Identification of phenotypic markers of B cells from patients with Chagas disease.

Chagas disease was discovered more than a hundred years ago, but its pathogenesis is still not completely understood. Autoimmunity is one of the mechanisms shown to contribute to its pathogenesis, which may indicate an important participation of B lymphocytes. Patients with Chagas disease have shown increased percentage of B cells producing IL-10. However, there are no reports of the phenotypic markers of B cells producing IL-10 in patients with Chagas disease. For the first time in the literature, we evaluated the phenotypic profile of distinct markers of B cells from peripheral blood of noninfected individuals and patients with Chagas disease. Our results showed that patients with Chagas disease had a higher expression of CD21 and CD24 on the surface of CD19+ B cells, while CD43 and CD23 were expressed equally in all groups. Moreover, the expression of MHC-II (HLA-DR), CD80, CD86, caspase-3, granzyme B and intracellular IL-10 and TGF-ß by CD19+ B cells was higher in patients with Chagas disease. The results of IL-10 production within CD19+ CD5+ CD1d+ B cells showed a higher percentage of this cytokine in patients with Chagas disease. Thus, our data bring a new knowledge about distinct markers of B cells in immune responses of Chagas disease.

Highly conserved CDR3 region in circulating CD4(+)Vß5(+) T cells may be associated with cytotoxic activity in Chagas disease.

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4(+) T cells from chagasic patients preferentially express T cell receptor (TCR) ß-chain variable region (Vß) 5. The aim of this work was to determine whether T cells expressing this particular Vß region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vß5·1 expressing CD4(+) T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jß region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4(+)Vß5·1(+) cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy.

Characterization of CD4⁺ cytotoxic lymphocytes and apoptosis markers induced by Trypanossoma cruzi infection.

Although the pathophysiology of Chagas disease is not completely understood, it is widely accepted that involvement of the immune response is critical in determining the outcome of the disease. In this context, CD4⁺ T cells may play an important role in generating different mechanisms of protection. In addition to effector and regulatory functions, CD4⁺ T cells may be also involved with lytic activities against the parasite and may have a relevant role on control of the infection. In this study, we have evaluated CD4⁺ T cells expressing cytotoxic and apoptosis markers in response to Trypanossoma cruzi infection in indeterminate (IND) and cardiac (CARD) patients with Chagas disease and non-infected individuals (NI). Our data demonstrated that: (1) CD4⁺ T cells presented higher ex vivo granzyme B expression in patients with Chagas disease compared with healthy individuals and that antigen induced a greater granzyme B expression in IND patients; (2) CD95L expression in CD4⁺ CD95⁺ T cells from IND patients is higher than in CARD and NI; (3) IND and CARD patients had an increased frequency of caspase-3 after in vitro stimulation and also expressed a high frequency of annexinV⁺ 7ADD⁺ within CD4⁺ T cells; (4) Lastly, a positive correlation was seen between cytotoxic molecules and CD45RO memory marker in CD4⁺ T cells and between caspase-3 and CD95L within CD4⁺ CD95⁺ T cells. These results suggest new insights into the functional competence of CD4⁺ T cells among the different clinical forms of Chagas disease, which will lead to a better understanding of their influence during immune responses against T. cruzi.

Association between myocardial mechanical dispersion and ventricular arrhythmogenicity in chagas cardiomyopathy.

Chagas disease is a major health concern in Latin America. Ventricular arrhythmia (VA) is a hallmark of Chagas cardiomyopathy (CCM), associated with worse prognosis. The present study aimed to verify the association between myocardial mechanical dispersion (MD) and ventricular arrhythmogenicity in CCM. In a cross-sectional study, 77 patients (55.8 ± 10.4 years) with CCM were evaluated. Global longitudinal strain (GLS) and MD were assessed by echocardiography, derived from the speckle tracking technique. Myocardial MD was measured from the onset of the Q/R wave on electrocardiogram to the peak longitudinal strain in 16 segments of the left ventricle. Frequency and complexity of ventricular extrasystoles (VES) were assessed by dynamic electrocardiography. The density and complexity of VES and the presence of non-sustained ventricular tachycardias (NSVTs) increase as MD increases. In logistic regression, MD was the only variable associated with the presence of paired VES and ventricular bigeminy. In addition, both MD and GLS were associated with the presence of NSVT (both, p < 0.01), and MD was independently associated with NSVT (OR 1.04, 95% CI 1.004-1.201, p = 0.031). In CCM, MD is associated with a higher density and complexity of VES, including NSVT.

Soluble inflammatory markers as predictors of liver histological changes in patients with chronic hepatitis C virus infection.

Host immune response seems to be mainly responsible for the progression of liver disease among patients with hepatitis C virus (HCV) infection. Immune activation involves the release of cytokines and their receptors that can be measured in plasma samples. The study aimed to evaluate the association between plasma levels of chemokines and soluble tumor necrosis factor receptors (sTNFR) and liver histological changes among patients with chronic HCV infection. Seventy-one treatment-naive patients were included. Plasma levels of CCL2, CCL3, CCL11, CCL24, CXCL9, CXCL10, sTNFR1, and sTNFR2 were measured and liver histological findings were reviewed. Plasma levels of CXCL9, sTNFR1, and sTNFR2 were significantly associated with liver fibrosis, with higher median levels found among patients with moderate/severe fibrosis (F >or= 2) if compared to those with no or mild fibrosis (p = 0.014; p = 0.012; p = 0.009, respectively). Plasma sTNFR2 levels were significantly associated with necroinflammatory activity, with higher median levels among patients with moderate/severe activity (A >or= 2) if compared to those with no or mild activity (2.34 ng/mL vs. 1.99 ng/mL; p = 0.019). In conclusion, plasma levels of CXCL9, sTNFR1, and sTNFR2 were independently associated with liver histological changes, suggesting a role of TNF activation and Th1-type cell-mediated immune response in the pathogenesis of HCV infection.

Mapping of the conserved antigenic domains shared between potato apyrase and parasite ATP diphosphohydrolases: potential application in human parasitic diseases.

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.

Comparative analysis of cell phenotypes in different severe clinical forms of Chagas' disease.

The understanding of the role of the immune response in the development of gastrointestinal and cardio-digestive (CD) forms of Chagas disease has received little attention. In this paper, the commitment of each leukocyte population of peripheral blood to the production of IFN-gamma, TNF-alpha, IL-12, IL-4, IL-5 and IL-10 was studied in patients with the CD form of Chagas disease. The data show that cells from patients with the CD form of the disease have distinct cytokine profiles when compared with the other clinical forms of Chagas disease and suggest that eosinophils are the major source of cytokine production in this clinical entity. The data presented in this paper demonstrate that patients with CD form can be distinguished from patients with gastrointestinal or cardiac forms of the disease by the distinct cytokine profile of peripheral blood cells.

Hepatitis C virus infection among Brazilian hemophiliacs: a virological, clinical and epidemiological study.

We determined and analyzed risk factors of hepatitis C virus (HCV)-infected Brazilian hemophiliacs according to their virological, clinical and epidemiological characteristics. A cross-sectional and retrospective study of 469 hemophiliacs was carried out at a Brazilian blood center starting in October 1997. The prevalence of HCV infection, HCV genotypes and factors associated with HCV RNA detection was determined. The seroprevalence of anti-HCV antibodies (ELISA-3.0) was 44.6% (209/469). Virological, clinical and epidemiological assessments were completed for 162 positive patients. There were seven (4.3%) anti-HCV seroconversions between October 1992 and October 1997. During the same period, 40.8% of the positive anti-HCV hemophiliacs had abnormal alanine transaminase (ALT) levels. Plasma HCV RNA was detected by nested-RT-PCR in 116 patients (71.6%). RFLP analysis showed the following genotype distribution: HCV-1 in 98 hemophiliacs (84.5%), HCV-3 in ten (8.6%), HCV-4 in three (2.6%), HCV-2 in one (0.9%), and not typeable in four cases (3.4%). Univariate analysis indicated that older age (P = 0.017) and abnormal ALT levels (P = 0.010) were associated with HCV viremia, while the presence of inhibitor antibodies (P = 0.024) and HBsAg (P = 0.007) represented a protective factor against the presence of HCV RNA. These findings may contribute to a better understanding of the relationship between HCV infection and hemophilia.

Spatial repolarization heterogeneity and survival in Chagas disease.

OBJECTIVES: We investigated if cardiac spatial repolarization heterogeneity might be associated with an increased risk of death in patients with chronic Chagas disease. METHODS: Repolarization heterogeneity was assessed using the V-index, a recently introduced metric founded on a biophysical model of the ECG. This metric provides an estimate of the standard deviation of the repolarization times across the heart. We analyzed 113 patients (aged 21- 67 years) enrolled between 1998 and 1999 who had a known serological status showing positive reactions to Trypanosoma cruzi. Fourteen subjects died during a 10-year follow-up period. RESULTS: The V-index was significantly lower in survivor (S) than in non-survivor (NS) subjects (S: 31.2 ± 13.3 ms vs NS: 41.2 ± 18.6 ms, single-tail t-test: p = 0.009, single-tail Wilcoxon rank sum test: p = 0.029). A V-index larger than 36.3 ms was related to a significantly higher risk of death in a univariate Cox proportional-hazards analysis (hazard ratio, HR = 5.34, p = 0.0046). In addition, V-index > 36.3 ms retained its prognostic value in a multivariate Cox proportional-hazards analysis after adjustment for other three clinical variables (left ventricular ejection factor < 0.50, QRS duration > 133 ms, ventricular tachycardia during stress testing or 24 hours Holter) and for T-wave amplitude variability > 30 µV, even using shrinkage, a statistical procedure that protects against over-fitting due to small sample size. CONCLUSIONS: The study showed that an increased dispersion of repolarization times in patients with Chagas disease, as measured by the V-index, is significantly correlated with the risk of death in a univariate survival analysis. The V-index captures prognostic information not immediately available from the analysis of other established risk factors.

Foxp3+CD25(high) CD4+ regulatory T cells from indeterminate patients with Chagas disease can suppress the effector cells and cytokines and reveal altered correlations with disease severity.

Immunoregulatory mechanisms are important to control the intense immune activity induced in Chagas disease. We evaluated the phenotypic profile and the mechanisms by which Treg cells function in patients with the indeterminate (IND) and cardiac (CARD) clinical forms of Chagas disease. The frequency of Foxp3(+)CD25(high) CD4(+)-T cells is augmented and correlated with the maintenance of a better cardiac function in IND. Treg cells from IND present suppressive activity, although the mechanism is not IL-10 or CTLA-4 dependent and are able to produce augmented levels of IL-17, IL-10 and granzyme B being its frequency correlated with percentage of Annexin V(+) CD4(+)-cells. In contrast, CARD presents higher frequency of IL-6(+), IFN-gamma(+), TNF-alpha(+) and CTLA-4(+) Treg-cells than IND. Thus, our data suggest that Treg cells have an important role in controlling the exacerbated immune response and morbidity in Trypanosoma cruzi infection, probably modulating the cytokine environment and/or killing effector cells.

Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1-specific immune response.

The role of interleukin 10 (IL-10) and gamma interferon (IFN-gamma) on the development of pathology in human Chagas' disease was investigated. Two categories of patients, low and high producers of IFN-gamma, were identified based on the levels of secretion of this cytokine in the supernatant of peripheral blood mononuclear cell (PBMC) cultures. Eighty-three percent of the patients presenting with cardiac disease (CARD) of different degrees and 59% of the patients with the indeterminate form of disease (IND) were identified as high IFN-gamma producers. PBMC from IND patients classified as low IFN-gamma producers secreted significantly higher amounts of IL-10 than did those from other groups. Flow cytometry analysis demonstrated that in PBMC from the IND group, the majority of the IL-10-producing cells were monocytes (CD14(High+) cells), whereas in the CARD group, the major sources of IFN-gamma were T lymphocytes (CD3(+) CD4(+) cells). These results suggest an association between the production of IFN-gamma by CD3(+) CD4(+) cells and morbidity in Chagas' disease, whereas the production of IL-10 by macrophages/monocytes leads to regulation of the immune response in IND patients. We hypothesize that an exacerbated production of IFN-gamma against Trypanosoma cruzi antigens favors the development of a strong Th1 response in CARD patients, which leads to progression of heart disease.

Phenotypic and functional characteristics of CD28+ and CD28- cells from chagasic patients: distinct repertoire and cytokine expression.

Chronic human Chagas' disease ranges from an asymptomatic to a severe cardiac clinical form. The involvement of the host's immune response in the development and maintenance of chagasic pathology has been demonstrated by several groups. We have shown that activated T-cells lacking CD28 expression are increased in the peripheral blood of chagasic patients (CP), suggesting a relationship between these cells and disease. In order to better characterize this cell population, determining their possible role in immunoregulation of human Chagas' disease, we evaluated the expression of TCR-Vbeta regions 2, 3.1, 5, 8 and 17, as well as the expression of IFN-gamma, TNF-alpha, IL-4 and IL-10 by CD28+ and CD28- cells from polarized indeterminate and cardiac CP. Flow cytometric analysis demonstrated equivalent TCR-Vbeta usage between CD4+CD28+ and CD4+CD28- cells from all groups (chagasic and healthy controls). However, there was a predominance of Vbeta5 expression in the CD28+ and CD28- populations in the CP groups (indeterminate and cardiac). Interestingly, CD8+CD28- cells from CP, but not from nonchagasic individuals, displayed a reduced frequency of most analysed Vbetas when compared with the CD8+CD28+ subpopulation. Comparison of V-beta expression in CD28+ or CD28- cell populations among individuals from different groups also showed several interesting differences. Functionally, cardiac CP displayed a higher frequency of IFN-gamma, TNF-alpha and IL-4 producing lymphocytes than indeterminate CP. Correlation analysis between the frequency of cytokine expressing cells, and the frequency of CD4+ T-cells with differential expression of CD28 demonstrated that CD4+CD28- T-cells were positively correlated with TNF-alpha in cardiac and with IL-10 in indeterminate CP, suggesting that these cells might have an important regulatory role in human Chagas' disease.

Hepatitis C virus infection among Brazilian hemophiliacs: a virological, clinical and epidemiological study

We determined and analyzed risk factors of hepatitis C virus (HCV)-infected Brazilian hemophiliacs according to their virological, clinical and epidemiological characteristics. A cross-sectional and retrospective study of 469 hemophiliacs was carried out at a Brazilian blood center starting in October 1997. The prevalence of HCV infection, HCV genotypes and factors associated with HCV RNA detection was determined. The seroprevalence of anti-HCV antibodies (ELISA-3.0) was 44.6 percent (209/469). Virological, clinical and epidemiological assessments were completed for 162 positive patients. There were seven (4.3 percent) anti-HCV seroconversions between October 1992 and October 1997. During the same period, 40.8 percent of the positive anti-HCV hemophiliacs had abnormal alanine transaminase (ALT) levels. Plasma HCV RNA was detected by nested-RT-PCR in 116 patients (71.6 percent). RFLP analysis showed the following genotype distribution: HCV-1 in 98 hemophiliacs (84.5 percent), HCV-3 in ten (8.6 percent), HCV-4 in three (2.6 percent), HCV-2 in one (0.9 percent), and not typeable in four cases (3.4 percent). Univariate analysis indicated that older age (P = 0.017) and abnormal ALT levels (P = 0.010) were associated with HCV viremia, while the presence of inhibitor antibodies (P = 0.024) and HBsAg (P = 0.007) represented a protective factor against the presence of HCV RNA. These findings may contribute to a better understanding of the relationship between HCV infection and hemophilia

IFN-gamma in human Chagas' disease: protection or pathology?

An apparently paradoxical role for IFN-gamma in human Chagas'disease was observed when studying the pattern of cytokine production by peripheral blood mononuclear cells (PBMC) obtained from two groups of chagasic patients after specific stimulation with Trypanosoma cruzi-derived antigens. The groups studied were 1) patients treated with bendnidazole during the acute phase of Trypanosoma cruzi infection and 2) chronically infected untreated patients. In the treated group, higher levels of IFN-gamma were produced by PBMC from individuals cured after treatment when compared to non-cured patients. In contrast, in the chronically infected group (not treated) higher levels of IFN-gamma were produced by PBMC from cardiac patients in comparison with asymptomatic (indeterminate) patients. This apparently paradoxical role for IFN-gamma in human Chagas'disease is discussed in terms of the possibility of a temporal difference in IFN-gamma production during the initial stages of the infection (acute phase) in the presence or absence of chemotherapy. The maintenance of an immune response with high levels of IFN-gamma production during the chronic phase of the infection may favor cure or influence the development of the cardiac form of the disease.