Role of sand fly saliva in human and experimental leishmaniasis: current insights.

Leishmaniases are wide spread diseases transmitted to their vertebrate host by infected sand fly. The saliva from these arthropods contains a vast repertoire of pharmacologically active molecules that hampers the host's haemostatic, inflammatory and immune responses. The early interactions between Leishmania and the host's immune response are closely linked to disease evolution or protection against the protozoan, and the ectoparasite saliva contributes directly to these interactions. Current studies have depicted these features, and these relations are being widely explored. There are concrete indications that the host response against sand fly saliva influences disease outcome in leishmaniasis. Additionally, there are demonstrations that immunization with whole sand fly saliva, or its components, leads to protection against leishmaniasis in different host species. The combination of these evidences opens up optimistic perspectives for improving vaccine development against Leishmania infection.

Influence of costimulatory molecules on immune response to Leishmania major by human cells in vitro.

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmania immune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmania immune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells of Leishmania-naive donors and cultured with or without Leishmania major in various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence of L. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. When L. major was present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, and L. major significantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response to L. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmania immune responses in humans.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65 percent smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75 percent in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57 percent by SC and 49 percent by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization

Parasite-driven in vitro human lymphocyte cytotoxicity against autologous infected macrophages from mucosal leishmaniasis.

Parasite-specific cytotoxicity in human leishmaniasis was evaluated in an autologous system. PBL from cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML) patients were exposed to Leishmania amazonensis-infected autologous macrophages for 7 days and then used as effector cells in a cytotoxic assay using 51Cr-labeled autologous infected macrophages as targets. Results are reported as LU per 10(7) PBMC. Cytotoxic activity is present in ML (9.7 +/- 2.1 LU/10(7) PBMC) but not in CL (1.5 +/- 2.4 LU/10(7) PBMC) patients' lymphocytes, and the differences were highly significant (p < 0.0001). Both CD8+ T cells and NK cells exhibited cytotoxic activity. Addition of rIL-12, but not of IFN-gamma, during the generation of effector cells increased cytotoxic responses against infected macrophages. On the other hand, addition of mAb against human IL-12 or IFN-gamma during the stimulation of PBL significantly decreased the cytotoxic responses. Addition of IL-10 led to diminished cytotoxic responses, whereas the addition of anti-IL-10 did not significantly increase the cytotoxic responses. The observation of parasite-driven autologous cytotoxic responses in patients with ML, the destructive form of leishmaniasis, but not in CL, suggests that this phenomenon is involved in tissue pathology rather than in protection. Understanding the regulation of cytotoxic responses in leishmaniasis may be relevant to strategies aimed at limiting pathologic tissue destruction.

Cytotoxicity in patients with different clinical forms of Chagas' disease.

There are few studies on cell-mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using 51Cr-labelled K562 cells as targets. Results are reported as lytic units (LU = number of cells required for 30% lysis) per million PBMC. Exposure of patients' PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8 +/- 5.0 LU/10(6) PBMC; mean +/- s.d.) than indeterminate (11.5 +/- 3.6 LU/10(6) and symptomatic cardiomyopathy (7.8 +/- 2.5 LU/10(6)). Normal control PBMC stimulated with T. cruzi antigen had 4.36 +/- 1.31 LU/10(6)) PBMC against K562. Addition of recombinant interferon-gamma (IFN-gamma) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL-12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL-12 and a neutralizing anti-IFN-gamma antibody did not change IL-12-induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up-regulated by IL-12.

Antigenic cross-reactivity of venoms obtained from snakes of genus Bothrops.

Antigenic cross-reactivity was studied among the components of venoms from nine species of the genus Bothrops using species-specific antivenoms. Sera titration by DOT-ELISA detected similar levels of antibody when either homologous or heterologous antigens were used. Transblotted antigens, after SDS-PAGE fractionation, were also revealed by homologous and heterologous antivenoms. Antigens with mol. wt greater than 30,000 seemed to be the most cross-reactive. Antigens of about 24,000 mol. wt were poorly immunogenic. Antigens between 14-18,000 mol. wt cross-reacted only with B. moojeni, B. jararacussu, B. neuwiedi and B. pradoi venoms. Neutralization of the lethality of B. jararaca venom was observed by homologous and heterologous antivenoms.

IgG subclasses responsible for immune clearance in mice infected with Trypanosoma cruzi.

To examine the role of different immunoglobulin subclasses in the immune clearance of Trypanosoma cruzi, mice containing bloodstream trypomastigotes were injected intravenously with immune serum, IgG-depleted serum, or with the IgG1 or IgG2 fractions and the rate of removal of the parasites from circulation was determined. Using IgG concentrations similar to those found in the immune serum, the rate of clearance mediated by IgG2 was six-fold higher than that obtained with IgG1. This difference did not appear to be due to differences in antibody specificity, as Western blotting showed that each isotype recognized a similar set of antigens extracted from the parasite. However, the T. cruzi specific antibody content of the IgG2 was approximately five-fold higher than IgG1. When the dose of IgG was adjusted to equalize the antibody content, the clearance ability of the IgG1 and IgG2 was very similar. It is concluded that the two subclasses have a similar clearance ability.

A comparative study of anti-Trypanosoma cruzi serum obtained in acute and chronic phase of infection in mice.

The IgG antibody content, specificity, lytic activity, clearance capacity and protective ability of mouse anti-Trypanosoma cruzi serum was determined during the course of infection. The IgG antibody content increased during the course of infection, reaching its highest level in the serum collected in the chronic phase of the infection. The T. cruzi antigens recognized by antibodies using the protein transfer technique also increased with time of infection. Antibodies present in day 22 post-infection (p.i.) serum were already able to recognize all the antigens detected by antibodies present in serum from the chronic phase. The lytic and clearance ability were not detected on day 7 p.i., but appeared on day 14 p.i. and reached their highest level on day 45 p.i. The protective ability was present in serum of the chronic phase, but was absent from the acute serum. The IgG antibody content of the acute serum was four times less than that of the chronic serum. When the IgG antibody concentration of the acute serum was equalized to that of the chronic serum, the acute serum was as able to protect the infected animals as the chronic serum. It is suggested that the disagreement between the protective ability of anti-T. cruzi antisera collected in the acute or in the chronic phase of the infection is due to a quantitative rather than a qualitative difference.

Differences in the antigenic profile of bloodstream and cell culture derived trypomastigotes of Trypanosoma cruzi.

A comparative study of the antigenic profile of bloodstream and cell culture derived trypomastigotes showed many differences in their components. Using mouse anti-T. cruzi antibodies the differences were located mostly in the 120 kDa band, whereas using chagasic patient sera the differences were located in the 85 and 52 kDa bands. These findings might explain known physiological differences between trypomastigotes obtained from cell culture and from infected blood. A brief report of this work has already been published.

Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi.

Humans and mice chronically infected with Trypanosoma cruzi present a strong humoral immune response mediated by specific antibodies. Passive transfer of homologous immune serum to normal mice containing circulating bloodstream trypomastigotes (Bts) induces a very fast clearance of the parasites. In order to find out the role of the different immunoglobulin classes in the clearance, mice containing a known number of these parasite forms in circulation were injected with total immune serum, IgG-free serum, IgG1, or IgG2 fractions and the speed of removal of the parasites from circulation was determined. The results of these experiments suggest that the immune clearance of T. cruzi is due to antibodies located in the IgG isotype, particularly in the IgG2 subclass.

Comparison between the antigenic composition of bloodstream and cell culture-derived trypomastigotes of Trypanosoma cruzi.

Antigens of bloodstream and cell culture-derived trypomastigotes of T. cruzi were compared by western blotting using sera of chronic chagasic patients as a source of antibodies. The immunoblots demonstrated that the two forms display extensive homology except for the 85- and 52-kDa bands. These antigens were more strongly stained in culture-derived trypomastigotes. Although the reported differences are not related to major antigens, these results might offer an explanation for previous studies showing that culture-derived trypomastigotes are more antigenic and infective in vitro than bloodstream trypomastigotes.

Comparison between the antigenic composition of bloodstream and cell culture-derived trypomastigotes of Trypanosoma cruzi

Antigens of bloodstream and cell culture-derived trypomastigotes of T. cruzi were compared by western blotting using sera of chronic chagasic patient as a source of antiobodies. The immunoblots demonstrated that the two forms display extensive homology except for the 85 - and 52 - kDa bands. These antigens were more strongly stained in culture - derived trypomastigotes. Although the reported differences are not related to major antigens, these results might offer an explanation for previous studies showing that culture - derived trypomastigotes are more antigenic and infective in vitro than bloodstream trypomastigotes

A new liquid medium without blood and serum for culture of hemoflagellates.

A liquid medium without blood or serum was developed for cultivation of hemoflagellates. To a basic LIT medium containing liver infusion broth and tryptose, a mixture of RPMI 1640 and Medium 199 was added. This combination permitted high parasite yields useful for biochemical and immunological studies.

Primary muscle disease: definition of a 25-kDa polypeptide myopathic specific chagas antigen.

The sera from patients with primary heart and skeletal muscle diseases, hospitalized patients without intrinsic muscle disease from an area endemic for Trypanosoma cruzi infections, and normal subjects (N = 693) were studied for the presence of immunoglobulin G (IgG) antisarcolemma activity using serologic methods. The prevalence of elevated serum IgG antisarcolemma activity from patients with chronic Chagas' cardiomyopathy, idiopathic cardiomyopathy, polymyositis, and Duchenne muscular dystrophy was 58.9 +/- 10.4% (N = 101) (P less than 0.001 when compared to normal subjects). Two of twelve (16.7%) patients with acute T. cruzi infection and parasitemia developed elevated antisarcolemma titers, and 9/46 (19.6%) patients with chronic T. cruzi infection without evidence of cardiomyopathy yielded high antisarcolemma titers. On the other hand, patients with chronic T. cruzi infection with advanced cardiomyopathy yielded high antisarcolemma titers in 35/74 (47.3%) (P less than 0.001 when compared to normal subjects). Radioimmunoprecipitation showed a circulating antibody to a 25-kDa T. cruzi polypeptide (P25) in 16/17 (94.1%) patients with advanced cardiomyopathy and T. cruzi infection. No such antibody was shown in 12 asymptomatic subjects with chronic T. cruzi infection.