Modulation of Regulatory T Cells Activity by Distinct CD80 and CD86 Interactions With CD28/CTLA-4 in Chagas Cardiomyopathy.

Chagas cardiomyopathy is the symptomatic cardiac clinical form (CARD) of the chronic phase of Chagas disease caused by Trypanosoma cruzi infection. It was described as the most fibrosing cardiomyopathies, affecting approximately 30% of patients during the chronic phase. Other less frequent symptomatic clinical forms have also been described. However, most patients who progress to the chronic form develop the indeterminate clinical form (IND), may remain asymptomatic for life, or develop some cardiac damage. Some mechanisms involved in the etiology of the clinical forms of Chagas disease have been investigated. To characterize the contribution of CD80 and CD86 co-stimulatory molecules in the activation of different CD4+ (Th1, Th2, Th17, and Treg) and CD8+ T lymphocyte subsets, we used blocking antibodies for CD80 and CD86 receptors of peripheral blood mononuclear cells (PBMC) in cultures with T. cruzi antigens from non-infected (NI), IND, and CARD individuals. We demonstrated a higher frequency of CD8+ CD25+ T lymphocytes and CD8+ Treg cells after anti-CD80 antibody blockade only in the CARD group. In contrast, a lower frequency of CD4+ Treg lymphocytes after anti-CD86 antibody blockade was found only in IND patients. A higher frequency of CD4+ Treg CD28+ lymphocytes, as well as an association between CD4+ Treg lymphocytes and CD28+ expression on CD4+ Treg cells in the CARD group, but not in IND patients, and once again only after anti-CD80 antibody blockade, was observed. We proposed that Treg cells from IND patients could be activated via CD86-CTLA-4 interaction, leading to modulation of the immune response only in asymptomatic patients with Chagas disease, while CD80 may be involved in the proliferation control of T CD8+ lymphocytes, as also in the modulation of regulatory cell activation via CD28 receptor. For the first time, our data highlight the role of CD80 in modulation of Treg lymphocytes activation in patients with CARD, highlighting a key molecule in the development of Chagas cardiomyopathy.

Increased frequencies of circulating CCR5+ memory T cells are correlated to chronic chagasic cardiomyopathy progression.

The infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease, a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, culminating in heart failure and high rates of sudden death. CCC pathogenesis is influenced by both host and parasite factors and is proposed to be mostly immune-driven. Chemokines are crucial players in orchestrating immune cell recruitment to infected tissues and inflammation. Herein, we investigated inflammatory chemokine receptor expression on circulating T cells in patients stratified by CCC severity. Compared to asymptomatic individuals, we found increased percentages of effector CD4+ T cells and central memory CD4+ and CD8+ T cells expressing CCR5 in patients with structural cardiopathy, but normal global ventricular function and no symptoms of chronic heart failure. Even naïve T cells expressed CCR5 in these patients. In contrast, reduced frequencies of CD4+ and CD8+ effector T cells expressing CXCR3 were observed in patients presenting with severe heart disease. Patients with increased left ventricular diameter, heart enlargement, and insufficiency had higher frequencies of CCR5+ effector and effector memory CD8+ T cells. Moreover, the percentage of effector CCR5+ CD8+ T cells was increased in patients with a reduced ejection fraction. Our results show that high expression CCR5 and low expression of CXCR3 on circulating T cells are associated with worse prognosis, possibly reflecting immune-mediated cardiac remodeling of CCC.

Evidence of Different IL-1ß Activation Pathways in Innate Immune Cells From Indeterminate and Cardiac Patients With Chronic Chagas Disease.

Background: Chagas cardiomyopathy is the main fibrosing myocarditis among known heart diseases. Development of cardiomyopathy has been related to extracellular matrix (ECM) remodeling, which are controlled by matrix metalloproteinases (MMPs) and cytokines, especially interleukin (IL)-1ß. The convertion of 31KDa inactive precursor, the proIL-1ß in 17KDa active IL-1ß peptide, is controlled by caspase-1-dependent pathway, associated with inflammasomes. Other caspase-1 independent mechanisms mediated by proteases, especially as MMPs, have already been described. Methods: We evaluated IL-1ß activation pathways in neutrophils and monocyte subsets from patients with different clinical forms of Chagas disease after T. cruzi antigen stimulation by multiparameter flow cytometry. Results: Our data demonstrated that Chagas patients with the indeterminate clinical form (IND) showed increased levels of IL-1ß post-stimulation as well as increased expression of MMP-2, NLRP3, and CASP1, which are associated with the classical caspase-1-dependent pathway. Conversely, patients with the cardiac clinical form (CARD) showed increased IL-1ß after stimulation associated with MMP-9 and alternative caspase-1-independent pathway. Conclusions: We suggest some distinct molecular mechanisms for production of IL-1ß in innate immune cells from patients with different clinical forms of Chagas disease. MMP-2 and MMP-9 gelatinases are associated with distinct disease outcomes and IL-1ß production.

Fostering novice students' diagnostic ability: the value of guiding deliberate reflection.

BACKGROUND: Deliberate reflection when practising the diagnosis of clinical cases has been shown to develop medical students' diagnostic competence. Adding guidance by cueing reflection or providing modelling of reflection increased the benefits of reflection for advanced (Years 5-6) students. The present study investigated whether we could replicate and extend these findings by comparing the effects of free, cued and modelled reflection on novice students' diagnostic competence. METHODS: A total of 80 third-year medical students participated in a two-phase experiment. In the learning phase, students diagnosed nine clinical cases under one of three conditions: free reflection; cued reflection, and modelled reflection. Two weeks later, all students diagnosed four new examples of the diseases studied in the learning phase and four cases of non-studied related diseases ('adjacent diseases'). The main outcome measurements were diagnostic accuracy scores (range 0-1) on studied and adjacent diseases. RESULTS: For studied diseases, there was a significant effect of experimental condition on diagnostic accuracy (p < 0.02), with the cued-reflection group (mean = 0.58, standard deviation [SD] = 0.23) performing significantly better than the free-reflection group (mean = 0.41, SD = 0.20; p < 0.02). The cued-reflection and modelled-reflection groups (mean = 0.54, SD = 0.22) did not differ in diagnostic accuracy (p > 0.05), nor did the modelled-reflection group perform better than the free-reflection group (p > 0.05). For adjacent diseases, the three groups scored extremely low, without significant differences in performance (p > 0.05). Cued reflection and free reflection were rated as requiring similar effort (p > 0.05) and both were more demanding than studying examples of reflection (both p < 0.001) in the learning phase. CONCLUSIONS: Simply cueing novice students' reflection to focus it on relevant diseases was sufficient to increase diagnostic performance relative to reflection without any guidance. Cued reflection and studying examples of reflection appear to be equally useful approaches for teaching clinical diagnosis to novice students. Students found studying examples of reflection required less effort but cued reflection will certainly demand much less investment from teachers.

Systems Biology Reveals Relevant Gaps in Fc-γR Expression, Impaired Regulatory Cytokine Microenvironment Interfaced With Anti-Trypanosoma cruzi IgG Reactivity in Cardiac Chagas Disease Patients.

The systems biology approach has become an innovative tool when it comes to shedding light on the complex immune response underlying the development/maintenance of distinct clinical forms of Chagas disease. The goal of this study was to describe an integrative overview of Fc-γR expression, cytokine microenvironment and anti-Trypanosoma cruzi IgG interface in indeterminate-(IND) and cardiac-(CARD) patients. Data demonstrated that IND displayed an overall higher Fcγ-R expression (CD16; CD32; CD64) on neutrophils-(NEU), along with (CD16; CD64) on monocytes-(MON) as compared to CARD. Additionally, CARD presented an increased expression of CD32 in B-cells. While preserved frequency of IL-10-producing cells was observed in IND, decreased levels of IL-10+ phagocytes and enhanced TNF+ MON and NK-cells were observed in CARD. T. cruzi-antigen recall in vitro induces a general decrease of Fc-γR expression in Chagas disease patients, especially in CARD. Moreover, T. cruzi-antigen stimuli triggered a concomitant increase of IFN-γ+NEU/TNF+NK-cells and IL-10+MON/IL-10+B-cells in IND. Biomarker signatures further emphasized the contrasting Fc-γR expression and cytokine microenvironment observed in Chagas disease patients with distinct clinical forms. Up-regulation of Fc-γR expression (CD16 on NEU;MON;NK) was observed in IND, whereas a general decrease was reported for CARD. Moreover, while a mixed cytokine microenvironment (TNF; IL-10) was observed in IND, CARD presented a contrasting profile with up-regulation of TNF+NEU and IL-12+NEU. Integrative network analysis revealed a distinct assemblage of biomarkers, with CARD presenting a large number of negative internode connectivity in comparison with IND. The relevant gaps in Fc-γR expression and impaired regulatory cytokine microenvironment interfaced with the anti-T. cruzi IgG reactivity throughout an exacerbated negative connectivity may account for the development/maintenance of the clinical status of cardiac Chagas disease.

CD86 Expression by Monocytes Influences an Immunomodulatory Profile in Asymptomatic Patients with Chronic Chagas Disease.

In the chronic phase of Chagas disease, 60% of the patients develop the asymptomatic form known as indeterminate (IND). The remaining 30% of the patients develop a life-threatening form in which digestive and/or cardiac (CARD) alterations take place. The mechanisms underlying the development of severe forms of Chagas disease remain poorly understood. It is well known that interactions between immune cells such as monocytes and lymphocytes drive immune responses. Further, the co-stimulatory molecules CD80 and CD86 expressed by monocytes and subsets induce lymphocyte activation, thereby triggering cellular immune response. Here, we revealed, for the first time, the functional-phenotypic profile of monocytes subsets in Chagas disease. Using flow cytometry, we evaluated the effect of in vitro stimulation with Trypanosoma cruzi antigens on the expression of the co-stimulatory molecules CD80 and CD86 in different monocyte subsets of patients with IND and CARD clinical forms of Chagas disease. We also assessed the expression of toll-like receptor (TLR)-2, TLR-4, TLR-9, HLA-DR, IL-10, and IL-12 in the monocyte subsets and of CTLA-4 and CD28, ligands of CD80 and CD86, in T lymphocytes. CD86 expression in all monocyte subsets was higher in IND patients when compared with non-infected (NI) individuals. After stimulation with T. cruzi, these patients also showed a higher frequency of CD4+CTLA-4+ T lymphocytes than NI individuals. We found an association between CD80 and CD28, and between CD86 and CTLA-4 expression, with a high frequency of regulatory T (Treg) cells in IND patients. We proposed that CD86 may be involved in immunoregulation by its association with CTLA-4 in asymptomatic patients. CD86 and CTLA-4 interaction may influence Treg activation, and this could represent a new strategy to control inflammation and tissue damage.

The development of self-regulated learning during the pre-clinical stage of medical school: a comparison between a lecture-based and a problem-based curriculum.

Society expects physicians to always improve their competencies and to be up to date with developments in their field. Therefore, an important aim of medical schools is to educate future medical doctors to become self-regulated, lifelong learners. However, it is unclear if medical students become better self-regulated learners during the pre-clinical stage of medical school, and whether students develop self-regulated learning skills differently, dependent on the educational approach of their medical school. In a cross-sectional design, we investigated the development of 384 medical students' self-regulated learning skills with the use of the Self-Regulation of Learning Self-Report Scale. Next, we compared this development in students who enrolled in two distinct medical curricula: a problem-based curriculum and a lectured-based curriculum. Analysis showed that more skills decreased than increased during the pre-clinical stage of medical school, and that the difference between the curricula was mainly caused by a decrease in the skill evaluation in the lecture-based curriculum. These findings seem to suggest that, irrespective of the curriculum, self-regulated learning skills do not develop during medical school.