T-cell repertoire analysis in acute and chronic human Chagas' disease: differential frequencies of Vbeta5 expressing T cells.

Here, we analysed the use of Vbeta-TCR regions by CD4+ and CD8+ T cells from acute and chronic chagasic patients using flow cytometry. We determined the Vbeta expression in cells freshly isolated from patients, as well as after in vitro stimulation with antigens derived from epimastigote (EPI) or trypomastigote (TRYPO) forms of Trypanosoma cruzi. Analysis of Vbeta-TCR expression of T cells freshly isolated from patients showed a decrease in Vbeta5 expression in the CD4+ T-cell population from acutely infected individuals, whereas CD4+Vbeta5+ T cells were found to be increased in chronic patients with the cardiac, but not indeterminate, clinical form. After culturing peripheral blood mononuclear cells (PBMC) from chronic patients with EPI or TRYPO, we found that both antigenic preparations led to a preferential expansion of CD4+Vbeta5+ T cells. EPI stimulation also led to the expansion of CD8+Vbeta5+ T cells, whereas TRYPO led to the expansion of this cell population only if PBMC were from cardiac and not indeterminate patients. We observed that TRYPO stimulation led to an increase in the frequency of CD4+Vbeta17+ T cells in cultures of PBMC from indeterminate patients, whereas an increase in the frequency of CD8+Vbeta17+ T cells was found upon TRYPO stimulation of PBMC from cardiac patients. Despite this increase in the frequency of Vbeta17+ T-cell populations upon TRYPO stimulation, the same antigenic preparation led to a much higher expansion of Vbeta5+ T cells. These results show a differential expression of Vbeta5-TCR in cells freshly isolated from chagasic patients in different stages of the disease and that parasite-specific antigens stimulate a portion of the T-cell repertoire with preferential usage of Vbeta5-TCR.

Self and nonself stimulatory molecules induce preferential expansion of CD5+ B cells or activated T cells of chagasic patients, respectively.

It has previously been demonstrated that Trypanosoma cruzi-derived antigens (TRP) and human parasite-specific antibodies (Id) stimulate proliferation of cells from Chagasic patients. More recently, we have shown that activated T cells and CD5+ B cells are present in elevated levels in the peripheral blood of Chagasic patients. Upon in vitro exposure to these two different types of stimulatory molecules (TRP, Id), we now show that each of these elevated populations respond differentially to TRP or Id. We found that stimulation with TRP led to preferential expansion of activated T cells, while Id preferentially stimulated CD5+ B cells and CD8+ T cells. Moreover, this expansion of CD5+ B cells by Id was even more pronounced in cultures of cells from Chagasic patients with the severe, cardiac form of the disease, as compared to indeterminate patients. CD8+ T cells comprise approximately 50% of the total T cells in cultures stimulated by Id while in TRP-stimulated cultures their frequency is proportionally lower. Since parasite antigens and antiparasite antibodies are always present in the host during the chronic phase of the disease, they may also be involved with differential activation mechanisms of these cell populations in vivo.

Flow cytometric study of blood leucocytes in clinical forms of human schistosomiasis.

The aim of this study was to examine three distinct groups of schistosomiasis patients and to determine whether cell phenotype profiles could be correlated with the different clinical forms of the disease. The data obtained indicate that Schistosoma mansoni infected patients have a lower percentage of CD3+ T cells than do non-infected individuals. Interestingly, infected patients presented more than twice the mean percentage of circulating activated T cells (CD3+HLA-DR+) when compared to the control group. Examination of T lymphocyte subpopulations showed that patients with the severe hepatosplenic form (HS) of the disease had lower levels of both CD8High+ and CD8Low+ cells when compared to the other groups of patients. All infected individuals had a higher percentage of circulating B cells, with an increase in the CD5+ B cell population that was more evident in the HS group. The data presented here are evidence to support a relationship between the hepatosplenic form of the disease, a decrease on the CD8+ cell population and an elevation on CD5+ B cells.

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

Cytokine mRNA profile of peripheral blood mononuclear cells isolated from individuals with Trypanosoma cruzi chronic infection.

Characterization of immunologic activities during chronic infection with Trypanosoma cruzi is critical for understanding the dynamics of human Chagas' disease. Since cytokine production is mainly regulated by transcription and mRNA stability, quantitative RT-PCR analysis gives an accurate picture of the influences of disease on cytokine profile. Using RT-PCR, the authors analysed the levels of message expression for several cytokines in peripheral blood mononuclear cells (PBMC) freshly isolated from chagasic patients (CP) and non-infected individuals (NI), and in in vitro-stimulated PBMC from CP. Ex vivo analysis showed that mean levels of expression of IL-5, IL-10, IL-13 and IFN gamma were dramatically increased in PBMC from CP, compared to NI. The levels of IL-2 and IL-4 were not significantly different between groups. Analysis of cytokine mRNA production after in vitro culture with parasite-derived antigens (EPI or TRP) or anti-epimastigote antibodies (Id) showed that these two classes of stimuli induced distinct cytokine responses. While EPI or TRP induced higher production of IFN gamma specific message and low IL-10, anti-Id cells produced higher levels of IL-10 and low IFN gamma. The simultaneous presence of antigenic and antibody stimulation in the host during the chronic phase of Chagas' disease could explain the existence of both inflammatory and anti-inflammatory cellular reactivity detected in most patients.

Influence of parasite presence on the immunologic profile of peripheral blood mononuclear cells from chagasic patients after specific drug therapy.

The aim of this study is to evaluate the effects of parasite clearance on the immunological profile of peripheral blood mononuclear cells (PBMC) from chagasic patients submitted to specific drug therapy. PBMC were examined by flow cytometry and proliferative responsiveness to Trypanosoma cruzi-related stimuli. Three groups of patients were studied: not treated (NT), treated not cured (TNC) and cured (C). All data were compared to values from uninfected individuals (NI). NT displayed a lower percentage of CD3+ cells as compared to NI, while TNC and C had mean values that were between those from NI and NT. Infected patients had double the percent of CD3+ HLA-DR+ cells, independent of the efficacy of the treatment. Thus, absence of circulating parasites did not reduce T cell activation in Chagas' disease. NT displayed a higher percentage of CD5+ B cells as compared to NI, while TNC and C had mean values between those from NI and NT. In contrast to the phenotypic data, the in vitro mean proliferative responses to parasite-related stimuli of PBMC from C were reduced to the low mean levels observed in NI. These striking differences were statistically different from the high responses seen in NT and TNC. Our data suggest that proliferative responses of PBMC from C reflect immunological changes due elimination of parasite. However, successful treatment did not alter the levels of peripheral T cell activation.