Elsa Vottero-Cima

Cruzipain induces autoimmune response against skeletal muscle and tissue damage in mice

The goal of the current study was to investigate whether cruzipain, a major Trypanosoma cruzi antigen, is able to induce in mice an autoimmune response and skeletal muscle damage. We demonstrate that immunization with cruzipain triggers immunoglobulin G antibody binding to a 210‐kDa antigen from a syngeneic skeletal muscle extract. The absorption of immune sera with purified myosin completely eliminated this reactivity, confirming that the protein identified is really myosin. We also found that spleen cells from immunized mice proliferated in response to a skeletal muscle extract rich in myosin and to purified myosin. Cells from control mice did not proliferate against any of the antigens tested. In addition, we observed an increase in plasma creatine kinase activity, a biochemical marker of muscle damage. Histological studies showed inflammatory infiltrates and myopathic changes in skeletal muscle of immunized animals. Electromyographic studies of these mice revealed changes such as are found in inflammatory or necrotic myopathy. Altogether, our results suggest that this experimental model provides strong evidence for a pathogenic role of anticruzipain immune response in the development of muscle tissue damage.

Humoral autoimmune response to rat male accessory glands. Effect of castration on the humoral response.

The ability to induce antibodies to rat male accessory glands in male and female rats was demonstrated, but a higher response with wider specificity was revealed in female animals. In order to investigate whether this different response may be influenced by sexual hormones we castrated male and female rats at 4 or 30 days after birth. After that we studied the course and specificity of their humoral response to male accessory glands comparing them with that of sham-operated, sex-matched, littermate controls. Orchidectomy in male or oophorectomy in female rats changed neither the course nor specificity of humoral immune response. We conclude, therefore, that hormonal factors do not play any important role in the experimental model under study.

Exoantigens from trypanosoma cruzi contain cruzipain.

This paper shows that human antibodies specific for exoantigens of pI 4.5 (Eas 4.5), released by the blood forms of the parasite, obtained from chagasic patients sera by immunoabsorption react with cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi. Sera from mice immunized with Eas 4.5 also recognize cruzipain. In addition, mouse antisera to cruzipain were reactive with Eas 4.5 as well as with total antigens excreted by culture-trypomastigotes. This reactivity was inhibited by cruzipain as revealed by enzyme-linked immunosorbent assay (ELISA). Furthermore, it was observed by immunoblot that the exoantigens recognized by mouse antisera to cruzipain have molecular weights between 50 and 60 kDa and human antibodies specific for Eas 4.5 recognize cruzipain with apparent molecular weight of 50 kDa. These findings suggest the presence of cruzipain in Eas and the subsequent release of this enzyme by the parasite.

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG against T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytotropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

The effect of cyclophosphamide on autoimmune response to rats immunized with modified accessory glands.

It was found that the organ specific hemagglutinating autoantibodies to rat male accessory glands can be suppressed by the injection of a single dose of cyclophosphamide applied 3 days after the first immunization. On the other hand, injection of the drug 3 days before immunization did not modify the response, that is, there were comparable incidence and titers in both treated and the control animals. Cyclophosphamide did not appear to act on the homocytotropic antibodies. These results indicate that cyclophosphamide acts only when it is administered after antigenic stimulation, to suppress hemagglutinating antibodies production.

Antibody isotypes profiles against Trypanosoma cruzi acidic antigens in two Amerindian populations from a Chagas' disease endemic area

The isotype distribution of the antibody response against one Trypanosoma cruzi antigenic fraction, FIV, and the putative association to heart disease were analyzed in patients of two apparently genetically distinct Amerindian populations, Mataco (M) and Toba (T), infected with this parasite. The isotypes profiles were analyzed by ELISA, and the antigen specificity of IgG immune response was determined by the immunoblot method. The percentages of infected individuals with abnormal electrocardiograms (GII) were 50% for population M and 10% for population T. Many individuals from both populations had measureable IgG2, IgG3 and IgG4 antibodies to FIV, but the level and frequency (%) of positive sera in population T was considerably higher than in population M (70% vs 15% for IgG2; 75% vs 40% for IgG3; 85% vs 20% for IgG4). The level and frequency of IgG1 reactivity against FIV were similar in the two populations. When the sera were titrated, the most remarkable difference in isotype levels between populations T and M was seen for IgG2 and IgG4, the T population showing the highest titer. No association between clinical state and a particular isotype profile was found by ELISA in any population. When the antigen specificity of antibody response was determined by immunoblot, the antigen patterns recognized by sera from the two clinical groups showed some differences only in population M. All sera assayed from GII of population M fixed more IgG than those with normal electrocardiograms (GI). Two bands of 36 and 43 kD were revealed only in GII of this population. Similar antigenic patterns between the two clinical groups from population T were observed, and they were comparable with those obtained with GI from population M.

IgG isotype profiles induced in mice by two Trypanosoma cruzi electronegative antigens

In this work we studied the IgG isotypes induced in mice immunized with two Trypanosoma cruzi acidic antigenic fractions (F IV and Eas 4.5) and the level of protection to a later infection with parasites. F IV is a cytosolic antigen from epimastigotes, and Eas 4.5 is an exoantigen released by trypomastigotes. The most relevant epitopes of Eas 4.5 are carbohydrates. A high prevalence of IgG1, low levels of IgG3 and no IgG2 antibodies against F IV and Eas 4.5 were found in sera obtained 2 weeks after the last antigen dose from animals immunized with F IV (group I) or Eas 4.5 (group II). Immunized mice from both groups were infected with trypomastigotes, and the parasitemias detected later on were significantly lower than in control groups (p less than 0.01, group I; p less than 0.001, group II). The amount of IgG2-specific antibodies, which was only detected using epimastigotes as antigen in ELISA, was significantly increased after the infection, but no major changes were seen in the profiles of other isotypes.

Trypanosoma cruzi: Involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5

In a previous work we demonstrated that Trypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgG1 antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgG1 and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33 degrees C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56 degrees C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgG1 and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgG1 retains most of this capacity. Purified immune IgG1 revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5.

Participation of different cellular types in the enhancement of autoimmune response of old animals to sex accessory glands in male rats: importance of macrophages

In a previous work, we showed that the immunization of male rats, 3 and 12 months old, with saline extract of rat male accessory glands chemically modified (MRAG) and human serum albumin (HSA) induced a higher humoral and cellular autoimmune response in old animals than in young ones. We have also demonstrated that the facilitation of the autoimmune response is transferred by spleen total cells of 12-month-old animals. The immune response to HSA was not modified. In this work, the cellular type involved in such facilitation was analyzed. For this transference experiment, cells enriched in T and B lymphocytes and macrophages were used. The results showed that the macrophage is the main cellular type involved. However, the transference was only total with the three cellular types together. The study, performed with macrophages pulsed in vivo with MRAG-HSA and then transferred to normal recipients, indicated that although the macrophages from young and old animals were capable of presenting the antigens, the latter did this with significantly greater efficiency for the autoantigen.

Effect of aging on the autoimmune response to sex accessory glands in male rats

The effect of aging on the immune response to autoantigen of rat male accessory glands (RAG) was studied in Wistar rats. Male and female rats, 3 and 12 months old, were immunized with chemically modified RAG and heterologous antigen (human serum albumin, HSA). The study of delayed type hypersensitivity (DTH) and antibodies against RAG revealed a higher response in 12-month-old animals than in 3-month-old animals (P less than 0.005), regardless of their sexes. No differences in DTH and humoral responses to HSA were observed. Experiments on the transfer of spleen cells showed an increase in response elicited by RAG immunization in young recipients of cells from normal or immunized old syngeneic donors. On the contrary, old recipients of spleen cells from normal or immunized young donors maintained their high response the same as non-transferred old rats. Therefore, both the lymphoid cells and the environment in which the response was elicited seem to be involved in the increase of the autoimmune response.