Cellular immunity in chronic Chagas'' disease.

The cellular immune response was assessed in 20 patients with chronic Chagas' disease (American trypanosomiasis). Thymus-derived lymphocyte function was determined in vivo by cutaneous reactivity to several antigens including a soluble preparation derived from Trypanosoma cruzi and sensitization to 2,4-dinitrochlorobenzene. The in vitro T-cell reactivity was investigated by the proliferative response to phytohemagglutinin and to T. cruzi antigen and by inhibition of leukocyte migration with the specific antigen. In addition, the proportion and absolute numbers of peripheral blood T and B-lymphocytes were determined by rosette formation. This research indicates that the general and specific cellular immune response, evaluated by the tests herein mentioned, is well preserved in patients, with Chagas' disease. We conclude that chronic Chagas' disease is not associated with deficiency in cellular immunity, nor does it lead to it. Conceivably, the active participation of delayed hypersensitivity may play an important role in the expression of the human chagasic lesions.     

Interleukin-2 receptors in experimental Chagas'' disease.

Mammals infected with the protozoan parasite Trypanosoma cruzi develop suppressed cellular and humoral immune responses. This immunosuppression has been correlated with reduced T-cell responses involving deficient interleukin-2 (IL-2) production and is apparently mediated primarily by suppressor macrophages. Various forms of immunosuppression in other systems have been associated with increased levels of soluble IL-2 receptors (sIL-2R), and in the present study levels of sIL-2R in the sera of T. cruzi-infected mice during the course of infection were examined in enzyme-linked immunosorbent assays. It was found that serum levels of sIL-2R were elevated only during the third week of acute infection, a time of intense immunosuppression. In addition, IL-2R on the surface of T cells were examined by flow cytometric analyses to determine whether there is an alteration in the number of IL-2R-positive cells and whether there is a change in expression of these receptors as infection progresses. The results revealed no significant change in the percentage of cells expressing IL-2R, nor did T cells become suppressed in their ability to express IL-2R in response to concanavalin A during the course of infection.     

In vitro cellular immunity in Chagas'' disease.

In vitro delayed hypersensitivity was studied in patients with either the chronic or the indeterminate phase of Chagas' disease. Normal healthy individuals were used as controls. The leucocyte migration inhibition test was applied using an antigen an extract of culture forms of T. cruzi or an extract of normal rat heart. The results showed that patients with the chronic phase of the disease reacted significantly with both antigens, whereas most patients with the indeterminate phase did not react with heart antigen and only five out of fourteen (35%) reacted with T. cruzi antigen. It is suggested that these individuals may represent patients about to develop the chronic phase of the disease. The relevance of these findings to the immune mechanism influencing the onset of the chronic phase of Chagas' disease is discussed.     

Elevated environmental temperature enhances immunity in experimental Chagas'' disease.

C3H mice are highly susceptible to the Brazil strain of Trypanosoma cruzi. These mice usually die during the acute phase of infection and develop a profound immunosuppression to heterologous and parasite antigen. In this study, we confirmed earlier reports that infected mice maintained at elevated environmental temperature (36 degrees C) are significantly more resistant to T. cruzi than are mice kept at 20 to 24 degrees C. To determine whether the benefits of increased environmental temperature were due to alterations in the host immune system, the production of antibody to heterologous antigen and the development of parasite-specific T-helper cells were examined in noninfected and T. cruzi-infected mice. Mice were immunized with either sheep erythrocytes (SRBC) or trinitrophenyl groups (TNP) conjugated to fixed culture forms of T. cruzi, and the splenic direct plaque-forming cell (DPFC) responses to SRBC and to TNP-conjugated SRBC were determined. The DPFC response to SRBC from infected mice maintained at elevated environmental temperature was much higher than the suppressed response of infected mice held at room temperature and slightly higher than the response of age-matched noninfected control mice. Likewise, maintaining infected mice at 36 degrees C significantly enhanced the parasite-specific responses of T-helper cells, as reflected by anti-TNP DPFC responses of mice immunized with TNP-conjugated TC.     

Immunopathologic and morphologic studies of skeletal muscle in Chagas'' disease.

Skeletal muscle biopsies from 21 individuals infected with Trypanosoma cruzi were studied my means of immunofluorescence, ultrastructural immunochemical, light and electron microscopic, and histochemical procedures. In 12 cases, definite morphologic alterations were found. These alterations were coincident with the presence of circulating antibodies against the plasma membrane of striated muscle fibers and endothelial cells (EVI antibodies). In almost all cases the lesions also presented autologous immunoglobulins bound to the plasma membrane of muscle fibers and endothelial cells. Interstitial inflammatory exudate was not observed in the diseased muscle. On the basis of these observations, it is suggested that the EVI antibody is related to some of the pathogenetic mechanism of skeletal muscle damage in Chagas' disease.     

Shift of excretory-secretory immunogens of Trypanosoma cruzi during human Chagas'' disease.

We studied secreted-excreted immunogens in human patients infected with Trypanosoma cruzi. A pair of 45- to 55-kDa antigens and a family of shed acute-phase antigens characterized the acute phase, while antibodies against a 160- to 170-kDa immunogen appeared at the chronic phase of the disease.     

Immunofluorescence as an adjunct to the histopathologic diagnosis of Chagas'' disease.

We have developed a new indirect immunofluorescence procedure for identifying Trypanosoma cruzi amastigotes in sections of Formalin-fixed, paraffin-embedded tissue pretreated with a 0.25% trypsin solution to enhance immunofluorescence. In sections of human and mouse myocardia infected with T. cruzi and stained by this procedure, both intact amastigotes and phagocytosed amorphous antigen were intensely fluorescent and easily detected; nonspecific background fluorescence was absent or minimal. No staining occurred in similarly treated sections of Formalin-fixed, paraffin-embedded tissue that contained Histoplasma capsulatum var. capsulatum, Toxoplasma gondii, or Leishmania donovani to control specificity. Because pathology laboratories usually receive Formalin-fixed tissues for evaluation, this rapid and reliable procedure can be used to extend the diagnostic capability of conventional histopathology.     

Antibodies to beta 1 and beta 2 adrenoreceptors in Chagas'' disease.

Evidence accumulated over the last decade concerning human and experimental models suggests that an immunopathological mechanism may be involved in the pathogenesis of chronic Chagas' disease. In this paper we demonstrate the existence of two different circulating IgG in chagasic patients which bind with myocardial beta 1 and spleen cell beta 2 adrenoceptors, acting as non-competitive inhibitors. Both chagasic IgG against beta 1 and beta 2 adrenoceptor increased intracellular levels of cAMP that could be blocked by specific beta 1 and beta 2 adrenoceptor antagonists. The specificity for beta 1 and beta 2 adrenoceptors and the independence of other tissue reactive antibodies was demonstrated by IgG absorption with turkey red blood cell (TRBC), human lymphocytes (HL) or guinea pig red blood cells (GPRBC). The F(ab')2 fraction acted similarly. This supports the specificity of beta 1 and beta 2 adrenoceptors to the chagasic IgG and the independence of the other tissue reactive antibodies, such as EVI system. The probable pathogenic role of both beta 1 and beta 2 adrenergic chagasic antibody is discussed.     

Tumour necrosis factor (cachectin) production during experimental Chagas'' disease.

Mice infected with the protozoan parasite Trypanosoma cruzi are primed for the production of tumour necrosis factor/cachectin. Active infection is not required for stimulation of TNF production as formalin-fixed, but not heat-killed, parasites can act as a priming stimulus. Both epimastigote and trypomastigote stages of the parasite can prime for TNF production but on a per cell basis, trypomastigotes are more potent stimulators than epimastigotes and live parasites prime more efficiently than killed preparations. Although the priming effect of epimastigotes and trypomastigotes is dose-dependent when assayed 10 days after parasite injection, parasitemia levels in the infected mice do not correlate with the level of TNF production. Spleen cells from infected mice are also primed for the production in vitro of TNF in response to LPS or T. cruzi. These results suggest that TNF may be constitutively produced in vivo and that the priming for TNF production, or the production itself, may be regulated during the course of the infection in mice.     

Induction of the acute-phase protein serum amyloid P in experimental Chagas'' disease.

Serum amyloid P protein (SAP), which shares several structural properties with C-reactive protein, has been recently identified as an acute-phase reactant in mice. In this study, the systemic inflammatory response of mice to infection with Trypanosoma cruzi was characterized with respect to induction of SAP as well as to stage-specific alterations on complement C3 and C4 levels. The SAP response depended on the dose and infectivity of the parasites. Kinetic data indicated a close temporal relationship between the onset of parasitemia and induction of SAP. The levels of SAP were maximally enhanced (1,050%) by the time parasitemia started to regress, and the response remained elevated as the infection entered the latency phase. The decline in parasitemia was paralleled by a significant reduction in C3 levels. A reciprocal relationship between the extent of parasitemia and SAP/C3 levels became apparent when these parameters were compared in individual inbred mice during the time of decreasing parasitemia.     

Restricted IgG isotype profiles in T. cruzi infected mice and Chagas'' disease patients.

IgG2 was the predominant specific antibody isotype in mice chronically infected with Y strain Trypanosoma cruzi; IgG1 and IgG3 antibodies were absent or present only at very low levels. Isotype analyses of the acute phase of infection confirmed no early production of IgG1 or IgG3 and no failure in the switch from IgM to IgG. In vivo passive transfer studies of immune serum fractions showed protection to be associated only with the IgG2 isotype. A characteristic specific anti-T. cruzi IgG isotype profile (IgG1, IgG3, greater than IgG2, IgG4) was detected in a majority (39 of 50) of sera from Chagas' disease patients.     

Interleukin 2 enhances specific and nonspecific immune responses in experimental Chagas'' disease.

Mice infected with Trypanosoma cruzi develop an early and profound immunosuppression of responses to heterologous antigens. Recently it has been demonstrated that this immunosuppression is linked, in part, to deficiency in the production of interleukin 2 (IL-2), and that the addition of IL-2 to cultures of normally unresponsive spleen cells from infected mice will restore responsiveness to sheep erythrocytes (SRBC) and enhance parasite-specific immune responses. In the present study, the effect of administration of ultrapure or recombinant IL-2 on immune responses to SRBC and parasite-specific responses in vivo was examined. It was found that a single injection of 1,500 U of IL-2 provided at the same time as SRBC more than doubled the number of direct plaque-forming cells to SRBC and that multiple injections of 1,500 U of IL-2 were no more restorative than a single injection. Anti-SRBC responses of normal mice were unaffected by injection of IL-2. Single or multiple injections of recombinant human IL-2, with and without gelatin, into highly susceptible C3H(He) mice induced greater parasite-specific immunity as reflected by significantly reduced levels of parasitemia and increased longevity. Three injections of 1,500 U each of recombinant human IL-2 on days 10, 14, and 18 was found to be the most efficacious in reducing parasitemia and increasing longevity. Injection of IL-2 with gelatin did not enhance the effect of IL-2 alone.     

Trypanosoma cruzi culture used as vaccine to prevent chronic Chagas'' disease in mice.

The development of chronic pathology in mice at 2 to 10 months after inoculation of 10(2) T. cruzi trypomastigotes can be prevented by preimmunization with live, attenuated culture parasites (strain TCC). Swiss mice received one or three immunizing inoculations of 10(6) TCC organisms and were challenged with 10(2) Tulahuén blood trypomastigotes. Control groups received only the immunizing or the challenge inoculations. Immunized groups as compared with nonimmunized controls had lower mortality rates at 2 months postchallenge (9% versus 23%; P = 0.059), lower early peaks of parasitemia, lower percentages of positive xenodiagnoses at 5.5 months (40 versus 80%; P = 0.061), and lower incidences of tissue lesions in the skeletal muscle (P less than 0.005) at 2,6, and 10 months postchallenge. Tissue lesions in the heart and smooth muscle were also reduced, reaching statistical significance after 10 months (P less than 0.02). Chronic pathology parameters were never enhanced in preimmunized groups. In spite of the putative role that autoimmunity may play in the development of chronic chagasic lesions, the preventive effect of vaccination is readily exerted upon the chronic murine model of Chagas' disease.     

Immunoglobulin A antibodies to Trypanosoma cruzi antigens in digestive forms of Chagas'' disease.

In an attempt to find a serological marker for the diagnosis of chronic digestive forms of Chagas' disease, we compared amastigote and trypomastigote antigens obtained from immunosuppressed mice infected with Trypanosoma cruzi (Y strain) with conventional epimastigote antigens to search for immunoglobulin A (IgA) antibodies. A total of 255 serum samples from patients with acute and chronic (indeterminate, digestive, and cardiac) forms of Chagas' disease and with nonchagasic diseases and from healthy individuals were studied. Amastigote antigens proved to be the most adequate for our purpose, since IgA antibodies could be detected in 23 of 25 serum samples from patients with digestive forms, with relative indices of sensitivity, specificity, and efficiency of 0.920, 0.911, and 0.912, respectively. These antigens also showed high reactivity with IgA antibodies, with a geometric mean titer of 16,635 (12.7 log2). IgA antibodies were detected in 16 of 28 serum samples from patients with the acute form as well, but this clinical form is easily distinguished from the chronic form by the demonstration of IgM antibodies. Poor results were seen with trypomastigote and epimastigote antigens. The finding of IgA antibodies in about 20% of indeterminate forms and 20% of cardiac forms, although in low titers, requires further investigation to ascertain their role as an early signal of gastrointestinal lesions. In addition, the amastigote antigens described here seem more convenient for use in endemic areas than those obtained from cell cultures because of their lower cost.     

Direct micromethod for diagnosis of acute and congenital Chagas'' disease

A microhematocrit concentration method (MH) for immediate diagnosis of Chagas' disease during the acute stage or in congenital cases was standardized. Parasitemia as low as 1,000 parasites per ml was detected, after centrifugation of six 50-microliters capillary tubes, by 10-min microscopic observation of each buffy coat spread between slide and cover glass. Operator's time was reduced by at least one-third when compared with a fresh blood observation (FB). In 12 of the 15 patients studied, diagnosis was performed in 4.9 +/- 3.08 min with MH, whereas 27.0 +/- 12.1 min were necessary when FB was used. In the three remaining patients whose FB results were negative, MH became positive after 13, 16, and 40 min. In our experience, FB proved to be more sensitive than previously reported. Suckling mouse inoculation also proved to be sensitive but, as in xenodiagnosis and in hemoculture, the delay in getting the final result was a limiting factor.     

Immunopathology of experimental Chagas'' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process.