluated: a) the correlation of the immune response with the presence of parasitemia; b) the suppression induced by Trypanosoma cruzi antigens using a costimulation assay; c) their production of IL2 when stimulated with antigens and mitogen; and d) the response of variable proportion of T cells subpopulations to parasite antigens. The immune response of INF was lower when parasitemia could be demonstrated. The negative regulation of the cell mediated immune response was higher in INF. The production of IL2 was lower in patients than in controls. CDM patients showed a tendency to produce less IL2 than INF. The outline of the response of variable proportions of subsets of T cells had a high concordance in CDM and no concordance in INF. These results show that there are differences in the cell mediated immune response of INF and CDM. The high concordance of the response of variable proportions of T cells, from CDM, suggests that this test could have a prognostic value.

Trypanosoma cruzi: infectivity and virulence of trypomastigotes incubated with leukocytes and serum from patients with Chagas' disease.

An important issue for the understanding of the host-parasite relationship in Chagas' disease is the extent of the action of the immune system on the parasite. To advance our understanding of this aspect, we evaluated the effect of leukocytes and/or sera from patients with Chagas' disease on trypomastigotes of Trypanosoma cruzi. We incubated, in vitro, leukocytes and sera, from patients with Chagas' disease without evidence of heart disease (INF), patients with Chagasic cardiopathy (CDM) and healthy controls (VOL), with trypomastigotes at 37 degrees C for three hours. Mice were inoculated with parasites at the end of the incubation. We kept a record of the survival time of each animal and every three days we evaluated their Parasitemia. INF (36.4%) and CDM (42.9%) prolonged the prepatent period, but not Vol. Only CDM (57.1%) extended the survival time. The sera from CDM patients that extended the survival of mice prolonged the prepatent period. However serum alone did not extend the survival time, providing evidence that leukocytes are required to decrease the virulence of the inoculum. The capacity of leukocytes and sera, from CDM, to prolong survival time shows that the immune system of patients with Chagasic cardiomyopathy can affect the parasite more intensely than IFN. On the other hand, the higher frequency of positive xenodiagnosis in CDM (11), suggests that, in vivo, occur a down regulation of the immune response.

Immunocytochemical characterization of immune cells in lesions of American cutaneous leishmaniasis using novel T cell markers.

Some recently defined lymphocyte immunophenotypes were determined in lesions of patients with American cutaneous leishmaniasis (ACL). New monoclonal antibodies have allowed the demonstration of cell surface antigens of T lymphocytes, such as CD45RA and CD45RO, which recognize different maturational stages of the same T CD4+ cell subgroup: 'virgin' (CD4+CD45RA+) and 'memory' (CD4+CD45RO+) T cells respectively. The CD4/CD8 cell ratios were higher in mucocutaneous leishmaniasis (MCL) than in localized cutaneous leishmaniasis (LCL) lesions. Diffuse cutaneous leishmaniasis (DCL) has the highest values of 'virgin' T cells; LCL and MCL patients have lower values, similar to each other. 'Memory' T cells were higher in MCL than in LCL or DCL. The ratio of 'memory'/'virgin' T cells was 7.9 for LCL, 9.6 for MCL and 2.5 for DCL. The highest value for IL-2 receptor positive cells (CD25) was observed in LCL, whereas single CD45RO-immunoreactive cells showed a peak value in DCL patients. HLA-DR+ cells were present in all three clinical forms of ACL. MCL patients showed a lack of epithelial Langerhans cell (CD1a+) in the nasal mucosa.

Cell mediated immunity in Chagas' disease. Trypanosoma cruzi antigens induce suppression of the in vitro proliferative response of mononuclear cells.

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). Mononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood bank donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative response of mononuclear cells, that might be relevant to the persistence of the parasite in the host.

Linfocitos T vírgenes y T memoria en la leishmaniasis cutánea americana / Tlyphovytes virgins and TY memory in American cutaneous leishmaniasis

La caracterización in situ de subpoblaciones leucocitarias ha permitido evaluar la participación de los diferentes componentes celulares en la respuesta inmunológica a distintos agentes patógenos. En lesiones de leishmaniasis cutánea americana se ha podido demostrar que parte de la falla inmunológica de los pacientes con leishmaniasis difusa recae sobre la subpoblación de lifocitos cooperadores inductores CD4+, los cuales no son capaces de producir Interleucina-2. Nuevos anticuerpos monoclonales permiten subdividir a los linfocitos T CD4+ en linfocitos T vírgenes CD4+ CD45RA+ y linfocitos T memoria CD4+CD45RA. En el presente estudio, se demostró que el número de linfocitos T memoria es mayor en pacientes muco-cutáneos (LCM) que localizados (LCL) y difusos (LCD). La relación de linfocitos T memoria/T vírgenes fue de 7,9 para LCM y 2,5 para LCD. Esta relación es una nueva forma de evaluar la condición inmunológica de los individuos, y pudiera ser útil en la evaluación de esquemas terapéuticos

Clinical trial with clofazimine for treating erythema dyschromicum perstans. Evaluation of cell-mediated immunity.

Eight patients were studied to determine the possible use of clofazimine for treating erythema dyschromicum perstans (EDP). The T-helper/T-suppressor cytotoxic ratio (CD-4/CD-8) and the in vitro lymphoproliferative response on stimulation with phytohemaglutin (PHA) and concanavalin A (Con A) were determined in peripheral blood before and after treatment. Of the eight patients studied, seven had excellent to good responses, whereas only one had a marginal response. The immunologic evaluation before and after treatment showed a significant change in the CD-4/CD-8 ratio, a decrease of the response to PHA, and no change in the response to Con A. The results obtained show that clofazimine is useful for treating this nosologic entity because of its cosmetic effect, and also because it induces changes in cell-mediated response, which could be very important therapeutically.

Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo.

Erythema dyschromicum perstans (EDP) and vitiligo are two cutaneous pigmentary dermatoses of unknown etiology. In the present study, the leukocyte infiltrates in the affected skin of EDP and vitiligo patients were studied using the avidin-biotin (ABC) immunoperoxidase technique and monoclonal antibodies which recognise the following mononuclear cell subgroups: T-suppressor/cytotoxic (CD8-Leu-2), T-helper (CD4 = OKT4), T-suppressor + macrophages (Leu-15), Pan T (CD3 = Leu-4), macrophages (Leu-M3) and Langerhans cells (CD1 = Leu-6), and other cellular markers such as Ia antigens and the Interleukin-2 receptor (CD25 = TAC). The immunocytochemical analysis showed a selective accumulation of CD3+, CD8+, Leu-15-, T-cytotoxic cells in the epidermis of both EDP and early lesions of vitiligo. In addition, an increase in the number of epidermal Langerhans cells (CD1+) was observed in some cases of EDP and vitiligo. The CD4/CD8 ratios in affected and uninvolved skin for both disorders were not significantly different, although values lower than unity were only observed in the infiltrates of affected skin. Ia antigen positivity was observed in the dendritic cells of the dermis and epidermis, as well as in most of the lymphoid cells within the infiltrates for both diseases. Macrophages (Leu-M3) in EDP dermal infiltrates were generally found adjacent to extracellular melanin pigment. Lymphocytes expressing TAC (CD25) surface antigens were also present in the dermal infiltrates. These morphological observations suggest a possible immune cell participation in the dyschromia of such cutaneous disorders.

Demonstration of an indomethacin-sensitive mechanism regulating immune reactivity in Chagas' disease patients.

We investigated some aspects of the regulation of the immune response that were sensitive to the effect of indomethacin, an inhibitor of prostaglandin synthesis, in two groups of patients in the chronic phase of Chagas' disease, and in normal controls. One group of patients was defined as being infected but with no clinical evidence of cardiac involvement, while the other showed electrocardiographic alterations that are characteristic of Chagasic cardiomyopathy. The in vitro responses to mitogens (phytohemagglutinin and concanavalin A) and Trypanosoma cruzi antigens were evaluated in the presence or absence of indomethacin. It was found that the in vitro mitogenic stimulation by both phytohemagglutinin and concanavalin A of peripheral blood mononuclear cells from normal controls and infected and cardiomyopathic patients was significantly increased by indomethacin. An inverse correlation was found between the initial response to concanavalin A and the subsequent increase caused by the presence of indomethacin, for both the patients and the controls. Considering specific responses to T. cruzi antigens, we showed that in the presence of indomethacin these were significantly increased in infected patients, but not in cases of cardiomyopathy. Again, a significant inverse correlation was found between the basal responsiveness and the indomethacin-induced change. In general, infected patients showed changes in the presence of indomethacin that were most comparable to those of normal individuals. It would appear, therefore, that normal indomethacin-sensitive (prostaglandin-dependent) suppressor mechanisms operate in Chagas' patients. In certain cardiomyopathy patients, however, these control mechanisms may not operate; a possible consequence of this could be tissue damage.

Evaluation of the interaction of leucocytes from Chagas disease patients with trypomastigotes of Trypanosoma cruzi.

We developed a method to asses the infectivity of Trypanosoma cruzi trypomastigotes for cultured Vero cells, after incubation under different conditions with human leucocyte populations. Total leucocytes, polymorphonuclear (PMN) and mononuclear (MN) cells, from either infected (INF) or cardiomyopathic (CDM) Chagas patients were tested in the presence or absence of autologous serum. Both patient groups had positive complement fixation tests (CFT) for T. cruzi and these were compared with a group of normal controls with negative CFT. Serum alone was found to cause reductions in the infectivity of the trypomastigotes, particularly in the case of CDM. However, in the control and INF groups, a significantly greater effect was observed with combinations of leucocytes and serum. The inhibition caused by cells plus serum was significantly greater in INF and CDM patients than in normal controls. In addition, we evaluated the penetration of trypomastigotes into the different types of leucocytes and found a significantly greater penetration of INF leucocytes at 15 min, compared to the controls, in the presence of both autologous and homologous normal serum. However, after 60 min of incubation the differences were not statistically significant.

Longitudinal study of immune response in human Chagas' disease.

Immune response, clinical status, and reactivity to heart tissue were studied longitudinally for 1 year in 42 patients with Chagas' disease (South American trypanosomiasis). The patients were divided into two groups. Group 1 was composed of patients with chagasic infection with no evidence of heart disease. Group 2 patients had chagasic infection and cardiomyopathy. Humoral immune response to Trypanosoma cruzi was measured serologically, and cell-mediated immune responses to T. cruzi and rat heart antigens were evaluated by lymphoblastogenesis. Parasitemia was detected by xenodiagnosis. Serological tests for anti-T. cruzi antibodies were positive in all patients of both groups, and the titers were significantly higher in group 2. A change of titer during the study period was more frequently associated with a positive xenodiagnosis in both groups. Lymphoblastogenesis in response to T. cruzi antigen was positive at least once in all patients of both groups. When rat heart antigen was used, 44.4% of the patients in group 1 and 40.0% of those in group 2 were positive on at least one occasion. Xenodiagnosis revealed that 20% of the patients in group 1 and 50% of those in group 2 (P = 0.01) had detectable circulating parasites during the course of the study. Positive xenodiagnosis was associated with lower lymphoblastogenic responses to T. cruzi in group 1 patients, suggesting the presence of a regulatory or modulatory mechanism which is lost in patients with chagasic cardiomyopathy. No relationship between positive xenodiagnosis and positive lymphoblastogenesis in response to heart antigen could be established. In addition, no correlation was found between clinical heart disease and reactivity to rat heart tissue.