Exploring T Cell Receptor Repertoires in Myocardial Diseases.

Mounting experimental and clinical evidence has revealed that adaptive immune mechanisms targeting myocardial antigens are triggered by different forms of cardiac injury and impact disease progression. B and T lymphocytes recognize specific antigens via unique adaptive immune receptors generated through a somatic rearrangement process that generates a potential repertoire of 1019 unique receptors. While the adaptive immune receptor repertoire diversity provides the basis for immunologic specificity, making sense of it can be a challenging task. In the present review, we discuss key aspects underlying the generation of TCRs (T cell receptors) and emerging tools for their study in the context of myocardial diseases. Moreover, we outline how exploring TCR repertoires could lead to a deeper understanding of myocardial pathophysiological principles and potentially serve as diagnostic tools.

Myocardial Milieu Favors Local Differentiation of Regulatory T Cells.

BACKGROUND: In the past years, several studies investigated how distinct immune cell subsets affects post-myocardial infarction repair. However, whether and how the tissue environment controls these local immune responses has remained poorly understood. We sought to investigate how antigen-specific T-helper cells differentiate under myocardial milieu's influence. METHODS: We used a transgenic T cell receptor (TCR-M) model and major histocompatibility complex-II tetramers, both myosin-specific, combined with single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and functional phenotyping to elucidate how the antigen-specific CD4+ T cells differentiate in the murine infarcted myocardium and influence tissue repair. Additionally, we transferred proinflammatory versus regulatory predifferentiated TCR-M-cells to dissect how they specially contribute to post-myocardial infarction inflammation. RESULTS: Flow cytometry and scRNA-/TCR-seq analyses revealed that transferred TCR-M cells acquired an induced regulatory phenotype (induced regulatory T cell) in the infarcted myocardium and blunted local inflammation. Myocardial TCR-M cells differentiated into 2 main lineages enriched with either cell activation and profibrotic transcripts (eg, Tgfb1) or suppressor immune checkpoints (eg, Pdcd1), which we also found in human myocardial tissue. These cells produced high levels of LAP (latency-associated peptide) and inhibited IL-17 (interleukin-17) responses. Endogenous myosin-specific T-helper cells, identified using genetically barcoded tetramers, also accumulated in infarcted hearts and exhibited a regulatory phenotype. Notably, TCR-M cells that were predifferentiated toward a regulatory phenotype in vitro maintained stable in vivo FOXP3 (Forkhead box P3) expression and anti-inflammatory activity whereas TH17 partially converted toward a regulatory phenotype in the injured myocardium. Overall, the myosin-specific Tregs dampened post-myocardial infarction inflammation, suppressed neighboring T cells, and were associated with improved cardiac function. CONCLUSIONS: These findings provide novel evidence that the heart and its draining lymph nodes actively shape local immune responses by promoting the differentiation of antigen-specific Tregs poised with suppressive function.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Role of CD4+ T-cells for regulating splenic myelopoiesis and monocyte differentiation after experimental myocardial infarction.

Myocardial infarction (MI) induces the generation of proinflammatory Ly6Chigh monocytes in the spleen and the recruitment of these cells to the myocardium. CD4+ Foxp3+ CD25+ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4+ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4+ T-cell deficient animals. Conventional CD4+ T-cells promoted myelopoiesis in vitro by cell-cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4+ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6Chigh monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4+ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.

Characterizing the immune response to myocardial infarction in pigs.

Though myocardial infarction (MI) in pigs is a well-established translational large animal model, it has not yet been widely used for immunotherapy studies, and a comprehensive description of the immune response to MI in this species is lacking. We induced MI in Landrace pigs by balloon occlusion of the left anterior descending artery over 90 min. Within 14 days, the necrotic myocardium was progressively replaced by scar tissue with involvement of myofibroblasts. We characterized the immune response in the heart ex vivo by (immuno)histology, flow cytometry, and RNA sequencing of myocardial tissue on days 3, 7, and 14 after MI. Besides a clear predominance of myeloid cells among heart-infiltrating leukocytes, we detected activated T cells and an increasing proportion of CD4+ Foxp3+ regulatory T cells (Treg), especially in the infarct core-findings that closely mirror what has been observed in mice and humans after MI. Transcriptome data indicated inflammatory activity that was persistent but markedly changing in character over time and linked to extracellular matrix biology. Analysis of lymphocytes in heart-draining lymph nodes revealed significantly higher proliferation rates of T helper cell subsets, including Treg on day 7 after MI, compared to sham controls. Elevated frequencies of myeloid progenitors in the spleen suggest that it might be a site of emergency myelopoiesis after MI in pigs, as previously shown in mice. We thus provide a first description of the immune response to MI in pigs, and our results can aid future research using the species for preclinical immunotherapy studies.

Quantitative risk assessment of the presence of fumonisin in corn produced in different regions of Brazil: Influence of climatic conditions.

In this study, the probability of occurrence of fumonisins in corn in the states of greatest production in Brazil was determined. The data were analyzed through quantitative risk analysis using the Monte Carlo simulation. The results indicated that there is a strong correlation between fumonisins contamination levels and the geographical region due to the influence of climatic characteristics, with temperature having the main influence. The Southern states presented higher risks of occurrence and concentration levels of fumonisins in corn due to the temperate climate with lower average temperature and higher relative humidity and precipitation indices. Cultivation in the best season indicates a significant reduction in the production of fumonisins when this period was evaluated, with average concentration levels up to 42% lower. The generated data are important for regulatory agencies and the agricultural sector, which needs to be aware that the chance of success in grain production depends on efficient planning of the growing season, mainly concerning the climatic conditions to which it is subject to minimize the risks.

Adult T-cells impair neonatal cardiac regeneration.

AIMS: Newborn mice and humans display transient cardiac regenerative potential that rapidly declines postnatally. Patients who survive a myocardial infarction (MI) often develop chronic heart failure due to the heart's poor regeneration capacity. We hypothesized that the cardiac 'regenerative-to-scarring' transition might be driven by the perinatal shifts observed in the circulating T-cell compartment. METHODS AND RESULTS: Post-MI immune responses were characterized in 1- (P1) vs. 7-day-old (P7) mice subjected to left anterior descending artery ligation. Myocardial infarction induced robust early inflammatory responses (36 h post-MI) in both age groups, but neonatal hearts exhibited rapid resolution of inflammation and full functional recovery. The perinatal loss of myocardial regenerative capacity was paralleled by a baseline increase in αß-T cell (CD4+ and CD8+) numbers. Strikingly, P1-infarcted mice reconstituted with adult T-cells shifted to an adult-like healing phenotype, marked by irreversible cardiac functional impairment and increased fibrosis. Infarcted neonatal mice harbouring adult T-cells also had more monocyte-derived macrophage recruitment, as typically seen in adults. At the transcriptome level, infarcted P1 hearts that received isolated adult T-cells showed enriched gene sets linked to fibrosis, inflammation, and interferon-gamma (IFN-γ) signalling. In contrast, newborn mice that received isolated Ifng-/- adult T-cells prior to MI displayed a regenerative phenotype that resembled that of its age-matched untreated controls. CONCLUSION: Physiological T-cell development or adoptive transfer of adult IFN-γ-producing T-cells into neonates contributed to impaired cardiac regeneration and promoted irreversible structural and functional cardiac damage. These findings reveal a trade-off between myocardial regenerative potential and the development of T-cell competence.

Identification of a novel cardiac epitope triggering T-cell responses in patients with myocardial infarction.

T-cells contribute to pathophysiological processes in myocardial diseases, including myocardial infarction (MI) and heart failure (HF). Antigen-specificity is a hallmark of T-cell responses but the cardiac antigens that trigger heart-directed T-cell responses in patients have not yet been uncovered, thus posing a roadblock to translation. In the present exploratory study, we identified a peptide fragment of the beta-1 adrenergic receptor (ADRB1) that elicits CD4+ T-cell responses after myocardial infarction in patients with a defined HLA haplotype. Our observations may advance the development of tools to monitor other antigen-specific immune responses in patients.

Effects of acute ischemia and hypoxia in young and adult calsequestrin (CSQ2) knock-out and wild-type mice.

Calsequestrin (CSQ2) is the main Ca2+-binding protein in the sarcoplasmic reticulum of the mammalian heart. In order to understand the function of calsequestrin better, we compared two age groups (young: 4-5 months of age versus adult: 18 months of age) of CSQ2 knock-out mice (CSQ2(-/-)) and littermate wild-type mice (CSQ2(+/+)). Using echocardiography, in adult mice, the basal left ventricular ejection fraction and the spontaneous beating rate were lower in CSQ2(-/-) compared to CSQ2(+/+). The increase in ejection fraction by ß-adrenergic stimulation (intraperitoneal injection of isoproterenol) was lower in adult CSQ2(-/-) versus adult CSQ2(+/+). After hypoxia in vitro (isolated atrial preparations) by gassing the organ bath buffer with 95% N2, force of contraction in electrically driven left atria increased to lower values in young CSQ2(-/-) than in young CSQ2(+/+). In addition, after global ischemia and reperfusion (buffer-perfused hearts according to Langendorff; 20-min ischemia and 15-min reperfusion), the rate of tension development was higher in young CSQ2(-/-) compared to young CSQ2(+/+). Finally, we evaluated signs of inflammation (immune cells, autoantibodies, and fibrosis). However, whereas no immunological alterations were found between all investigated groups, pronounced fibrosis was found in the ventricles of adult CSQ2(-/-) compared to all other groups. We suggest that in young mice, CSQ2 is important for cardiac performance especially in isolated cardiac preparations under conditions of impaired oxygen supply, but with differences between atrium and ventricle. Lack of CSQ2 leads age dependently to fibrosis and depressed cardiac performance in echocardiographic studies.

Effect of the COVID-19 pandemic on food habits and perceptions: A study with Brazilians.

BACKGROUND: Following the COVID-19 pandemic (caused by the SARS-CoV-2 coronavirus) at the beginning of 2020, containment measures have been taken by different countries around the globe. Citizens were forced to stay in quarantine, affecting their food consumption habits and food sector. These impacts have not yet been properly understood. Thus, it is important to describe the consequences of COVID-19 on food consumption habits globally, especially in the context of developing countries, such as Brazil. SCOPE AND APPROACH: In this study, the Brazilian's food consumption habits and perceptions during the COVID-19 pandemic were assessed, highlighting the food consumption changes and selection of food products. Consumer perceptions about issues related to food safety and food marketing were also assessed. An online survey was performed and data were analyzed by descriptive analysis; independence and per cell chi-square test; and factor analysis. KEY FINDINGS AND CONCLUSIONS: Brazilians perceptions indicated that the COVID-19 pandemic context (assessed in May 2020) changed their food consumption and purchase. Respondents stated that they are eating and buying a greater amount of food, indicating a perception of a less healthy diet, mainly by women. On other hand, they are prioritizing homemade preparations and fresh food. Moreover, they reduced their shopping trips to markets and are starting to use delivery services and shopping platforms. Basic products of animal, vegetable, and bakery origin are being preferred during this period, in addition to economical packaging and products. Brazilians also indicated that they are more concerned with food safety and hygienic practices. However, at a time when global health is threatened, government agencies must create measures that ensure the food supply and consumer's awareness, in order to guarantee the country's food security during the current crisis.

Pleiotropic cardiac functions controlled by ischemia-induced lncRNA H19.

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) nanodispersions as potential anti-inflammatory therapeutics: Comparison of in vitro activity and impact of pegylation.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with putative anti-inflammatory potential. However, studies comparing PS and PG are rare and were mainly conducted with phospholipid-dispersions of large size and broad distributions. Thus, we prepared small-sized PS- and PG-loaded liposomes exhibiting narrow distribution, and additionally studied the impact of liposome-pegylation on the reduction of the TNFα-production caused by the PS- and PG-liposomes. These PS- and PG-containing nanodispersions had a small size around 100nm and a narrow distribution (PDI<0.1). The liposome-dispersions showed no toxicity in NHDF- and 3T3-cells and virtually no hemolytic activity. They decreased the TNFα-production of LPS-(lipopolysaccharide)-stimulated mouse peritoneal macrophages in vitro. PG-liposomes always decreased the TNFα-levels more potently than PS-liposomes. Pegylation of PS- and PG-liposomes caused different Zeta potentials, but did not change biological activity. The results of the current study indicate a high potential of the tested formulations for phospholipid-based anti-inflammatory therapies.

Effect of Lactobacillus rhamnosus on growth of Listeria monocytogenes and Staphylococcus aureus in a probiotic Minas Frescal cheese.

This study aimed to evaluate the effect of Lactobacillus rhamnosus GG on growth of Staphylococcus aureus and Listeria monocytogenes, inoculated alone or in combination on surface of Minas Frescal cheeses, during storage for 21 days at 7 °C. Survival percentages of each individual bacterial species after exposure to in vitro simulated gastrointestinal conditions (SGC) were also determined. The addition of L. rhamnosus did not affect (P > 0.05) pH, moisture, fat, protein and texture profile of Minas Frescal cheeses. L. rhamnosus was able to survive in suitable counts (>6 Log CFU/g) in cheeses from the 7th day of storage, with high survival (>74.6-86.4%) after SGC. An inhibitory effect of L. rhamnosus on L. monocytogenes was observed in cheeses (decrease of 1.1-1.6 Log CFU/g) and after SGC (20% reduction in the survival). No inhibitory effect of L. rhamnosus was observed on S. aureus counts (P > 0.05), and this microorganism did not survive the exposure to SGC. In conclusion, the addition of L. rhamnosus in Minas Frescal cheese has a potential for L. monocytogenes inhibition. Further studies are necessary to elucidate the mechanisms involved in the inhibition process and determine the survival ability of the bacterial species evaluated in in vivo experiments.

Myocardial aging as a T-cell-mediated phenomenon.

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3- (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.

Antibodies aggravate the development of ischemic heart failure.

Heart-specific antibodies have been widely associated with myocardial infarction (MI). However, it remains unclear whether autoantibodies mediate disease progression or are a byproduct of cardiac injury. To disambiguate the role of immunoglobulins in MI, we characterized the development of ischemic heart failure in agammaglobulinemic mice (AID-/-µS-/-). Although these animals can produce functional B cells, they cannot synthesize secretory IgM (µS-/-) or perform Ig class switching (AID-/-), leading to complete antibody deficiency. Agammaglobulinemia did not affect overall post-MI survival but resulted in a significant reduction in infarct size. Echocardiographic analyses showed that, compared with wild-type infarcted control mice, AID-/-µS-/- mice exhibited improved cardiac function and reduced remodeling on day 56 post-MI. These differences remained significant even after animals with matched infarct sizes were compared. Infarcted AID-/-µS-/- mice also showed reduced myocardial expression levels of transcripts known to promote adverse remodeling, such as matrix metalloproteinase-9, collagen type I a1, collagen type III a1, and IL-6. An unbiased screening of the heart reactivity potential in the plasma of wild-type MI animals revealed the presence of antibodies that target the myocardial scar and collagenase-sensitive epitopes. Moreover, we found that IgG accumulated within the scar tissues of infarcted mice and remained in close proximity with cells expressing Fcγ receptors (CD16/32), suggesting the existence of an in situ IgG-Fcγ receptor axis. Collectively, our study results confirm that antibodies contribute to ischemic heart failure progression and provide novel insights into the mechanisms underlying this phenomenon. NEW & NOTEWORTHY Our study sheds some light on the long-standing debate over the relevance of autoantibodies in heart failure and might stimulate future research in the field. The observation of extracellular matrix-specific antibodies and the detection of Fcγ receptor-expressing cells within the scar provide novel insights into the mechanisms by which antibodies may contribute to adverse remodeling.

Congenital Posterior Urethral Fistulae: Literature Review and Case Report.

Congenital posterior urethral-perineal fistula is an abnormal communication extending from the posterior urethra to the perineal skin. We present the case of an 11-year-old boy who had recurrent febrile urinary tract infections and abnormal dribbling of urine from the perineum. Fistulogram showed a paraescrotal fistula tract, which was then surgically excised. During the 10 months of follow-up, the patient remained asymptomatic without recurrence of urinary pathology.

Zika-prevention Knowledge among Hispanic Women Living in Puerto Rico: A Cross-sectional Study.

OBJECTIVE: To describe the level of knowledge about Zika virus exposure, symptoms, complications, and transmission prevention in Hispanic women living in Puerto Rico. METHODS: A sample of 168 women aged 21 to 64 at the general community, consented to complete a self-administered questionnaire of 112 questions from October 2016 to July 2017. RESULTS: A univariate analysis showed that the participating women recognized that the Zika virus was transmitted through mosquito bites (95.2%), sexual intercourse (78.0%), and from a mother to her fetus (41.1%); participants also believed that other, incorrect, routes of transmission were plausible. Regarding their knowledge of Zika infection prevention, the participants' correct answers included the following: using mosquito repellent (94.1%), eliminating standing water (83.9%), and using condoms (83.3%). When asked about fetal risks associated with Zika, they believed that the fetus would not develop normally (75.6%), would be born with a disability (69.6%), or would experience nervous system problems (54.2%). Only 22.6% of the participants had taken precautions to prevent pregnancy during the Zika outbreak, of which 65.8% reported that they had used condoms during all sexual relationships. CONCLUSION: There is a need to educate the general population about the Zika virus to reduce the misconceptions about disease prevention and transmission as well as about the complications associated with Zika during pregnancy. Awareness efforts should emphasize the prevention of infections during pregnancy and the use of available contraceptive methods.