Isolation and molecular characterization of circulating extracellular vesicles from blood of chronic Chagas disease patients.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encase several types of molecules. Their contents mostly reflect their cell origin and possible targets at other locations in the organism and can be modified in pathological conditions to interfere with intercellular communication, thus promoting disease establishment and development. These characteristics, in addition to their presence in virtually all body fluids, make such vesicles ideal for biomarker discovery in human diseases. Here, we describe the effect of different anticoagulants and the combination of two purification methods for isolation and characterization of circulating EVs from blood of chronic Chagas disease (CCD) patients. We illustrated this procedure by studying a population of patients with Chagas disease at the indeterminate chronic stage, in which the Trypanosoma cruzi is very scarce in circulation. EVs were harvested from blood collected without or with different anticoagulants. Protein and nanoparticle tracking analysis was used to measure EVs size and concentration. The EVs were purified by ultracentrifugation, followed by size-exclusion chromatography and characterized by chemiluminescent enzyme-linked immunosorbent assay and dot blot using antibodies that recognized parasite-derived EVs, such as hyperimmune sera, polyclonal and monoclonal antibodies against trans-sialidase and mucins. In parallel, antibodies against classical human EV markers CD9, CD63, CD81, and CD82, were also analyzed. The results showed that anticoagulants did not interfere with the analyzed parameters and circulating EVs from CCD patients contain T. cruzi antigens and classical human exosomal markers. Overall, our protocol is adequate for the isolation of the total circulating EVs and can serve as an important basis for further studies on biomarker discovery in Chagas' disease.

Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease.

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy. METHODS: We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY. RESULTS: We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related - most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction. CONCLUSION: Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.

Effect of exercise training on cardiovascular autonomic and muscular function in subclinical Chagas cardiomyopathy: a randomized controlled trial.

PURPOSE: Patients with chronic chagasic cardiomyopathy with preserved ventricular function present with autonomic imbalance. This study evaluated the effects of exercise training (ET) in restoring peripheral and cardiac autonomic control and skeletal muscle phenotype in patients with subclinical chronic chagasic cardiomyopathy. METHODS: This controlled trial (NCT02295215) included 24 chronic chagasic cardiomyopathy patients who were randomized www.random.org/lists/ into two groups: those who underwent exercise training (n = 12) and those who continued their usual activities (n = 12). Eight patients completed the exercise training protocol, and 10 patients were clinically followed up for 4 months. Muscular sympathetic nerve activity was measured by microneurography and muscle blood flow (MBF) using venous occlusion plethysmography. The low-frequency component of heart rate variability in normalized units (LFnuHR) reflects sympathetic activity in the heart, and the low-frequency component of systolic blood pressure variability in normalized units reflects sympathetic activity in the vessels. The infusion of vasoactive drugs (phenylephrine and sodium nitroprusside) was used to evaluate cardiac baroreflex sensitivity, and a vastus lateralis muscle biopsy was performed to evaluate atrogin-1 and MuRF-1 gene expression. RESULTS: The baroreflex sensitivity for increases (p = 0.002) and decreases (p = 0.02) in systolic blood pressure increased in the ET group. Muscle blood flow also increased only in the ET group (p = 0.004). Only the ET group had reduced resting muscular sympathetic nerve activity levels (p = 0.008) and sympathetic activity in the heart (LFnu; p = 0.004) and vessels (p = 0.04) after 4 months. Regarding skeletal muscle, after 4 months, participants in the exercise training group presented with lower atrogin-1 gene expression than participants who continued their activities as usual (p = 0.001). The reduction in muscular sympathetic nerve activity was positively associated with reduced atrogin-1 (r = 0.86; p = 0.02) and MuRF-1 gene expression (r = 0.64; p = 0.06); it was negatively associated with improved baroreflex sensitivity both for increases (r = -0.72; p = 0.020) and decreases (r = -0.82; p = 0.001) in blood pressure. CONCLUSIONS: ET improved cardiac and peripheral autonomic function in patients with subclinical chagasic cardiomyopathy. ET reduced MSNA and sympathetic activity in the heart and vessels and increased cardiac parasympathetic tone and baroreflex sensitivity. Regarding peripheral muscle, after 4 months, patients who underwent exercise training had an increased cross-sectional area of type I fibers and oxidative metabolism of muscle fibers, and decreased atrogin-1 gene expression, compared to participants who continued their activities as usual. In addition, the reduction in MSNA was associated with improved cardiac baroreflex sensitivity, reduced sympathetic cardiovascular tone, and reduced atrogin-1 and MuRF-1 gene expression. TRIAL REGISTRATION: ID: NCT02295215. Registered in June 2013.

Dysregulation of insulin levels in Chagas heart disease is associated with altered adipocytokine levels.

Metabolic, inflammatory, and autonomic nervous system (ANS) dysfunction are present in patients with heart failure. However, whether these changes are due to left ventricular dysfunction or heart failure etiology is unknown. We evaluated metabolism and inflammatory activity in patients with idiopathic dilated cardiomyopathy (IDC) and Chagas cardiomyopathy (CHG) and their correlation with the ANS. Forty-six patients were divided into 3 groups: IDC, CHG, and control. We evaluated adiponectin, leptin, insulin, interleukin-6, and tumor necrosis factor-alpha. ANS were analyzed by heart rate variability in time and frequency domains on a 24-hour Holter monitor. Levels of glucose, cholesterol, leptin, and adiponectin did not show differences between groups. Insulin levels were lower in CHG group (5.4 ± 3.3 µU/mL) when compared with control (8.0 ± 4.9 µU/mL) and IDC (9.9 ± 5.0 µU/mL) groups (p = 0.007). Insulin was positively associated with LFr/HFr ratio (r = 0.562; p = 0.029) and with the LFr component (r = 0.562; p = 0.029) and negatively associated with adiponectin (r = -0.603; p = 0.017) in CHG group. The addition of an adiponectin unit reduced average insulin by 0.332 µg/mL. Insulin levels were decreased in the CHG group when compared with the IDC group and were associated with ANS indexes and adiponectin levels.

Chagas' heart disease: gender differences in myocardial damage assessed by cardiovascular magnetic resonance.

BACKGROUND: Since a male-related higher cardiovascular morbidity and mortality in patients with Chagas' heart disease has been reported, we aimed to investigate gender differences in myocardial damage assessed by cardiovascular magnetic resonance (CMR). METHODS AND RESULTS: Retrospectively, 62 seropositive Chagas' heart disease patients referred to CMR (1.5 T) and with low probability of having significant coronary artery disease were included in this analysis. Amongst both sexes, there was a strong negative correlation between LV ejection fraction and myocardial fibrosis (male r = 0.64, female r = 0.73, both P < 0.001), with males showing significantly greater myocardial fibrosis (P = 0.002) and lower LV ejection fraction (P < 0.001) than females. After adjustment for potential confounders, gender remained associated with myocardial dysfunction, and 53% of the effect was mediated by myocardial fibrosis (P for mediation = 0.004). Also, the transmural pattern was more prevalent among male patients (23.7 vs. 9.9%, P < 0.001) as well as the myocardial heterogeneity or gray zone (2.2 vs. 1.3 g, P = 0.003). CONCLUSIONS: We observed gender-related differences in myocardial damage assessed by CMR in patients with Chagas' heart disease. As myocardial fibrosis and myocardial dysfunction are associated to cardiovascular outcomes, our findings might help to understand the poorer prognosis observed in males in Chagas' disease.

Functional IL18 polymorphism and susceptibility to Chronic Chagas Disease.

BACKGROUND: Chronic Chagas Disease cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi, the rest of the infected subjects remaining asymptomatic (ASY). The Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis. Local expression of IL-18 in CCC myocardial tissue has recently been described. IL-18 could potentially amplify the process by inducing increased expression of IFN-γ which in turn can increase the production of IL-18, thereby creating a positive feedback mechanism. In order to assess the contribution of the IL-18 to susceptibility to Chronic Chagas Disease, we investigated the association between a single nucleotide polymorphism (SNP) located in the IL-18 gene with the risk of developing Chagas cardiomyopathy. METHODS AND RESULTS: We analyzed the rs2043055 marker in the IL18 gene in a cohort of Chagas disease cardiomyopathy patients (n=849) and asymptomatic subjects (n=202). We found a significant difference in genotype frequencies among moderate and severe CCC patients with ventricular dysfunction. CONCLUSIONS: Our analysis suggests that the IL18 rs2043055 polymorphism- or a SNP in tight linkage disequilibrium with it- may contribute to modulating the Chagas cardiomyopathy outcome.

Myocardial tissue characterization in Chagas' heart disease by cardiovascular magnetic resonance.

BACKGROUND: Chagas' heart disease is an important public health problem in South America. Several aspects of the pathogenesis are not fully understood, especially in its subclinical phases. On pathology Chagas' heart disease is characterized by chronic myocardial inflammation and extensive myocardial fibrosis. The latter has also been demonstrated by late gadolinium enhancement (LGE) by cardiovascular magnetic resonance (CMR). In three clinical phases of this disease, we sought to investigate the presence of LGE, myocardial increase in signal intensity in T2-weighted images (T2W) and in T1-weighted myocardial early gadolinium enhancement (MEGE), previously described CMR surrogates for myocardial fibrosis, myocardial edema and hyperemia, respectively. METHODS: Fifty-four patients were analyzed. Sixteen patients with the indeterminate phase (IND), seventeen patients with the cardiac phase with no left ventricular systolic dysfunction (CPND), and twenty-one patients with the cardiac phase with left ventricular systolic dysfunction (CPD). All patients underwent 1.5 T CMR scan including LGE, T2W and MEGE image sequences to evaluate myocardial abnormalities. RESULTS: Late gadolinium enhancement was present in 72.2 % of all patients, in 12.5 % of IND, 94.1 % of the CPND and 100 % of the CPD patients (p < 0.0001). Myocardial increase in signal intensity in T2-weighted images (T2W) was present in 77.8 % of all patients, in 31.3 % of the IND, 94.1 % of the CPND and 100 % of the CPD patients (p < 0.0001). T1-weighted myocardial early gadolinium enhancement (MEGE) was present in 73.8 % of all patients, in 25.0 % of the IND, 92.3 % of the CPND and 94.1 % of the CPD (p < 0.0001). A good correlation between LGE and T2W was observed (r = 0.72, and p < 0.001). CONCLUSIONS: Increase in T2-weighted (T2W) myocardial signal intensity and T1-weighted myocardial early gadolinium enhancement (MEGE) can be detected by CMR in patients throughout all phases of Chagas' heart disease, including its subclinical presentation (IND). Moreover, those findings were parallel to myocardial fibrosis (LGE) in extent and location and also correlated with the degree of Chagas' heart disease clinical severity. These findings contribute to further the knowledge on pathophysiology of Chagas' heart disease, and might have therapeutic and prognostic usefulness in the future.

Ten-year incidence of Chagas cardiomyopathy among asymptomatic Trypanosoma cruzi-seropositive former blood donors.

BACKGROUND: Very few studies have measured disease penetrance and prognostic factors of Chagas cardiomyopathy among asymptomatic Trypanosoma cruzi-infected persons. METHODS AND RESULTS: We performed a retrospective cohort study among initially healthy blood donors with an index T cruzi-seropositive donation and age-, sex-, and period-matched seronegatives in 1996 to 2002 in the Brazilian cities of São Paulo and Montes Claros. In 2008 to 2010, all subjects underwent medical history, physical examination, ECGs, and echocardiograms. ECG and echocardiogram results were classified by blinded core laboratories, and records with abnormal results were reviewed by a blinded panel of 3 cardiologists who adjudicated the outcome of Chagas cardiomyopathy. Associations with Chagas cardiomyopathy were tested with multivariate logistic regression. Mean follow-up time between index donation and outcome assessment was 10.5 years for the seropositives and 11.1 years for the seronegatives. Among 499 T cruzi seropositives, 120 (24%) had definite Chagas cardiomyopathy, and among 488 T cruzi seronegatives, 24 (5%) had cardiomyopathy, for an incidence difference of 1.85 per 100 person-years attributable to T cruzi infection. Of the 120 seropositives classified as having Chagas cardiomyopathy, only 31 (26%) presented with ejection fraction <50%, and only 11 (9%) were classified as New York Heart Association class II or higher. Chagas cardiomyopathy was associated (P<0.01) with male sex, a history of abnormal ECG, and the presence of an S3 heart sound. CONCLUSIONS: There is a substantial annual incidence of Chagas cardiomyopathy among initially asymptomatic T cruzi-seropositive blood donors, although disease was mild at diagnosis.

Exercise-induced decrease in myocardial high-energy phosphate metabolites in patients with Chagas heart disease.

BACKGROUND: The influence of exercise on cardiac metabolic response in patients with Chagas disease is incompletely understood. METHODS AND RESULTS: Changes in cardiac energetic metabolism were investigated in Chagas disease patients before and during isometric handgrip exercise with (31)P magnetic resonance spectroscopy (MRS). Twenty-eight patients (10 with systolic dysfunction: group I; 10 with normal systolic function and electrocardiogram (ECG) abnormalities: group II; and 8 asymptomatic without ECG abnormalities: group III) and 8 healthy control subjects (group C) were evaluated by electrocardiogram, echocardiogram, functional tests for coronary artery disease, and image-selected localized cardiac (31)P-MRS. The myocardial phosphocreatine to [ß-phosphate]adenosine triphosphate ratio (PCr/ß-ATP) was measured at rest and during isometric handgrip exercise. Exercise testing or 99mTc-sestamibi scintigraphy were negative for myocardial ischemia in all individuals. At rest, cardiac PCr/ß-ATP was decreased in all Chagas groups (1.23 ± 0.37) versus group C (1.88 ± 0.08; P < .001) and was lower in group I (0.89 ± 0.24) versus groups II (1.44 ± 0.23) and III (1.40 ± 0.37; P < .001). There was no stress-induced change in cardiac PCr/ß-ATP (1.88 ± 0.08 at rest vs 1.89 ± 0.08 during exercise; P = NS) in group C. Mean cardiac PCr/ß-ATP was 0.89 ± 0.24 and 0.56 ± 0.21 at rest and during exercise, respectively, in group I (37% decrease; P < .001). In group II, PCr/ß-ATP was 1.44 ± 0.23 at rest and 0.97 ± 0.37 during exercise (33% decrease; P < .001). In group III, PCr/ß-ATP was 1.40 ± 0.37 at rest and 0.60 ± 0.19 during exercise (57% decrease; P < .001). CONCLUSIONS: Myocardial high-energy phosphates are reduced at rest in Chagas heart disease patients, and the reduction is greater in patients with left ventricular dysfunction. Regardless of left ventricular function, Chagas patients exhibit an exercise-induced decline in cardiac high-energy phosphates consistent with myocardial ischemia, suggesting the possibility that this metabolic approach may offer a tool to probe new interventions in Chagas disease patients.

Genetic susceptibility to Chagas disease cardiomyopathy: involvement of several genes of the innate immunity and chemokine-dependent migration pathways.

BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America. Thirty percent of infected individuals develop chronic Chagas cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy that is, by far, the most important clinical consequence of T. cruzi infection. The others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Migration of Th1-type T cells play a major role in myocardial damage. METHODS: Our genetic analysis focused on CCR5, CCL2 and MAL/TIRAP genes. We used the Tag SNPs based approach, defined to catch all the genetic information from each gene. The study was conducted on a large Brazilian population including 315 CCC cases and 118 ASY subjects. RESULTS: The CCL2rs2530797A/A and TIRAPrs8177376A/A were associated to an increase susceptibility whereas the CCR5rs3176763C/C genotype is associated to protection to CCC. These associations were confirmed when we restricted the analysis to severe CCC, characterized by a left ventricular ejection fraction under 40%. CONCLUSIONS: Our data show that polymorphisms affecting key molecules involved in several immune parameters (innate immunity signal transduction and T cell/monocyte migration) play a role in genetic susceptibility to CCC development. This also points out to the multigenic character of CCC, each polymorphism imparting a small contribution. The identification of genetic markers for CCC will provide information for pathogenesis as well as therapeutic targets.

Detection of Early Diffuse Myocardial Fibrosis and Inflammation in Chagas Cardiomyopathy with T1 Mapping and Extracellular Volume.

Purpose: To evaluate myocardial T1 mapping and extracellular volume (ECV) parameters in different stages of Chagas cardiomyopathy and determine whether they are predictive of disease severity and prognosis. Materials and Methods: Prospectively enrolled participants (July 2013 to September 2016) underwent cine and late gadolinium enhancement (LGE) cardiac MRI and T1 mapping with a precontrast (native) or postcontrast modified Look-Locker sequence. The native T1 and ECV values were measured among subgroups that were based on disease severity (indeterminate, Chagas cardiomyopathy with preserved ejection fraction [CCpEF], Chagas cardiomyopathy with midrange ejection fraction [CCmrEF], and Chagas cardiomyopathy with reduced ejection fraction [CCrEF]). Cox proportional hazards regression and the Akaike information criterion were used to determine predictors of major cardiovascular events (cardioverter defibrillator implant, heart transplant, or death). Results: In 107 participants (90 participants with Chagas disease [mean age ± SD, 55 years ± 11; 49 men] and 17 age- and sex-matched control participants), the left ventricular (LV) ejection fraction and the extent of focal and diffuse or interstitial fibrosis were correlated with disease severity. Participants with CCmrEF and participants with CCrEF showed significantly higher global native T1 and ECV values than participants in the indeterminate, CCpEF, and control groups (T1: 1072 msec ± 34 and 1073 msec ± 63 vs 1010 msec ± 41, 1005 msec ± 69, and 999 msec ± 46; ECV: 35.5% ± 3.6 and 35.0% ± 5.4 vs 25.3% ± 3.5, 28.2% ± 4.9, and 25.2% ± 2.2; both P < .001). Remote (LGE-negative areas) native T1 and ECV values were also higher (T1: 1056 msec ± 32 and 1071 msec ± 55 vs 1008 msec ± 41, 989 msec ± 96, and 999 msec ± 46; ECV: 30.2% ± 4.7 and 30.8% ± 7.4 vs 25.1% ± 3.5, 25.1% ± 3.7, and 25.0% ± 2.2; both P < .001). Abnormal remote ECV values (>30%) occurred in 12% of participants in the indeterminate group, which increased with disease severity. Nineteen combined outcomes were observed (median follow-up time: 43 months), and a remote native T1 value greater than 1100 msec was independently predictive of combined outcomes (hazard ratio, 12 [95% CI: 4.1, 34.2]; P < .001). Conclusion: Myocardial native T1 and ECV values were correlated with Chagas disease severity and may serve as markers of myocardial involvement in Chagas cardiomyopathy that precede LGE and LV dysfunction.Keywords: MRI, Cardiac, Heart, Imaging Sequences, Chagas Cardiomyopathy Supplemental material is available for this article. © RSNA, 2023.

Effect of colchicine on myocardial injury induced by Trypanosoma cruzi in experimental Chagas disease.

BACKGROUND: The hallmark of Chagas disease (CD) is multifocal myocarditis and extensive fibrosis. We investigated the potential effect of colchicine on myocardial remodeling in experimental CD. METHODS AND RESULTS: One hundred Syrian hamsters were randomly divided into noninfected untreated control (CG), noninfected control treated with colchicine (COLG 0.4 mg kg(-1) d(-1) by gavage), infected (IG), and infected treated with colchicine (ICOLG, 0.4 mg kg(-1) d(-1)) groups. The interstitial collagen volume fraction (ICVF) was evaluated by videomorphometry with picrosirius red staining. The gelatinolytic activities of matrix metalloproteinase (MMP) 2 were examined with the use of zymography. Myocarditis was described according to the Dallas criteria. Statistical comparisons were performed with parametric analysis of variance and Tukey test. ICVF (%) accumulation was attenuated in infected colchicine-treated animals in the left (CG 0.81 ± 0.13, COLG 0.85 ± 0.13, IG: 1.35 ± 0.31,* ICOLG 1.06 ± 0.19; *P < .05 compared with ICOLG) and right ventricles (CG 1.4 ± 0.36, COLG 1.26 ± 0.14, IG 1.97 ± 0.058,* ICOLG: 1.52 ± 0.23; *P < .05 compared with ICOLG). A significant increase in MMP-2 enzymatic activity (UA) was observed in ICOLG (17,432.8*) compared with GC (3731.6), COLG (2,792.6), and IG (4,286.3; *P < .001). In IG, 66% of animals had myocarditis compared with only 49% in ICOLG. CONCLUSIONS: Colchicine had a protective effect on myocardium, indicated by decreased interstitial myocardial fibrosis, increased intensity of MMP-2, and attenuated myocardial inflammation.

Sex Differences in Heart Failure Mortality with Preserved, Mildly Reduced and Reduced Ejection Fraction: A Retrospective, Single-Center, Large-Cohort Study.

BACKGROUND: Heart failure (HF) is one of the leading causes of death worldwide. Studies show that women have better survival rates than men despite higher hospitalizations. However, little is known about differences in mortality and predictors of death in women and men with HF with preserved (HFpEF), mildly reduced (HFmrEF), and reduced ejection fraction (HFrEF). METHODS: From February 2017 to September 2020, mortality and predictors of death were analyzed in women and men with HF. Baseline data included clinical characteristics and echocardiographic findings. RESULTS: A total of 11,282 patients, 63.9 ± 14.4 years, including 6256 (55.4%) males, were studied. Females were older, had a higher baseline mean left ventricular ejection fraction (LVEF) and lower left ventricular diastolic diameter. During follow-ups, 1375 (22%) men and 925 (18.4%) women died. Cumulative incidence of death was higher in men with HFrEF but similar for HFmrEF and HFpEF. Cox regression for death showed renal dysfunction, stroke, diabetes, atrial fibrillation, age, LVEF, valve disease, MI, and hypertensive CMP as independent death predictors for all HF patients. CONCLUSIONS: Women had a better prognosis than men in HFrEF and similar mortality for HFmrEF and HFpEF, but sex was not an independent predictor of death for all HF subtypes.

Air Pollution's Impact on Cardiac Remodeling in an Experimental Model of Chagas Cardiomyopathy.

Background: Chagas disease is characterized by intense myocardial fibrosis stimulated by the exacerbated production of inflammatory cytokines, oxidative stress, and apoptosis. Air pollution is a serious public health problem and also follows this same path. Therefore, air pollution might amplify the inflammatory response of Chagas disease and increase myocardial fibrosis. Methods: We studied groups of Trypanosoma cruzi infected Sirius hamsters (Chagas=CH and Chagas exposed to pollution=CH+P) and 2 control groups (control healthy animals=CT and control exposed to pollution=CT+P). We evaluated acute phase (60 days post infection) and chronic phase (10 months). Echocardiograms were performed to assess left ventricular systolic and diastolic diameter, in addition to ejection fraction. Interstitial collagen was measured by morphometry in picrosirius red staining tissue. The evaluation of inflammation was performed by gene and protein expression of cytokines IL10, IFN-γ, and TNF; oxidative stress was quantified by gene expression of NOX1, MnSOD, and iNOS and by analysis of reactive oxygen species; and apoptosis was performed by gene expression of BCL2 and Capsase3, in addition to TUNEL analysis. Results: Chagas groups had increased collagen deposition mainly in the acute phase, but air pollution did not increase this deposition. Also, Chagas groups had lower ejection fraction in the acute phase (p = 0.002) and again air pollution did not worsen ventricular function or dilation. The analysis of the inflammation and oxidative stress pathways were also not amplified by air pollution. Apoptosis analysis showed increased expression of BCL2 and Caspase3 genes in chagasic groups in the acute phase, with a marginal p of 0.054 in BCL2 expression among infected groups, and TUNEL technique showed amplified of apoptotic cells by pollution among infected groups. Conclusions: A possible modulation of the apoptotic pathway was observed, inferring interference from air pollution in this pathway. However, it was not enough to promote a greater collagen deposition, or worsening ventricular function or dilation caused by air pollution in this model of Chagas cardiomyopathy.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

Defining Normal Healthy Term Newborn Automated Hematologic Reference Intervals at 24 Hours of Life.

CONTEXT.­: Automated analyzers have advanced the field of clinical hematology, mandating updated complete blood count (CBC) reference intervals (RIs) to be clinically useful. Contemporary newborn CBC RI publications are mostly retrospective, which some authors have cited as one of their cardinal limitations and recommended future prospective studies. OBJECTIVE.­: To prospectively establish accurate hematologic RIs for normal healthy term newborns at 24 hours of life given the limitations of the current medical literature. DESIGN.­: This prospective study was conducted at an academic tertiary care center, and hematology samples were collected from 120 participants deemed to be normal healthy term newborns. Distributions were assessed for normality and tested for outliers. Reference intervals were values between the 2.5th percentile and 97.5th percentile. RESULTS.­: The novel RIs obtained for this study population are as follows: absolute immature granulocyte count, 80/µL to 1700/µL; immature granulocyte percentage, 0.6% to 6.1%; reticulocyte hemoglobin equivalent, 31.7 to 38.4 pg; immature reticulocyte fraction, 35.9% to 52.8%; immature platelet count, 4.73 × 103/µL to 19.72 × 103/µL; and immature platelet fraction, 1.7% to 9.8%. CONCLUSIONS.­: This prospective study has defined hematologic RIs for this newborn population, including new advanced clinical parameters from the Sysmex XN-1000 Automated Hematology Analyzer. These RIs are proposed as the new standard and can serve as a strong foundation for continued research to further explore their value in diagnosing and managing morbidities such as sepsis, anemia, and thrombocytopenia.

New Biomarker in Chagas Disease: Extracellular Vesicles Isolated from Peripheral Blood in Chronic Chagas Disease Patients Modulate the Human Immune Response.

Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.

Galectin-3 Associated with Severe Forms and Long-term Mortality in Patients with Chagas Disease. / Galectina-3 Associada a Formas Graves e Mortalidade em Longo Prazo em Pacientes com Doença de Chagas.

BACKGROUND: The histopathological characteristics of Chagas disease (ChD) are: presence of myocarditis, destruction of heart fibers, and myocardial fibrosis. Galectin-3 (Gal-3) is a biomarker involved in the mechanism of fibrosis and inflammation that may be useful for risk stratification of individuals with ChD. OBJECTIVES: We sought to evaluate whether high Gal-3 levels are associated with severe forms of Chagas cardiomyopathy (CC) and whether they are predictive of mortality. METHODS: We studied anti-T. cruzi positive blood donors (BD): Non-CC-BD (187 BD without CC with normal electrocardiogram [ECG] and left ventricular ejection fraction [LVEF]); CC-Non-Dys-BD (46 BD with CC with abnormal ECG but normal LVEF); and 153 matched serum-negative controls. This cohort was composed of 97 patients with severe CC (CC-Dys). We used Kruskall-Wallis and Spearman's correlation to test hypothesis of associations, assuming a two-tailed p<0.05 as significant. RESULTS: The Gal-3 level was 12.3 ng/mL for Non-CC-BD, 12.0 ng/mL for CC-Non-Dys-BD, 13.8 ng/mL for controls, and 15.4 ng/mL for CC-Dys. LVEF<50 was associated with higher Gal-3 levels (p=0.0001). In our linear regression adjusted model, we found association between Gal-3 levels and echocardiogram parameters in T. cruzi-seropositive subjects. In CC-Dys patients, we found a significant association of higher Gal-3 levels (≥15.3 ng/mL) and subsequent death or heart transplantation in a 5-year follow-up (Hazard ratio - HR 3.11; 95%CI 1.21-8.04; p=0.019). CONCLUSIONS: In ChD patients, higher Gal-3 levels were significantly associated with severe forms of the disease and more long-term mortality, which means it may be a useful means to identify high-risk patients. (Arq Bras Cardiol. 2021; 116(2):248-256).

FUNDAMENTO: As características histopatológicas da doença de Chagas (DCC) são: presença de miocardite, destruição das fibras cardíacas e fibrose miocárdica. A Galectina-3 (Gal-3) é um biomarcador envolvido no mecanismo de fibrose e inflamação que pode ser útil para a estratificação de indivíduos com DCC por risco. OBJETIVOS: Nosso objetivo foi avaliar se níveis elevados de Gal-3 estão associados a formas graves de cardiomiopatia chagásica (CC) e são preditivos de mortalidade. MÉTODOS: Estudamos doadores de sangue (DS) positivos para anti-T. cruzi: não-CC-DS (187 DS sem CC com eletrocardiograma [ECG] e fração de ejeção do ventrículo esquerdo [FEVE] normais); CC-Não-Dis-DS (46 DS com CC e apresentando ECG anormal, mas FEVE normal); e 153 controles negativos correspondentes. Esta amostra foi composta por 97 pacientes com CC grave (CC-Dis). Usamos as correlações de Kruskall-Wallis e Spearman para testar a hipótese de associações, assumindo um p bicaudal <0,05 como significativo. RESULTADOS: O nível de Gal-3 foi de 12,3 ng/mL para não-CC-DS, 12,0 ng/mL para CC-Não-Dis-DS, 13,8 ng/mL para controles e 15,4 ng/mL para CC-Dis. FEVE <50 foi associada a níveis mais elevados de Gal-3 (p=0,0001). Em nosso modelo de regressão linear ajustado, encontramos associação entre os níveis de Gal-3 e os parâmetros do ecocardiograma em indivíduos positivos para T. cruzi. Nos pacientes CC-Dis, encontramos uma associação significativa de níveis mais elevados de Gal-3 (≥15,3 ng/mL) e morte ou transplante cardíaco em acompanhamento de cinco anos (Hazard ratio ­ HR 3,11; IC95% 1,21­ 8,04; p=0,019). CONCLUSÕES: Em pacientes com CC, níveis mais elevados de Gal-3 estiveram significativamente associados a formas graves da doença e maior taxa de mortalidade em longo prazo, o que significa que pode ser um meio efetivo para identificar pacientes de alto risco. (Arq Bras Cardiol. 2021; 116(2):248-256).

Lower Prevalence and Severity of Coronary Atherosclerosis in Chronic Chagas' Disease by Coronary Computed Tomography Angiography. / Menor Prevalência e Extensão da Aterosclerose Coronária na Doença de Chagas Crônica por Angiotomografia Coronária.

BACKGROUND: In Chagas' disease endemic regions, there has been for many years a recurrent empirical observation that coronary artery disease (CAD) is uncommon in patients with Chagas' disease. Previous pathological and invasive coronary angiography studies led to controversial results. OBJECTIVE: We sought to investigate whether CAD is less prevalent and less severe in patients with chronic Chagas' disease when compared with a matched population with a similar CAD risk profile. METHODS: A total of 86 participants, 43 consecutive patients with chronic Chagas' disease and 43 asymptomatic individuals, without any prior history of cardiac disease or known CAD (control group), were included. Patients and controls were matched according to gender, age, and Framingham risk score. All participants underwent coronary calcium scoring and coronary computed tomography angiography on a 320-row detector scanner. Statistical significance level adopted was p < 0.05. RESULTS: The coronary artery calcium score (CACS) was significantly lower in patients with Chagas' disease than in controls (p<0.05). The presence of coronary atherosclerotic plaques was significantly less frequent in patients with Chagas' disease than in controls (20.9% versus 41.9%, p=0.037). After adjustment for the Framingham score, the odds ratio for the presence of any coronary artery calcium (CAC) in Chagas patients was 0.26 (95%CI: 0.07-0.99, p=0.048). The pattern is similar for CACS > 10 (OR: 0.11, 95%CI: 0.01-0.87, p=0.04) and for the presence of any stenosis (OR: 0.06, 95%CI: 0.01-0.47, p=0.001). Propensity score matching also indicated an effect of Chagas disease on the CACS (-21.6 points in the absolute score and 25% less of patients with CACS >10, p=0.015). CONCLUSIONS: CAD is less prevalent and less severe in patients with chronic Chagas' disease when compared with a matched population with a similar CAD risk profile. (Arq Bras Cardiol. 2020; 115(6):1051-1060).

FUNDAMENTO: Em regiões endêmicas da doença de Chagas, por muitos anos, existe uma observação empírica recorrente de que a doença arterial coronariana (DAC) é incomum em pacientes com doença de Chagas. Estudos anteriores baseados em análise patológica ou angiografia coronária invasiva apresentam resultados controversos. OBJETIVO: Investigar se a DAC é menos prevalente e menos grave em pacientes com doença de Chagas crônica em comparação a uma população pareada controle, com perfil de risco para DAC similar. MÉTODOS: Um total de 86 participantes, 43 pacientes com doença de Chagas crônica consecutivos e 43 indivíduos assintomáticos, sem qualquer história prévia de doença cardíaca ou doença DAC conhecida (grupo controle), foram incluídos no estudo. Pacientes e controles foram pareados quanto sexo, idade e escore de risco de Framingham. Todos os pacientes foram analisados quanto ao escore de cálcio coronário (ECC) e submetidos à angiotomografia coronária usando um tomógrafo de 320 detectores. O nível de significância estatística adotado foi de p < 0,05. RESULTADOS: O ECC foi significativamente mais baixo em pacientes com doença de Chagas em comparação aos controles (p<0,05). A presença de placas ateroscleróticas coronárias foi significativamente menos frequente em pacientes com doença de Chagas que nos controles (20,9% versus 41,9%, p=0,037). Após ajuste quanto ao escore de Framingham, o odds ratio para a presença de qualquer calcificação coronária foi de 0,26 (IC95%: 0,07-0,99, p=0,048). O padrão é similar para escore de cálcio coronário (ECC) > 10 (OR: 0,11, IC95%: 0,01-0,87, p=0,04), e para a presença de estenose (OR: 0,06, IC95%: 0,01-0,47, p=0,001). O pareamento por escore de propensão também mostrou um efeito da doença de Chagas no ECC (-21,6 pontos no escore absoluto e 25% menos pacientes com ECC > 10; p=0,015). CONCLUSÕES: A prevalência e a gravidade da DAC são mais baixas nos pacientes com doença de Chagas crônica em comparação a uma população pareada e perfil de risco para DAC similar. (Arq Bras Cardiol. 2020; 115(6):1051-1060).