Strategy to assess the overall cytokine profile of circulating leukocytes and its association with distinct clinical forms of human Chagas disease.

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate-IND and cardiac-CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine-positive leukocytes of our study sample to establish, for each cytokine-positive cell population, the cut-off edge that would segregate 'low' and 'high' cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4+ T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4+ T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL-10-producing CD4+ T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.

[The use of insecticide fumigant canister to protect an insectarium and its residual effect against triatomine bugs, in laboratory conditions]. / Emprego de pote fumígeno para proteção de insetário e sua ação residual contra triatomíneos, em condições de laboratório.

An insecticide fumigant canister based on synthetic pyrethroids and dichlorvos was employed against cockroaches and ants which were invading an insectarium used for rearing triatominae. After removal of the Triatominae, the canister was activated and found to kill all the invading insects within 48 hours. Possible residual action against triatomines was then monitored by a 24-hour exposure of eggs, nymphs and adults of Triatoma infestans, Panstrongylus megistus and Rhodnius neglectus in the treated insectarium. No ovicidal action was observed but some mortality of adults and nymphs of the three species was observed up to 72 hours after the fumigation.

A study of experimental reinfection by Trypanosoma cruzi in dogs.

The role of reinfection in the evolution of Chagas' disease was evaluated in dogs alternately infected with the 147 and SC-1 strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain 147, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas' disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas' disease. All infected dogs developed this form of the disease, which was independent of the number of infections.

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.

Analysis of anti-Trypanosoma cruzi antibody isotype specificities by western blot in sera from patients with different forms of Chagas' disease.

Infection of humans with Trypanosoma cruzi leads to either a lifelong asymptomatic infection or to symptomatic presentations such as cardiomyopathy, mega-syndromes, or both. The reasons for the different clinical manifestations are not understood. We have previously studied a group of chronically infected individuals with different clinical forms of Chagas' disease and found that the levels of some anti-T. cruzi antibody isotypes, analyzed by enzyme-linked immunosorbent assay, differed among patients with different clinical presentations. We have expanded these studies to examine the antigen specificity of these patients' IgG1, 2, 3, IgM, and IgA by western blot. We observed that binding of particular antigens by some antibody isotypes were more prevalent in some clinical groups as compared to others. For example, IgG3 from 13 of 19 (68%) individuals with digestive manifestations bound a 68-kDa antigen, but only 3 of 31 (9%) individuals with cardiac involvement detected this same moiety. We also found that, regardless of the clinical group, the profiles of antigens recognized by each antibody isotype differs dramatically from the profiles recognized by each other isotype. Together with our previous observations demonstrating that the levels of anti-parasite antibody isotypes correlates with the clinical form, these data suggest that overall anti-T. cruzi antibody reactivities may indeed be skewed toward different antigens in individuals with different clinical presentations.

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.

Chagasic patients lack CD28 expression on many of their circulating T lymphocytes.

A balanced host-parasite interaction during Trypanosoma cruzi infection allows for the establishment of a chronic infection that can last for many years. T cells are a major element responsible for parasite specific and non-specific immunity during the complex immune response of the host. However, the subpopulations of T cells involved in the response, as well as the exact mechanisms through which those cells are activated or rendered unresponsive, are not well defined. It is known that co-stimulatory signals, some of which are mediated via CD28, are of critical importance in the triggering of appropriate T cell responses. In this study the authors performed double-labelling studies to determine the frequency of expression of CD28 by CD4+ and CD8+ T lymphocytes in the peripheral blood of patients with Chagas' disease. The results show that chagasic patients throughout the spectrum of chronic clinical forms of the infection have significantly higher mean frequencies of CD4+CD28- and CD8+CD28-T cells, as compared with non-chagasic individuals. Considering the importance of CD28 for T-cell activation, the observed down-regulation or loss of CD28 during infection may indicate a possible basis for observed immunoregulatory events or distinct stages of T-cell activation in this infection. Recent evidence from patients with HIV/AIDS indicates that CD28- cell populations are more likely to undergo apoptosis, and increased apoptosis has been observed in experimental Chagas disease.

Activated T and B lymphocytes in peripheral blood of patients with Chagas' disease.

Whole blood preparations from patients with either the indeterminate (asymptomatic) or cardiac clinical forms of chronic Trypanosoma cruzi infection were analyzed by flow cytometry using double-labeling to identify subsets of circulating lymphocytes. Several significant differences were demonstrated between the blood lymphocyte profiles of chagasic patients and non-chagasic controls. Clear increase in the percentages and actual numbers of double-positive cells of the phenotype CD3+/HLA-DR+, as well as decrease in the percentage of CD45RA+/CD4+ and CD45RA+/CD8+ T cells, indicate greater numbers of activated T cells circulating in the blood of infected patients. Consistent parallel increases were seen also in the B lymphocyte subset which stained double-positive for CD19/CD5. There were no significant differences in the circulation of these chronic chagasic patients in the CD4:CD8 ratios. Also, no substantive phenotypic differences were observed in the lymphocyte populations between the two ends of the clinical spectrum (indeterminate versus cardiac) in chronic human Chagas' disease. These observations demonstrate that increased levels of activated T cells and CD5+ B cells are present in the circulation of people with chronic Chagas' disease. These are cell phenotypes that have been associated in other conditions with autoimmune, polyclonal, and hyperimmune responses. The specificities of these activated cells and the roles they may play in resistance or pathogenesis during chronic Chagas' disease need now to be determined.