The specificity of B lymphocytes activated in the acute phase of murine Trypanosoma cruzi infection was analysed in a panel of immunoglobulin-secreting hybridomas derived by fusion of lymph node cells 7 days after intraperitoneal parasite inoculation. The immunoglobulin isotype distribution of the hybrids reflected the total plaque-forming cell (PFC) response in the animal at this point, with a predominance of IgG2a, IgM, and IgG2b. Screening of the hybridoma antibodies on parasite antigens by three independent methods (western blot, ELISA, and immunofluorescence) revealed only one (out of a total of 51) that bound a parasite molecule with an apparent molecular mass of 180 kDa. In contrast, antibodies of both IgM and IgG classes were found to react with a panel of autologous antigens. These results establish that most B cells activated by T. cruzi infection are not specific for parasite antigens and therefore indicate the relevance of analysing the totality of host responses to infection.

A Trypanosoma cruzi Xgtll cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE

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Different tissues and organs of mice infected with Trypanosoma cruzi trypomastigotes have been examined for the presence of parasites and parasitic antigens during both the acute and the chronic phases of infection. Specimens of skeletal and cardiac muscles, spleen, liver, brain and sciatic nerves were studied by histological and immunological methods. During the acute phase of infection, the parasites were commonly observed in these tissues. In the chronic phase of the experimental infection, pseudocysts filled with amastigotes were seen in less than 1% of the tissue sections, while immunohistological methods showed that T. cruzi antigens were present in 11% of the inflammatory infiltrates. These findings suggest that antigenic stimulation persists throughout the chronic phase, even though the parasites are not morphologically detectable.

Changes in thymic T-cell subsets in mice acutely infected with Trypanosoma cruzi have been studied in both C3H/HeJ and C57BL/6 mice. The significant decrease in thymocyte number, observed in both mouse strains on day 14 post-infection correlated with a drastic decrease in CD4+CD8+ cell number, whereas the number of CD4-CD8-, CD4+CD8- and CD4-CD8+ cells remained essentially unchanged. The important increase in CD3hi cell frequency confirmed that resistant thymocytes during Chagas' disease development were mostly medullary thymocytes, whereas the thymic cortex was largely depleted, as previously observed on thymus sections. This involution of the thymus could have been due to the increase of circulating glucocorticoid levels observed after infection. However, similar cell modifications were found in infected adrenalectomized mice whose serum corticosterone levels were only slightly augmented. Thus, the thymic alterations appear not to be linked to stress responses, at least those dependent on high levels of circulating glucocorticoids.

During the chronic phase of infection with the parasite Trypanosoma cruzi, mice develop inflammatory lesions in the heart and skeletal muscles, as well as in peripheral nerves and the liver. We demonstrated the presence, in the blood of chronically infected mice, of L3T4+ T cells able to transfer a specific T. cruzi delayed-type hypersensitivity (DTH) reaction. Transfer of the chronic inflammatory lesions was obtained by injecting Lyt-2+-depleted lymphocytes from either lymph node or blood of infected mice. T cell lines were established from the chronically infected mice by culturing peripheral blood lymphocytes or lymph node cells with either T. cruzi extracts (TC) or mouse peripheral nerve extracts (PN). Those cell lines that presented an L3T4+ phenotype were also able to specifically transfer a local DTH reaction to naive recipients. Examination of the antigen specificities of these TDTH lines revealed three types: those that mediated a DTH reaction to TC, those that responded to both TC and PN and those that provoked a DTH response when injected with subinflammatory doses of an irrelevant antigen. Some of the lines, when injected into the sciatic nerve of naive recipients, provoked demyelination of the type observed in chronically infected animals. These results suggest that T. cruzi and the host nervous system share common epitopes that can be recognized by TDTH cells.

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Previous observations demonstrated severe thymocyte depletion in mice undergoing acute Chagas' disease. These data led us to investigate the status of the thymic microenvironment in these animals. Young adult C57BL/6 and C3H/HeJ mice were infected i.p. with 10(5) blood-derived trypomastigote forms of Trypanosoma cruzi (CL strain) and killed 7-14 days after infection. Sera were then analyzed for thymic hormone (thymulin) levels, and frozen thymus sections were studied by immunohistochemistry for the expression of functional antigens (thymulin and Ia), the distribution of distinct thymic epithelial cell subsets and extracellular matrix components. Infected mice exhibited a transient decrease in thymulin production and those with severe thymic atrophy showed a denser Ia-bearing cellular network. In addition, an abnormal localization of the TR5 and CK18 antigens restricted to the medullary and cortical TEC subsets, respectively, was observed. Furthermore, an increase in the basement membrane proteins was detected within thymic lobules. We suggest that the thymic microenvironment is also affected during T. cruzi infection, extending the concept that the thymus should be regarded as a target in Chagas' disease.