Gene expression network analyses during infection with virulent and avirulent Trypanosoma cruzi strains unveil a role for fibroblasts in neutrophil recruitment and activation.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that has a heterogeneous population composed of a pool of strains with distinct characteristics, including variable levels of virulence. In previous work, transcriptome analyses of parasite genes after infection of human foreskin fibroblasts (HFF) with virulent (CL Brener) and non-virulent (CL-14) clones derived from the CL strain, revealed a reduced expression of genes encoding parasite surface proteins in CL-14 compared to CL Brener during the final steps of the intracellular differentiation from amastigotes to trypomastigotes. Here we analyzed changes in the expression of host genes during in vitro infection of HFF cells with the CL Brener and CL-14 strains by analyzing total RNA extracted from cells at 60 and 96 hours post-infection (hpi) with each strain, as well as from uninfected cells. Similar transcriptome profiles were observed at 60 hpi with both strains compared to uninfected samples. However, at 96 hpi, significant differences in the number and expression levels of several genes, particularly those involved with immune response and cytoskeleton organization, were observed. Further analyses confirmed the difference in the chemokine/cytokine signaling involved with the recruitment and activation of immune cells such as neutrophils upon T. cruzi infection. These findings suggest that infection with the virulent CL Brener strain induces a more robust inflammatory response when compared with the non-virulent CL-14 strain. Importantly, the RNA-Seq data also exposed an unexplored role of fibroblasts as sentinel cells that may act by recruiting neutrophils to the initial site of infection. This role for fibroblasts in the regulation of the inflammatory response during infection by T. cruzi was corroborated by measurements of levels of different chemokines/cytokines during in vitro infection and in plasma from Chagas disease patients as well as by neutrophil activation and migration assays.

Systemic cytokines, chemokines and growth factors reveal specific and shared immunological characteristics in infectious cardiomyopathies.

Heart disease is a major cause of death worldwide. Chronic Chagas cardiomyopathy (CCC) caused by infection with Trypanosoma cruzi leading to high mortality in adults, and rheumatic heart disease (RHD), resulting from infection by Streptococcus pyogenes affecting mainly children and young adults, are amongst the deadliest heart diseases in low-middle income countries. Despite distinct etiology, the pathology associated with both diseases is a consequence of inflammation. Here we compare systemic immune profile in patients with these cardiopathies, to identify particular and common characteristics in these infectious heart diseases. We evaluated the expression of 27 soluble factors, employing single and multivariate analysis combined with machine-learning approaches. We observed that, while RHD and CCC display higher levels of circulating mediators than healthy individuals, CCC is associated with stronger immune activation as compared to RHD. Despite distinct etiologies, univariate analysis showed that expression of TNF, IL-17, IFN-gamma, IL-4, CCL4, CCL3, CXCL8, CCL11, CCL2, PDGF-BB were similar between CCC and RHD, consistent with their inflammatory nature. Network analysis revealed common inflammatory pathways between CCC and RHD, while highlighting the broader reach of the inflammatory response in CCC. The final multivariate model showed a 100% discrimination power for the combination of the cytokines IL-12p70, IL-1Ra, IL-4, and IL-7 between CCC and RHD groups. Thus, while clear immunological distinctions were identified between CCC and RHD, similarities indicate shared inflammatory pathways in these infectious heart diseases. These results contribute to understanding the pathogenesis of CCC and RHD and may impact the design of immune-based therapies for these and other inflammatory cardiopathies that may also share immunological characteristics.

High interleukin 17 expression is correlated with better cardiac function in human Chagas disease.

This study was designed to investigate whether the expression of interleukin 17 (IL-17) is associated with the indeterminate or cardiac clinical forms of Chagas disease and whether IL-17 expression can be correlated with patients' cardiac function. Our results demonstrated that cardiac Chagas patients have a lower intensity of expression of IL-17 by total lymphocytes and lower frequency of circulating T helper 17 cells. Correlative analysis showed that high IL-17 expression was associated with better cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Therefore, IL-17 expression can be a protective factor to prevent myocardial damage in human Chagas disease.

"Subclinical atherosclerosis in acromegaly: Possible association with cardiovascular risk factors rather than disease activity".

OBJECTIVE: Cardiovascular (CV) disease is still a major cause of excessive morbidity and mortality in patients with active acromegaly, which may be attributed to a high prevalence of associated pro-atherosclerotic risk factors. However, a direct effect of GH/IGF-1 excess on the vasculature has been previously suggested, warranting further investigation. The present study was designed to investigate whether chronic GH/IGF-1 excess is associated with an increased prevalence of subclinical atherosclerosis in patients with acromegaly. DESIGN: We measured carotid intima-media thickness (cIMT) and assessed carotid plaques by ultrasonography along with classical CV risk factors in 54 acromegaly patients (34 females, 50 ± 12 years and compared those with 62 (42 females, 53 ± 13 years) age-, sex- and CV risk factors- matched controls. In order to compare cIMT measurements between patients and controls we analyzed common carotid artery far wall data as well as a combined measurement result, which consisted of the mean value of the six different measurements, three at each side. RESULTS: mean ± SD serum GH and IGF-1 levels were 2.76 ± 4.65 ng/mL and 1.7 ± 1.25 x ULN, respectively, in all acromegaly patients. Age, body mass index, blood pressure, lipid levels, fasting glucose and Framingham's global cardiovascular risk score classification were similar comparing patients and controls. Combined median [IQR] cIMT measurements were similar in acromegaly patients and matched controls (0.59 [0.52-0.66] mm vs. 0.59 [0.52-0.69] mm; P = 0.872) as well as in acromegaly patients with active and controlled disease (0.59 [0.51-0.68] mm vs. 0.60 [0.54-0.68] mm; P = 0.385). No significant correlations were observed between cIMT measurements and GH (Spearman r = 0.1, P = 0.49) or IGF-1 (Spearman r = 0.13, P = 0.37) levels in patients with acromegaly. Carotid atherosclerotic plaques prevalence was similar in patients and controls (26% vs. 32%; P = 0.54) as well as in patients with active and controlled acromegaly (22% vs. 30%; P = 0.537). CONCLUSIONS: Our data suggest that GH/IGF-1 excess itself is not one of the main drivers of subclinical morphological atherosclerosis changes in patients with acromegaly and that optimal control of acromegaly-associated CV risk factors may preserve vasculature structure even when strict biochemical control is not achieved.

T-Cell Subpopulations Exhibit Distinct Recruitment Potential, Immunoregulatory Profile and Functional Characteristics in Chagas versus Idiopathic Dilated Cardiomyopathies.

Chronic Chagas cardiomyopathy (CCC) is one of the deadliest cardiomyopathies known and the most severe manifestation of Chagas disease, which is caused by infection with the parasite Trypanosoma cruzi. Idiopathic dilated cardiomyopathies (IDC) are a diverse group of inflammatory heart diseases that affect the myocardium and are clinically similar to CCC, often causing heart failure and death. While T-cells are critical for mediating cardiac pathology in CCC and IDC, the mechanisms underlying T-cell function in these cardiomyopathies are not well-defined. In this study, we sought to investigate the phenotypic and functional characteristics of T-cell subpopulations in CCC and IDC, aiming to clarify whether the inflammatory response is similar or distinct in these cardiomyopathies. We evaluated the expression of systemic cytokines, determined the sources of the different cytokines, the expression of their receptors, of cytotoxic molecules, and of molecules associated with recruitment to the heart by circulating CD4+, CD8+, and CD4-CD8- T-cells from CCC and IDC patients, using multiparameter flow cytometry combined with conventional and unsupervised machine-learning strategies. We also used an in silico approach to identify the expression of genes that code for key molecules related to T-cell function in hearts of patient with CCC and IDC. Our data demonstrated that CCC patients displayed a more robust systemic inflammatory cytokine production as compared to IDC. While CD8+ T-cells were highly activated in CCC as compared to IDC, CD4+ T-cells were more activated in IDC. In addition to differential expression of functional molecules, these cells also displayed distinct expression of molecules associated with recruitment to the heart. In silico analysis of gene transcripts in the cardiac tissue demonstrated a significant correlation between CD8 and inflammatory, cytotoxic and cardiotropic molecules in CCC transcripts, while no correlation with CD4 was observed. A positive correlation was observed between CD4 and perforin transcripts in hearts from IDC but not CCC, as compared to normal tissue. These data show a clearly distinct systemic and local cellular response in CCC and IDC, despite their similar cardiac impairment, which may contribute to identifying specific immunotherapeutic targets in these diseases.

The duration of the use of imatinib mesylate is only weakly related to elevated BNP levels in chronic myeloid leukaemia patients.

Cardiotoxicity has been feared as a potential side effect of imatinib therapy. Studies with short-term follow-up failed to identify an excess of cardiac events, but longer-term observations are needed to more definitely exclude this adverse effect. This study was designed to assess the cardiac effects of imatinib in patients under long-term treatment. We included 90 chronic myeloid leukaemia (CML) patients under imatinib therapy for a median treatment time of 3.3 years (mean age 48.9 ± 15.1 years). Patients underwent clinical evaluation, electrocardiography, echocardiography (two-dimensional, colour flow, tissue Doppler and strain imaging), brain natiuretic peptide (BNP) and troponin I measurements. Twenty healthy volunteers were included as a control group for strain measurements. The mean ejection fraction was 68 ± 7% and the median BNP level was 9.6 pg/ml (interquartile range [IQR] 5.7-17.0 pg/ml). Two patients had either an elevated BNP or a depressed ejection fraction (2.2%; 90%CI 0.9-6.8%). Most of troponin I measurements were lower than the detection limit, except for two patients. Longitudinal strain was similar to measurements in healthy controls. A weak relation was observed between log BNP and imatinib treatment duration and dose. There was no relation between these variables and left ventricle ejection fraction. In conclusion, matinib-related cardiotoxicity is an uncommon event in CML patients, even during long-term treatment. Therefore, its use should not be cause of great concern, and the usefulness of regular cardiac monitoring all patients while on imatinib therapy is questionable.

Assessment of functional performance in Chagas heart disease by Human Activity Profile questionnaire.

PURPOSE: To verify the association between Human Activity Profile and functional capacity, functional class and systolic function of the patients with Chagas heart disease (CHD). METHODS: Sixty-two patients with CHD were evaluated by echocardiography, maximal exercise testing and Human Activity Profile questionnaire. The sample was stratified, according to the values of peak oxygen uptake (low or normal), functional class (symptomatic or asymptomatic), and left ventricular ejection fraction (preserved or systolic dysfunction). Linear regression and two-group comparisons analyses were used. Receiver-operating characteristic analysis was used to determine different cutoff values of the Human Activity Profile for low peak oxygen uptake prediction. RESULTS: Peak oxygen uptake was an independent predictor of Human Activity Profile (R2-adjusted = 0.27). Patients with low peak oxygen uptake had lower scores in Human Activity Profile [difference of 6.9 (95%CI 2.5-11.4)] than those with normal peak oxygen uptake. Symptomatic patients also showed lower scores when compared to the asymptomatic [difference of 6.2 (95%CI 1.7-10.8)]. There was no difference between left ventricular ejection fraction classes. The Human Activity Profile score of 76.5 was the optimal cut point value in predicting low peak oxygen uptake (sensitivity = 66.0% and specificity = 71.8%). CONCLUSION: The Human Activity Profile questionnaire is associated with functional capacity of patients with CHD and is able to identify individuals with low peak oxygen uptake.Implications for rehabilitationFunctional impairment is one of the most common clinical findings in all stages and is an important predictor of poor prognosis of the Chagas heart disease;A patient-derived measure of functional capacity is potentially useful in the setting of the Chagas heart disease;The Human Activity Profile questionnaire is effective in the identification of patients with Chagas heart disease with functional impairment and may be a valid method for functional evaluation.

Correlation between BNP levels and Doppler echocardiographic parameters of left ventricle filling pressure in patients with Chagasic cardiomyopathy.

UNLABELLED: Cardiomyopathy is the most important manifestation of Chagas' disease. Brain natriuretic peptide (BNP) level and Doppler echocardiographic parameters for diastolic dysfunction have shown correlation with left ventricle (LV) filling pressures. OBJECTIVES: The purpose of this study is to compare BNP levels with Doppler echocardiographic parameters in patients with chagasic cardiomyopathy. METHODS: Forty-three patients (69.8% men; mean age 41.0 +/- 10.4 years) were submitted to an echocardiographic study and 39 had their BNP levels measured. RESULTS: BNP levels increased with the deterioration of the diastolic function (P=0.025). Pulmonary venous flow parameters were correlated with BNP levels, but E/E'ratio (E'measured at the inferior mitral annulus) was the only diastolic parameter that remained an independent predictor of elevated BNP levels in the multivariate analysis. The area under the receiver-operating curve for BNP to detect E/E' >15 was 0.875. A BNP value of 280.4 pg/ml had a sensitivity of 96% and a specificity of 75% for predicting E/E' >15. CONCLUSIONS: In a group of patients with chagasic cardiomyopathy, BNP levels correlated with diastolic function patterns regardless of systolic function. The E/E'ratio (inferior wall) was the only isolated parameter of diastolic function that was independently associated with BNP levels.

An evaluation of the cardiotoxicity of imatinib mesylate.

We studied 103 consecutive patients with chronic myeloid leukaemia on treatment with imatinib (IM) and 57 patients with chronic myeloproliferative disorders not treated with IM in order to evaluate its cardiotoxicity. There was no statistical difference regarding cardiac symptoms and signs, BNP levels and echocardiographic measurements for IM and control groups, except for peripheral oedema, more frequent in the IM group. Four patients in the IM group presented a BNP level >100pg/ml, one of them with depressed LVEF. IM was not related to systematic deterioration of cardiac function, but there is still a possibility of isolated cases of cardiotoxicity.

Telehealth solutions to enable global collaboration in rheumatic heart disease screening.

Background The global burden of rheumatic heart disease is nearly 33 million people. Telemedicine, using cloud-server technology, provides an ideal solution for sharing images performed by non-physicians with cardiologists who are experts in rheumatic heart disease. Objective We describe our experience in using telemedicine to support a large rheumatic heart disease outreach screening programme in the Brazilian state of Minas Gerais. Methods The Programa de Rastreamento da Valvopatia Reumática (PROVAR) is a prospective cross-sectional study aimed at gathering epidemiological data on the burden of rheumatic heart disease in Minas Gerais and testing of a non-expert, telemedicine-supported model of outreach rheumatic heart disease screening. The primary goal is to enable expert support of remote rheumatic heart disease outreach through cloud-based sharing of echocardiographic images between Minas Gerais and Washington. Secondary goals include (a) developing and sharing online training modules for non-physicians in echocardiography performance and interpretation and (b) utilising a secure web-based system to share clinical and research data. Results PROVAR included 4615 studies that were performed by non-experts at 21 schools and shared via cloud-telemedicine technology. Latent rheumatic heart disease was found in 251 subjects (4.2% of subjects: 3.7% borderline and 0.5% definite disease). Of the studies, 50% were preformed on full functional echocardiography machines and transmitted via Digital Imaging and Communications in Medicine (DICOM) and 50% were performed on handheld echocardiography machines and transferred via a secure Dropbox connection. The average time between study performance date and interpretation was 10 days. There was 100% success in initial image transfer. Less than 1% of studies performed by non-experts could not be interpreted. Discussion A sustainable, low-cost telehealth model, using task-shifting with non-medical personal in low and middle income countries can improve access to echocardiography for rheumatic heart disease.

Differential Expression of Matrix Metalloproteinases 2, 9 and Cytokines by Neutrophils and Monocytes in the Clinical Forms of Chagas Disease.

Dilated cardiomyopathy, the most severe manifestation in chronic phase of Chagas disease, affects about 30% of patients and is characterized by myocardial dysfunction and interstitial fibrosis due to extracellular matrix (ECM) remodeling. ECM remodeling is regulated by proteolytic enzymes such as matrix metalloproteinases (MMPs) and cytokines produced by immune cells, including phagocytes. We evaluated by flow cytometry the expression of MMP-2, MMP-9, IL-1ß, TNF-α, TGF-ß and IL-10 by neutrophils and monocytes from patients with indeterminate (IND) and cardiac (CARD) clinical forms of Chagas disease and non-infected individuals (NI), before and after in vitro stimulation with Trypanosoma cruzi antigens. Our results showed an important contribution of neutrophils for MMPs production, while monocytes seemed to be involved in cytokine production. The results showed that neutrophils and monocytes from IND and CARD patients had higher intracellular levels of MMP-2 and MMP-9 than NI individuals. On the other hand, T. cruzi derived-antigens promote a differential expression of MMP-2 and MMP-9 in patients with Chagas disease and may regulate MMPs expression in neutrophils and monocytes, mainly when a cardiac alteration is not present. Our data also showed that in the presence of T. cruzi derived-antigens the production of cytokines by neutrophils and monocytes, but mainly by monocytes, may be intensified. Correlation analysis demonstrated that MMP-2 had a positive correlation with IL-10 and a negative correlation with IL-1ß, whereas MMP-9 showed a negative correlation with IL-10. We also observed that IND patients presented a greater percentage of high producer cells of regulatory molecules when compared to CARD patients, indicating a different pattern in the immune response. Our data suggest that MMPs and cytokines produced by neutrophils and monocytes are important contributors for cardiac remodeling and may be an interesting target for new biomarker research.

Efficacy of a Standardized Computer-Based Training Curriculum to Teach Echocardiographic Identification of Rheumatic Heart Disease to Nonexpert Users.

The ability to integrate echocardiographic for rheumatic heart disease (RHD) into RHD prevention programs is limited because of lack of financial and expert human resources in endemic areas. Task shifting to nonexperts is promising; but investigations into workforce composition and training schemes are needed. The objective of this study was to test nonexperts' ability to interpret RHD screening echocardiograms after a brief, standardized, computer-based training course. Six nonexperts completed a 3-week curriculum on image interpretation. Participant performance was tested in a school-screening environment in comparison to the reference approach (cardiologists, standard portable echocardiography machines, and 2012 World Heart Federation criteria). All participants successfully completed the curriculum, and feedback was universally positive. Screening was performed in 1,381 children (5 to 18 years, 60% female), with 397 (47 borderline RHD, 6 definite RHD, 336 normal, and 8 other) referred for handheld echo. Overall sensitivity of the simplified approach was 83% (95% CI 76% to 89%), with an overall specificity of 85% (95% CI 82% to 87%). The most common reasons for false-negative screens (n = 16) were missed mitral regurgitation (MR; 44%) and MR ≤1.5 cm (29%). The most common reasons for false-positive screens (n = 179) included identification of erroneous color jets (25%), incorrect MR measurement (24%), and appropriate application of simplified guidelines (39.4%). In conclusion, a short, independent computer-based curriculum can be successfully used to train a heterogeneous group of nonexperts to interpret RHD screening echocardiograms. This approach helps address prohibitive financial and workforce barriers to widespread RHD screening.

Assessing pre-clinical ventricular dysfunction in obese children and adolescents: the value of speckle tracking imaging.

AIMS: Obesity has become a major health problem worldwide. Cardiovascular abnormalities have been described not only in obese adults but also in obese children and adolescents. The aim of the present study was to investigate left and right, systolic and diastolic ventricular dysfunction in obese paediatric patients without comorbidities using 2D speckle tracking longitudinal strain. METHODS AND RESULTS: Doppler echocardiogram was performed on 50 obese children and adolescents with body mass index (BMI) above the 95th percentile (OG) and 46 non-obese sex- and age-matched controls (CG). Systolic and diastolic functions of both ventricles were investigated through conventional Doppler echocardiography. Tissue Doppler imaging (TDI), colour Doppler myocardial imaging (CDMI), and two-dimensional (2D) speckle tracking were also used to analyse ventricular performance in both groups. Left-ventricular (LV) ejection fraction was similar between groups (68.2 ± 6.2 vs. 68.3 ± 5.3, P = 0.931). Left-ventricular diastolic parameters did not differ between groups, except for a lower mitral A wave (61.6 ± 13.0 vs. 51.9 ± 10.0 cm/s, P > 0.001) and higher E/A ratio (1.8 ± 0.5 vs. 2.1 ± 0.4, P = 0.007) in the controls. Left-ventricular global strain was lower in the OG by both methods (CDMI: 22.0 ± 2.8 vs. 24.6 ± 2.7%, P = 0.020; 2D speckle tracking: 18.4 ± 1.6 vs. 20.4 ± 1.7%, P < 0.001). In multivariate analysis, 2D longitudinal global strain correlated negatively with BMI, r = -0.433, p = 0.002. CONCLUSION: Although EF was not different between the two groups, LV 2D speckle tracking longitudinal strain was lower in the obese group, even in the absence of other comorbidities, indicating that obesity effects on LV function is an early finding in obesity.

NT-proBNP levels in patients with non-ST-segment elevation acute coronary syndrome.

BACKGROUND: Non-ST-segment elevation acute coronary syndrome is associated with elevation of brain natriuretic peptide and markers of myocardial necrosis, although its relationship with the TIMI score and left ventricular function are largely unknown. OBJECTIVE: To evaluate the correlation between plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) and markers of myocardial necrosis [creatine phosphokinase muscle-brain fraction (CK-MB) and troponin I], TIMI risk score and left ventricular ejection fraction in patients with non-ST-segment elevation acute coronary syndrome. METHODS: Eighty-seven patients with non-ST-segment elevation acute coronary syndrome were divided into two groups: 37 (42.5%) with unstable angina and 50 (57.5%) with non-ST-segment elevation myocardial infarction. RESULTS: Left ventricular ejection fraction more than 40% was found in 86.2% of the total sample. Serum levels of NT-proBNP was higher in patients with non-ST elevation myocardial infarction than in those with unstable angina (p<0.001). Increased levels of NT-proBNP were associated with increases in troponin I (rs=0.425, p<0.001), peak CK-MB (rs=0.458, p<0.001) and low left ventricular ejection fraction (rs=-0.345, p=0.002); no correlation was found with the TIMI risk score (rs=0.082, p=0.44). Multivariate analysis revealed that left ventricular ejection fraction and troponin I levels were independently correlated with NT-proBNP levels (p=0.017 and p=0.002, respectively). CONCLUSION: Increased levels of NT-proBNP in patients with non-ST-segment elevation acute coronary syndrome are not related exclusively to low left ventricular ejection fraction, but can also be caused by the presence of myocardial ischemia and necrosis.

The use of imatinib mesylate has no adverse effects on the heart function. Results of a pilot study in patients with chronic myeloid leukemia.

To investigate cardiac effects of imatinib at an extended follow-up (median 12.4 months), 12 chronic myeloid leukemia patients underwent cardiac screening. No significant changes on the frequency of cardiovascular signs and symptoms, electrocardiographic abnormalities, echocardiographic measurements and BNP levels were observed. Median ejection fraction was 67% at baseline versus 68% at follow-up (median intra-patient change 0.5%). Median BNP levels were 8.3 versus 7.3pg/mL (median intra-patient change 0.2pg/mL). Troponin I measures were below the lower limit of detection, whereas strain measures were similar to healthy control. This pilot study suggests that it is probably safe to perform cardiac monitoring on an annual basis.

Dobutamine-stress echocardiography in asymptomatic patients with aortic regurgitation.

BACKGROUND: Decreased contractile reserve may already be present in asymptomatic patients with aortic regurgitation and normal ejection fraction (EF), thus indicating the need for frequent and accurate assessments of the left ventricular function for the early detection of systolic dysfunction. OBJECTIVE: To analyze if increments in EF with low dose dobutamine could predict surgery and/or death in patients with aortic regurgitation. METHODS: Dobutamine-stress echocardiography was performed in 24 patients with aortic regurgitation in order to analyze whether EF increments at low dobutamine doses could predict the need for surgery and/or death in this group of patients. RESULTS: Mean age was 37.8+/-16.8 years and 16 patients (66%) were male. EF increased from a mean baseline value of 62.3+/-7.9% to 71.5+/-10.5% at a dobutamine dose of 20 microg/kg/min (p<0.001). The patients were followed-up for 36.6+/-20.1 months; two patients died (one of cardiovascular death) and five underwent cardiac surgery. Baseline EF was correlated with surgery and death in the follow-up of patients. CONCLUSION: Baseline EF was correlated with surgery or death in the follow-up of young patients with aortic regurgitation. However, the percentage increase in EF at low dobutamine doses did not allow us to predict events in these patients.