Blood leukocytes from benznidazole-treated indeterminate chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with Trypanosoma cruzi antigens.

BACKGROUND: Benznidazole (Bz)-chemotherapy is recommended to prevent Chagas disease progression, despite its limited efficacy during chronic disease. However, the host mechanisms underlying these benefits still remain to be elucidated. METHODS: In this study, we have used short-term whole blood cultures to describe the cytokine profile of Bz-treated Indeterminate Chagas disease patients-(INDt) as compared to untreated patients-(IND). RESULTS: Our findings showed that IND presented increased levels of IL-10+neutrophils, IL-12⁺ and IL-10⁺ monocytes and IFN-γ⁺NK-cells. Moreover, IND showed slight increase of IL-4⁺CD4⁺T-cells and enhanced levels of IL-10⁺CD8⁺T-cells and B-cells. Additional analysis of cytokine Low and High producers also highlighted the presence of High cytokine producers within IND, including IL-10 from CD4⁺ T-cells and IFN-γ from CD8⁺ T-cells, as compared to NI. The Bz-treatment lead to an overall cytokine down-regulation in the innate and adaptive compartments, including low levels of IL-12⁺ and IL-10⁺ neutrophils and monocytes, IFN-γ⁺NK-cells, IL-12⁺, TNF-α⁺, IFN-γ⁺ and IL-5⁺CD4⁺T-cells and IL-10⁺B-cells, along with basal levels of cytokine-expressing CD8⁺T-cells in INDt as compared to IND. The in vitro antigen stimulation shifted the cytokine profile toward a type 1-modulated profile, with increased levels of IL-12⁺ and IL-10⁺ monocytes, IFN-γ⁺ and IL-4⁺NK-cells along with TNF-α⁺ and IFN-γ⁺CD8⁺T-cells. Analysis of Low and High cytokine producers, upon in vitro antigen stimulation, further confirm these data. CONCLUSION: Together, our findings showed that the Bz treatment of Indeterminate Chagas' disease patients shifts the cytokine patterns of peripheral blood monocytes, NK-cells and CD8⁺ T-cells towards a long-lasting Type-1-modulated profile that could be important to the maintenance of a non-deleterious immunological microenvironment.

Phenotypic and Functional Signatures of Peripheral Blood and Spleen Compartments of Cynomolgus Macaques Infected With T. cruzi: Associations With Cardiac Histopathological Characteristics.

We performed a detailed analysis of immunophenotypic features of circulating leukocytes and spleen cells from cynomolgus macaques that had been naturally infected with Trypanosoma cruzi, identifying their unique and shared characteristics in relation to cardiac histopathological lesion status. T. cruzi-infected macaques were categorized into three groups: asymptomatic [CCC(-)], with mild chronic chagasic cardiopathy [CCC(+)], or with moderate chronic chagasic cardiopathy [CCC(++)]. Our findings demonstrated significant differences in innate and adaptive immunity cells of the peripheral blood and spleen compartments, by comparison with non-infected controls. CCC(+) and CCC(++) hosts exhibited decreased frequencies of monocytes, NK and NKT-cell subsets in both compartments, and increased frequencies of activated CD8+ T-cells and GranA+/GranB+ cells. While a balanced cytokine profile (TNF/IL-10) was observed in peripheral blood of CCC(-) macaques, a predominant pro-inflammatory profile (increased levels of TNF and IFN/IL-10) was observed in both CCC(+) and CCC(++) subgroups. Our data demonstrated that cardiac histopathological features of T. cruzi-infected cynomolgus macaques are associated with perturbations of the immune system similarly to those observed in chagasic humans. These results provide further support for the validity of the cynomolgus macaque model for pre-clinical research on Chagas disease, and provide insights pertaining to the underlying immunological mechanisms involved in the progression of cardiac Chagas disease.

Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate 'what must be understood' to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern.

Pro-inflammatory immune response is usually associated with Chagas disease pathogenesis, but is also relevant to treatment effectiveness. Cross-sectional studies have suggested that this activated state may persist for years after therapeutic intervention. However, short-term longitudinal investigation has suggested that the Benznidazole treatment (Bz-treatment) leads to decreased immunological activation. In order to elucidate this issue, we performed a longitudinal study to evaluate the immunological status following Bz-treatment during early indeterminate Chagas disease. Our results demonstrated that Bz-treatment led to higher activation status of circulating monocytes but was negatively associated with the number of IL-12(+)CD14(+) cells. Moreover, Bz-treatment triggered a high frequency of circulating CD3(-)CD16(+)CD56(-) NK cells, in addition to elevated activation status associated with a type 1-modulated cytokine pattern. Bz-treatment induced substantial T and B-cell activation status associated with an overall IL-10 modulated type 1 cytokine profile. In summary, these findings provide new information regarding immune activation status following the etiological treatment of Chagas disease. These results suggest that in addition to the increased number of activated leukocytes in the peripheral blood, Bz-treatment may also involve a qualitative change in their functional capacity that drives their activation state toward a modulated cytokine profile. These changes may account for the benefits of etiological treatment of Chagas disease.

Variation rhythms of lymphocyte subsets during healthy aging.

Immunological alterations associated with aging (immunosenescence) do not represent a simple unidirectional decline in all functions but develop as a complex remodeling of the immune system, involving multiple reorganization and developmentally regulated changes. In general, most data available about aging were obtained at particular age intervals and most of them come from Caucasian individuals from either Europe or the United States. Here, we report the frequencies of major lymphocyte subsets in healthy Brazilian individuals from 2 distinct geographic regions (Southeast and South) at several age intervals spanning a lifetime period (0-86 years). Overall, we demonstrated that changes in the frequencies of cells related to both innate and adaptive immunity clearly occur with aging in these individuals. These changes were not progressive and equally steady for all cell populations tested but instead showed an oscillatory or rhythmic behavior that was distinctive of each population at different age intervals. We also observed that abrupt changes in the frequencies of immune cells may occur in healthy individuals over 75 years old, suggesting there is an impaired flexibility of the immune system at late stages of life to sustain homeostasis via immune mechanisms. We presented reference ranges for healthy Brazilian individuals at all ages. The knowledge of these parameters in further detail will allow interventions to optimize immune function in advanced age and to improve the quality of life in the elderly.

Non-conventional flow cytometry approaches to detect anti-Trypanosoma cruzi immunoglobulin G in the clinical laboratory.

We have recently developed a flow cytometric approach to detect anti-live trypomastigote and anti-fixed epimastigote IgG antibodies (FC-ALTA and FC-AFEA) in sera from individuals infected by Trypanosoma cruzi. Here, we present the first evaluation of the applicability of FC-AFEA-IgG as a diagnostic tool for Chagas disease. Performance analysis demonstrated that FC-AFEA-IgG has a sensitivity of 82% and a specificity of 100%. The assessment for prognosis performed by FC-ALTA-IgG1 and FC-AFEA-IgG, after classification of chagasic patients as belonging to indeterminate (IND), cardiac (CARD) or digestive (DIG) clinical forms, showed that most of IND have higher amounts of IgG than individuals' carrying CARD or DIG Chagas disease. FC-AFEA-IgG was also evaluated as a method to monitor chemotherapy efficacy in individuals classified into three distinct categories: not treated (NT), treated but not cured (TNC), and treated and cured (TC). Performance analysis demonstrated that FC-AFEA-IgG has an extraordinary capacity as a serological criterion to assess cure after therapeutic intervention in Chagas disease. These results represent a great advance in the application of serological techniques for clinical investigations on Chagas disease, and they clearly define new directions and perspectives. We intend to continue this field research focusing our attention on the influence of the degree of clinical damage on the FC-ALTA-IgG1 and FC-AFEA-IgG reactivity.

Cynomolgus macaques naturally infected with Trypanosoma cruzi-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease.

BACKGROUND: Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease. METHODS AND FINDINGS: In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges. CONCLUSIONS: Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease.

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease.

BACKGROUND: Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations. METHODS AND FINDINGS: Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications. CONCLUSIONS: Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.

FC-TRIPLEX Chagas/Leish IgG1: a multiplexed flow cytometry method for differential serological diagnosis of chagas disease and leishmaniasis.

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.

Preeclampsia: integrated network model of platelet biomarkers interaction as a tool to evaluate the hemostatic/immunological interface.

BACKGROUND: Preeclampsia (PE) is associated with platelet activation, which may be involved in its pathogenesis promoting coagulation and mediating inflammation. We investigated whether the platelet activation status together with the frequency of platelet-leukocyte aggregates/PLA and monocyte tissue factor/TF expression could be used as laboratorial biomarkers for PE diagnosis and prognosis. METHODS: Ninety-seven women were evaluated including severe PE/sPE (N=15), mild PE/mPE (N=20), normotensive pregnant/NP (N=31) and non-pregnant women/nonP (N=31). Platelet markers were analyzed by flow cytometry. RESULTS: Platelet counts and CD41a expression by platelets were lower in NP and sPE vs nonP. The expression of CD61 was lower during pregnancy. Altered balance of platelet marker expression was also observed in NP and sPE vs nonP. No significant differences in the PLA and TF expression by monocytes were observed among the groups. There are several correlations between platelet activation markers, especially in sPE, which suggest a relevant role of the hemostatic/immunological cross-talk in this disease. CONCLUSIONS: PE is not associated with increased platelet activation markers. It cannot rule out a role of platelet activation in the PE pathophysiology. Despite those correlations, we did not find a putative laboratorial biomarker that could be useful by itself for PE diagnosis and prognosis.

The use of IgG antibodies in conventional and non-conventional immunodiagnostic tests for early prognosis after treatment of Chagas disease.

Treatment success of chronically infected Chagas disease patients is laborious and a positive prognosis often is made only after repetitive serological and/or parasitological examinations with continuous negative results. Recently, we have developed a non-conventional flow-cytometric method in order to detect immunoglobulin G antibodies against live trypomastigote forms of Trypanosoma cruzi and showed its usefulness in the prognosis of treatment success. In the present study, we investigated the performance of flow-cytometric anti-live trypomastigote IgG antibodies (FC-ALTA) and flow-cytometric anti-fixed epimastigote IgG antibodies (FC-AFEA), as well as conventional serological methods, for early monitoring of benznidazole treated Chagas disease patients, e.g. 5years after treatment. The analysis of individual FC-ALTA reactivity along the titration curve before and after treatment, we were able to show, that between 4% and 13% of treated patients under evaluation presented with reduced serological reactivity and segregated from the other patient groups. Similar results were obtained with semi-quantitative, conventional indirect hemagglutination or indirect immunofluorescence. Our data therefore suggest that the combined use of conventional and non-conventional serological methods could provide more suitable cure criteria in early post-therapeutic prognosis of Chagas disease.

Regulatory T cells phenotype in different clinical forms of Chagas' disease.

CD25(High) CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25(High)CD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25(High) CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections.

Applicability of an optimized non-conventional flow cytometry method to detect anti-Trypanosoma cruzi immunoglobulin G for the serological diagnosis and cure assessment following chemotherapeutic treatment of Chagas disease.

One of the challenges on immunodiagnostic of Chagas disease in endemic areas has been the search for more practical and safe antigenic preparation that provides tests with higher sensitivity and specificity, with low cross-reactivity. A new approach using fixed Trypanosoma cruzi epimastigotes to detect IgG reactivity was investigated previously. In order to continue this investigation, this study aimed at optimizing the flow cytometry-based method to the diagnosis of Chagas disease patients after specific chemotherapy. To achieve our goal, serum samples from 93 subjects - 52 adults chronically infected by T. cruzi, and 41 uninfected controls were tested by flow cytometry. Secondly, serum samples from patients Treated Cured and Treated Uncured from Chagas disease were also tested to evaluate the potential of the method on assessing cure. After establishing the ideal serum dilution and cut off, 121 serum samples from patients with other endemic infections were tested to check cross-reactivity. The results showed that parasite staining with Evan's blue dye eliminated debris, allowing trustworthy analysis of anti-fixed epimastigote IgG reactivity. The applicability of the method to diagnose Chagas disease was confirmed by the high sensitivity (98.1%) and specificity (100%) found. This method also contributed for post-therapeutic assessment of cure, identifying 94.1% of Treated Uncured and 83.3% of Treated Cured patients. Cross-reactivity was observed in a very low number (6.7%). On the whole, these data highly recommend the use of anti-fixed T. cruzi epimastigote IgG reactivity by flow cytometry to the diagnosis and cure monitoring of Chagas disease in endemic areas.

Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate "what must be understood" to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

Aspectos imunológicos celulares e humorais na fase crônica da doença de Chagas

Diversos estudos que avaliam os mecanismos imunológicos associados à manifestação de diferentes formas clínicas da doença de Chagas têm sugerido eventos multifatoriais. Com esse estudo, nosso objetivo foi adicionar novos elementos à complexa rede imunológica que envolve a interação parasita-hospedeiro e as distintas manifestações clínicas da doença de Chagas crônica, indeterminada (IND), cardíaca (CARD) e digestiva (DIG). Para esse objetivo, realizamos uma análise detalhada de características fenotípicas da resposta imune com ênfase na análise imunofenotípica de células mononucleares do sangue periférico, no perfil de citocinas intracitoplasmáticas de leucócitos circulantes bem como na magnitude da resposta de IgG anti-Trypanosoma cruzi. Nossos principais achados demonstraram elevadas freqüências de células reguladoras CD4+CD25high e NKT, associadas com níveis aumentados de células NK citotóxicas circulantes em IND, enquanto percentuais aumentados de linfócitos T CD8+HLA-DR+ foram observados em CARD e DIG. Adicionalmente, nossos dados demonstraram valores aumentados de células pré-NK, além de elevados valores de monócitos pró-inflamatórios e linfócitos B ativados, contrastando com a ativação diminuída de células T e diminuição da freqüência de células T reguladoras CD4+CD25high marcos do estágio recente (E-IND) da doença de Chagas crônica. Análise comparativa transversal entre E-IND, IND e CARD ainda sugeriu que uma mudança em direção a altos valores de “macrófagos-like” e níveis basais de monócitos próinflamatórios além de elevados valores de células NK maduras, NKT e CD4+CD25high podem resultar na forma clínica IND. Por outro lado, altos níveis de células CD8+HLA-DR+ paralelo a níveis basais de células NK maduras, NKT e CD4+CD25high podem levar a doença cardíaca.

Além. disso, freqüência aumentada de linfócitos totais alto-produtores de IL-4, IL-10 e IL-13 e um perfil misto de citocinas derivadas de NK e neutrófilos foram marcos do grupo IND, enquanto freqüências aumentadas de monócitos alto-produtores de TNF- e baixas freqüências de linfócitos T CD8+IL-10+ foram as principais características de CARD. A avaliação do perfil de citocinas de leucócitos circulantes aplicando a estratégia de visão panorâmica demonstrou que enquanto a maioria dos IND apresentam um perfil de citocinas reguladoras, a maioria dos CARD apresentam um micro-ambiente de citocinas inflamatórias. Apesar dos nossos achados corroborarem com relatos prévios de perfis distintos de anticorpos IgG anti-T. cruzi entre IND e CARD, eles indicaram que marcadores da resposta imune humoral são, sem dúvida, mais aplicáveis para diagnóstico sorológico e monitoração de critério de cura. Em suma, nossos dados demonstram que, mais que uma mudança em direção a um padrão polarizado, um fino balanço é relevante para direcionar os mecanismos imuno-mediados essenciais para a definição da doença de Chagas

Aspectos imunológicos celulares e humorais na fase crônica da doença de Chagas / Humoral and Cellular immunological aspects of chronic Chagas disease

Diversos estudos que avaliam os mecanismos imunológicos associados à manifestação de diferentes formas clínicas da doença de Chagas têm sugerido eventos multifatoriais. Com esse estudo, nosso objetivo foi adicionar novos elementos à complexa rede imunológica que envolve a interação parasita-hospedeiro e as distintas manifestações clínicas da doença de Chagas crônica, indeterminada (IND), cardíaca (CARD) e digestiva (DIG). Para esse objetivo, realizamos uma análise detalhada de características fenotípicas da resposta imune com ênfase na análise imunofenotípica de células mononucleares do sangue periférico, no perfil de citocinas intracitoplasmáticas de leucócitos circulantes bem como na magnitude da resposta de IgG anti-Trypanosoma cruzi. Nossos principais achados demonstraram elevadas freqüências de células reguladoras CD4+CD25high e NKT, associadas com níveis aumentados de células NK citotóxicas circulantes em IND, enquanto percentuais aumentados de linfócitos T CD8+HLA-DR+ foram observados em CARD e DIG. Adicionalmente, nossos dados demonstraram valores aumentados de células pré-NK, além de elevados valores de monócitos pró-inflamatórios e linfócitos B ativados, contrastando com a ativação diminuída de células T e diminuição da freqüência de células T reguladoras CD4+CD25high marcos do estágio recente (E-IND) da doença de Chagas crônica. Análise comparativa transversal entre E-IND, IND e CARD ainda sugeriu que uma mudança em direção a altos valores de “macrófagos-like” e níveis basais de monócitos próinflamatórios além de elevados valores de células NK maduras, NKT e CD4+CD25high podem resultar na forma clínica IND. Por outro lado, altos níveis de células CD8+HLA-DR+ paralelo a níveis basais de células NK maduras, NKT e CD4+CD25high podem levar a doença cardíaca.(AU)

Além. disso, freqüência aumentada de linfócitos totais alto-produtores de IL-4, IL-10 e IL-13 e um perfil misto de citocinas derivadas de NK e neutrófilos foram marcos do grupo IND, enquanto freqüências aumentadas de monócitos alto-produtores de TNF- e baixas freqüências de linfócitos T CD8+IL-10+ foram as principais características de CARD. A avaliação do perfil de citocinas de leucócitos circulantes aplicando a estratégia de visão panorâmica demonstrou que enquanto a maioria dos IND apresentam um perfil de citocinas reguladoras, a maioria dos CARD apresentam um micro-ambiente de citocinas inflamatórias. Apesar dos nossos achados corroborarem com relatos prévios de perfis distintos de anticorpos IgG anti-T. cruzi entre IND e CARD, eles indicaram que marcadores da resposta imune humoral são, sem dúvida, mais aplicáveis para diagnóstico sorológico e monitoração de critério de cura. Em suma, nossos dados demonstram que, mais que uma mudança em direção a um padrão polarizado, um fino balanço é relevante para direcionar os mecanismos imuno-mediados essenciais para a definição da doença de Chagas(AU)

Aspectos imunológicos celulares e humorais na fase crônica da doença de Chagas / Humoral and Cellular immunological aspects of chronic Chagas disease

Diversos estudos que avaliam os mecanismos imunológicos associados à manifestação de diferentes formas clínicas da doença de Chagas têm sugerido eventos multifatoriais. Com esse estudo, nosso objetivo foi adicionar novos elementos à complexa rede imunológica que envolve a interação parasita-hospedeiro e as distintas manifestações clínicas da doença de Chagas crônica, indeterminada (IND), cardíaca (CARD) e digestiva (DIG). Para esse objetivo, realizamos uma análise detalhada de características fenotípicas da resposta imune com ênfase na análise imunofenotípica de células mononucleares do sangue periférico, no perfil de citocinas intracitoplasmáticas de leucócitos circulantes bem como na magnitude da resposta de IgG anti-Trypanosoma cruzi. Nossos principais achados demonstraram elevadas freqüências de células reguladoras CD4+CD25high e NKT, associadas com níveis aumentados de células NK citotóxicas circulantes em IND, enquanto percentuais aumentados de linfócitos T CD8+HLA-DR+ foram observados em CARD e DIG. Adicionalmente, nossos dados demonstraram valores aumentados de células pré-NK, além de elevados valores de monócitos pró-inflamatórios e linfócitos B ativados, contrastando com a ativação diminuída de células T e diminuição da freqüência de células T reguladoras CD4+CD25high marcos do estágio recente (E-IND) da doença de Chagas crônica. Análise comparativa transversal entre E-IND, IND e CARD ainda sugeriu que uma mudança em direção a altos valores de “macrófagos-like” e níveis basais de monócitos próinflamatórios além de elevados valores de células NK maduras, NKT e CD4+CD25high podem resultar na forma clínica IND. Por outro lado, altos níveis de células CD8+HLA-DR+ paralelo a níveis basais de células NK maduras, NKT e CD4+CD25high podem levar a doença cardíaca.

Além. disso, freqüência aumentada de linfócitos totais alto-produtores de IL-4, IL-10 e IL-13 e um perfil misto de citocinas derivadas de NK e neutrófilos foram marcos do grupo IND, enquanto freqüências aumentadas de monócitos alto-produtores de TNF- e baixas freqüências de linfócitos T CD8+IL-10+ foram as principais características de CARD. A avaliação do perfil de citocinas de leucócitos circulantes aplicando a estratégia de visão panorâmica demonstrou que enquanto a maioria dos IND apresentam um perfil de citocinas reguladoras, a maioria dos CARD apresentam um micro-ambiente de citocinas inflamatórias. Apesar dos nossos achados corroborarem com relatos prévios de perfis distintos de anticorpos IgG anti-T. cruzi entre IND e CARD, eles indicaram que marcadores da resposta imune humoral são, sem dúvida, mais aplicáveis para diagnóstico sorológico e monitoração de critério de cura. Em suma, nossos dados demonstram que, mais que uma mudança em direção a um padrão polarizado, um fino balanço é relevante para direcionar os mecanismos imuno-mediados essenciais para a definição da doença de Chagas