Differential cytokine profiling in Chagasic patients according to their arrhythmogenic-status.

BACKGROUND: Chagas disease is caused by the protozoan Trypanosoma cruzi and is characterized by heart failure and sudden death. Identifying which factors are involved in evolution and treatment response is actually challenging. Thus, the aim of this work was to determine the Th1/Th17 (IL-6, IL-2, TNF, IL-17 and IFN-γ) and Th2 (IL-4 and IL-10) serum profile in Venezuelan Chagasic patients stratified according amiodarone treatment, hypertension and arrhythmias. METHODS: Sera from 38 chagasic patients were analyzed to determine the level of cytokines by Multiplexed Bead-Based Immunoassays. ANOVA test was applied to determine differences for each group. Additionally, a Linear Discriminant Analysis (LDA) was applied to observe the accuracy of different cytokines to discriminate between the groups. RESULTS: The levels of several cytokines were significantly higher in the high-risk of sudden death and untreated group. LDA showed that IL-2, IFN-γ and IL-10 were the best cytokines for discriminating between high-risk of sudden death and untreated patients versus low-risk of sudden death, treated and control groups. CONCLUSIONS: High IL-2 levels seem to identify patients with high-risk of sudden death and seems adequate as treatment efficacy marker. To our knowledge, this is the first report about the anti-inflammatory role of the amiodarone in Chagas disease, suggesting an inmunomodulatory effect that may be exploited as coadjutant therapy in chronic Chagas disease.

The efficacy of the ultraviolet C pathogen inactivation system in the reduction of Babesia divergens in pooled buffy coat platelets.

BACKGROUND: Babesia spp. is an intraerythrocytic parasite that causes human babesiosis and its transmission by transfusion has been extensively demonstrated. The aim of this study was to ascertain the efficacy of an ultraviolet C (UVC)-based pathogen inactivation system in the reduction of Babesia divergens-infected platelet (PLT) concentrates and to determine the parasite's ability to survive in PLT concentrates stored under blood bank conditions. STUDY DESIGN AND METHODS: This study was conducted using in vitro cultures of B. divergens. The detection limit of the culture assay was established and, subsequently, 15 buffy coat-derived PLT concentrates (BC-PCs) were inoculated with 10(7) B. divergens-infected red blood cells. Infected BC-PCs were irradiated with 0.2 J/cm(2) UVC light using the THERAFLEX UV-Platelets method (Macopharma). Viability and parasite growth were evaluated before and after inactivation. Culture growth kinetics were monitored by DNA incorporation of [(3) H]thymidine. The ability of B. divergens to survive in PLT concentrates was also analyzed. RESULTS: The limit of detection in cultures was established at 0.1 × 10(-6) % parasites. The THERAFLEX UV-Platelets system inactivated B. divergens to below the limit of detection in 12 of 15 BC-PCs (log reduction, >6.0) and to the limit of detection (log reduction, 5.0) in three of 15. It was also demonstrated that B. divergens remains viable in BC-PCs stored up to 7 days. CONCLUSION: Since B. divergens can survive in PLT concentrates and given the performance of UVC, this system could be considered as an alternative to prevent B. divergens and other Babesia species from being transmitted through PLT transfusions.

Discovery of circulating miRNAs as biomarkers of chronic Chagas heart disease via a small RNA-Seq approach.

Chagas disease affects approximately 7 million people worldwide in Latin America and is a neglected tropical disease. Twenty to thirty percent of chronically infected patients develop chronic Chagas cardiomyopathy decades after acute infection. Identifying biomarkers of Chagas disease progression is necessary to develop better therapeutic and preventive strategies. Circulating microRNAs are increasingly reliable biomarkers of disease and therapeutic targets. To identify new circulating microRNAs for Chagas disease, we performed exploratory small RNA sequencing from the plasma of patients and performed de novo miRNA prediction, identifying potential new microRNAs. The levels of the new microRNAs temporarily named miR-Contig-1519 and miR-Contig-3244 and microRNAs that are biomarkers for nonchagasic cardiomyopathies, such as miR-148a-3p and miR-224-5p, were validated by quantitative reverse transcription. We found a specific circulating microRNA signature defined by low miR-Contig-3244, miR-Contig-1519, and miR-148a-3 levels but high miR-224-5p levels for patients with chronic Chagas disease. Finally, we predicted in silico that these altered circulating microRNAs could affect the expression of target genes involved in different cellular pathways and biological processes, which we will explore in the future.

Myeloid-derived suppressor cells infiltrate the heart in acute Trypanosoma cruzi infection.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed in the acute and chronic phases of the disease, is characterized by a mononuclear cell inflammatory infiltrate. We previously identified a myeloid cell population in the inflammatory heart infiltrate of infected mice that expressed arginase I. In this study, we purified CD11b(+) myeloid cells from the heart and analyzed their phenotype and function. Those CD11b(+) cells were ∼70% Ly6G(-)Ly6C(+) and 25% Ly6G(+)Ly6C(+). Moreover, purified CD11b(+)Ly6G(-) cells, but not Ly6G(+) cells, showed a predominant monocytic phenotype, expressed arginase I and inducible NO synthase, and suppressed anti-CD3/anti-CD28 Ab-induced T cell proliferation in vitro by an NO-dependent mechanism, activity that best defines myeloid-derived suppressor cells (MDSCs). Contrarily, CD11b(+)Ly6G(+) cells, but not CD11b(+)Ly6G(-) cells, expressed S100A8 and S100A9, proteins known to promote recruitment and differentiation of MDSCs. Together, our results suggest that inducible NO synthase/arginase I-expressing CD11b(+)Ly6G(-) myeloid cells in the hearts of T. cruzi-infected mice are MDSCs. Finally, we found plasma l-arginine depletion in the acute phase of infection that was coincident in time with the appearance of MDSCs, suggesting that in vivo arginase I could be contributing to l-arginine depletion and systemic immunosuppression. Notably, l-arginine supplementation decreased heart tissue parasite load, suggesting that sustained arginase expression through the acute infection is detrimental for the host. This is, to our knowledge, the first time that MDSCs have been found in the heart in the context of myocarditis and also in infection by T. cruzi.

Sec22b-dependent antigen cross-presentation is a significant contributor of T cell priming during infection with the parasite Trypanosoma cruzi.

Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b-/-) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b-/- mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival.

Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo.

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice.

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.

TCFL5 deficiency impairs the pachytene to diplotene transition during spermatogenesis in the mouse.

Spermatogenesis is a complex, multistep process during which spermatogonia give rise to spermatozoa. Transcription Factor Like 5 (TCFL5) is a transcription factor that has been described expressed during spermatogenesis. In order to decipher the role of TCFL5 during in vivo spermatogenesis, we generated two mouse models. Ubiquitous removal of TCFL5 generated by breeding TCFL5fl/fl with SOX2-Cre mice resulted in sterile males being unable to produce spermatozoa due to a dramatic alteration of the testis architecture presenting meiosis arrest and lack of spermatids. SYCP3, SYCP1 and H1T expression analysis showed that TCFL5 deficiency causes alterations during pachytene/diplotene transition resulting in a meiotic arrest in a diplotene-like stage. Even more, TCFL5 deficient pachytene showed alterations in the number of MLH1 foci and the condensation of the sexual body. In addition, tamoxifen-inducible TCFL5 knockout mice showed, besides meiosis phenotype, alterations in the spermatids elongation process resulting in aberrant spermatids. Furthermore, TCFL5 deficiency increased spermatogonia maintenance genes (Dalz, Sox2, and Dmrt1) but also increased meiosis genes (Syce1, Stag3, and Morc2a) suggesting that the synaptonemal complex forms well, but cannot separate and meiosis does not proceed. TCFL5 is able to bind to the promoter of Syce1, Stag3, Dmrt1, and Syce1 suggesting a direct control of their expression. In conclusion, TCFL5 plays an essential role in spermatogenesis progression being indispensable for meiosis resolution and spermatids maturation.

Isoform-specific effects of transcription factor TCFL5 on the pluripotency-related genes SOX2 and KLF4 in colorectal cancer development.

Colorectal cancer (CRC) is a very common life-threatening malignancy. Transcription factor-like 5 (TCFL5) has been suggested to be involved in CRC. Here, we describe the expression of four alternative transcripts of TCFL5 and their relevance in CRC. Complete deletion of all isoforms drastically decreased pro-tumoural properties such as spheroids formation and in vivo tumour growth, although increased migration in CRC cell lines. Overexpression of the two main isoforms, TCFL5_E8 and CHA, had opposite effects: TCFL5_E8 reduced proliferation and spheroids formation, while CHA increased them. TCFL5_E8 reduced in vivo tumour formation, while CHA had no effect. In addition, TCFL5_E8 and CHA have different roles in the regulation of the pluripotency-related genes SOX2 and KLF4. Both isoforms bind directly to their promoters; however, TCFL5_E8 induced SOX2 and reduced KLF4 mRNA levels, whereas CHA did the opposite. Together, our results show that TCFL5 plays an important role in the development of CRC, being however isoform-specific. This work also points to the need to analyse separately TCFL5 isoforms in cancer, due to their different and opposite functions.

Myeloid-Derived Suppressor Cells in Trypanosoma cruzi Infection.

Myeloid-derived suppressor cells (MDSCs) are immature heterogeneous myeloid cells that expand in pathologic conditions as cancer, trauma, and infection. Although characterization of MDSCs is continuously revisited, the best feature is their suppressor activity. There are many markers for MDSC identification, it is distinctive that they express inducible nitric oxide synthase (iNOS) and arginase 1, which can mediate immune suppression. MDSCs can have a medullary origin as a result of emergency myelopoiesis, but also can have an extramedullary origin. Early studies on Trypanosoma cruzi infection showed severe immunosuppression, and several mechanisms involving parasite antigens and host cell mediators were described as inhibition of IL-2 and IL-2R. Another mechanism of immunosuppression involving tumor necrosis factor/interferon γ-dependent nitric oxide production by inducible nitric oxide synthase was also described. Moreover, other studies showed that nitric oxide was produced by CD11b+ Gr-1+ MDSCs in the spleen, and later iNOS and arginase 1 expressed in CD11b+Ly6C+Ly6Glo monocytic MDSC were found in spleen and heart of T. cruzi infected mice that suppressed T cell proliferation. Uncontrolled expansion of monocytic MDSCs leads to L-arginine depletion which hinders nitric oxide production leading to death. Supplement of L-arginine partially reverts L-arginine depletion and survival, suggesting that L-arginine could be administered along with anti-parasitical drugs. On the other hand, pharmacological inhibition of MDSCs leads to death in mice, suggesting that some expansion of MDSCs is needed for an efficient immune response. The role of signaling molecules mediating immune suppression as reactive oxygen species, reactive nitrogen species, as well as prostaglandin E2, characteristics of MDSCs, in T. cruzi infection is not fully understood. We review and discuss the role of these reactive species mediators produced by MDSCs. Finally, we discuss the latest results that link the SLAMF1 immune receptor with reactive oxygen species. Interaction of the parasite with the SLAMF1 modulates parasite virulence through myeloid cell infectivity and reactive oxygen species production. We discuss the possible strategies for targeting MDSCs and SLAMF1 receptor in acute Trypanosoma cruzi infection in mice, to evaluate a possible translational application in human acute infections.

The Complete Mitochondrial DNA of Trypanosoma cruzi: Maxicircles and Minicircles.

The mitochondrial DNA of Trypanosomatids, known as the kinetoplast DNA or kDNA or mtDNA, consists of a few maxicircles and thousands of minicircles concatenated together into a huge complex network. These structures present species-specific sizes, from 20 to 40 Kb in maxicircles and from 0.5 to 10 Kb in minicircles. Maxicircles are equivalent to other eukaryotic mitochondrial DNAs, while minicircles contain coding guide RNAs involved in U-insertion/deletion editing processes exclusive of Trypanosomatids that produce the maturation of the maxicircle-encoded transcripts. The knowledge about this mitochondrial genome is especially relevant since the expression of nuclear and mitochondrial genes involved in oxidative phosphorylation must be coordinated. In Trypanosoma cruzi (T. cruzi), the mtDNA has a dual relevance; the production of energy, and its use as a phylogenetic marker due to its high conservation among strains. Therefore, this study aimed to assemble, annotate, and analyze the complete repertoire of maxicircle and minicircle sequences of different T. cruzi strains by using DNA sequencing. We assembled and annotated the complete maxicircle sequence of the Y and Bug2148 strains. For Bug2148, our results confirm that the maxicircle sequence is the longest assembled to date, and is composed of 21 genes, most of them conserved among Trypanosomatid species. In agreement with previous results, T. cruzi minicircles show a conserved structure around 1.4 Kb, with four highly conserved regions and other four hypervariable regions interspersed between them. However, our results suggest that the parasite minicircles display several sizes and numbers of conserved and hypervariable regions, contrary to those previous studies. Besides, this heterogeneity is also reflected in the three conserved sequence blocks of the conserved regions that play a key role in the minicircle replication. Our results using sequencing technologies of second and third-generation indicate that the different consensus sequences of the maxicircles and minicircles seem to be more complex than previously described indicating at least four different groups in T. cruzi minicircles.

Molecular Remodeling of Cardiac Sinus Node Associated with Acute Chagas Disease Myocarditis.

Chagas disease principally affects Latin-American people, but it currently has worldwide distribution due to migration. Death among those with Chagas disease can occur suddenly and without warning, even in those who may not have evidence of clinical or structural cardiac disease and who are younger than 60 years old. HCN4 channels, one of the principal elements responsible for pacemaker currents, are associated with cardiac fetal reprogramming and supraventricular and ventricular arrhythmias, but their role in chagasic arrhythmias is not clear. We found that a single-dose administration of ivabradine, which blocks HCN4, caused QTc and QRS enlargement and an increase in P-wave amplitude and was associated with ventricular and supraventricular arrhythmias in mice challenged with isoproterenol, a chronotropic/ionotropic positive agent. Continuous treatment with ivabradine did not alter the QTc interval, but P-wave morphology was deeply modified, generating supraventricular arrhythmias. In addition, we found that repolarization parameters improved with ivabradine treatment. These effects could have been caused by the high HCN4 expression observed in auricular and ventricular tissue in infected mice. Thus, we suggest, for the first time, that molecular remodeling by overexpression of HCN4 channels may be related to supraventricular arrhythmias in acute Chagas disease, causing ivabradine over-response. Thus, ivabradine treatment should be administered with caution, while HCN4 overexpression may be an indicator of heart failure and/or sudden death risk.

Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy.

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

Trypanosoma Cruzi Genome: Organization, Multi-Gene Families, Transcription, and Biological Implications.

Chagas disease caused by the parasite Trypanosoma cruzi affects millions of people. Although its first genome dates from 2005, its complexity hindered a complete assembly and annotation. However, the new sequencing methods have improved genome annotation of some strains elucidating the broad genetic diversity and complexity of this parasite. Here, we reviewed the genomic structure and regulation, the genetic diversity, and the analysis of the principal multi-gene families of the recent genomes for several strains. The telomeric and sub-telomeric regions are sites with high recombination events, the genome displays two different compartments, the core and the disruptive, and the genome plasticity seems to play a key role in the survival and the infection process. Trypanosoma cruzi (T. cruzi) genome is composed mainly of multi-gene families as the trans-sialidases, mucins, and mucin-associated surface proteins. Trans-sialidases are the most abundant genes in the genome and show an important role in the effectiveness of the infection and the parasite survival. Mucins and MASPs are also important glycosylated proteins of the surface of the parasite that play a major biological role in both insect and mammal-dwelling stages. Altogether, these studies confirm the complexity of T. cruzi genome revealing relevant concepts to better understand Chagas disease.

Interaction of Signaling Lymphocytic Activation Molecule Family 1 (SLAMF1) receptor with Trypanosoma cruzi is strain-dependent and affects NADPH oxidase expression and activity.

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease.

Impact of fascioliasis reinfection on Fasciola hepatica egg shedding: relationship with the immune-regulatory response.

Fascioliasis is a disease caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm. Control strategies in humans require the use of egg count techniques to calculate the appropriate treatment dose for colic risk prevention. The present study investigates how fascioliasis reinfection affects liver fluke egg shedding and its relationship with the immune-regulatory response. The experimental design reproduced the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 4 weeks (R4), 8 weeks (R8), 12 weeks (R12), and negative control rats. In a longitudinal study (0-20 weeks post-infection, p.i.), serical IgG1 levels and eggs per gram of faeces (epg) were analyzed. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. In R8 and R12, transiently higher averages of epg and epg/worm in reinfected groups vs PI group were detected at least in the weeks following reinfection. The kinetics of IgG1 levels shows that reinfected groups followed a pattern similar to the one in the PI group, but transiently higher averages of IgG1 levels in reinfected groups vs the PI group were detected in the weeks following reinfection. Epg correlated with IgG1 levels and also with systemic Il10 and thymic Ifng, and Il10 expression levels. These results suggest that epg depends on the Th1 and Treg phenotype and that the determination of the fluke burden by epg is likely to be an overestimation in cases of recent reinfection in low burden situations. A strategy to facilitate the implementation of epg count techniques and the subsequent decision on the appropriate treatment dose for each patient to prevent colic risk is required.

Multi-Parametric Evaluation of Trypanosoma cruzi Infection Outcome in Animal Models.

Biomarkers of infection with consistent discriminating power for diagnosis and/or prognosis are keystones for efficient therapeutic management of diseases. The protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease (CD), exhibits high clinical and genetic diversity, making it difficult to define biomarkers. In animal models of infection, as well as in patients, many different outcomes have been described. Thus, pathophysiogenesis parameters were highly variable in patients and even in inbred animals, which impeded reliable one-dimensional diagnosis/prognosis. To help solve those problems, an in-depth analysis of the similarities and differences in the CD caused by different parasite strains or different patient conditions is needed. Multidimensional statistics may overcome the high variability for each individual parameter in patients and even in inbred animals, revealing some pathophysiological patterns that accurately allow diagnosis of clinical and physiopathological characteristics. Here, we describe this type of method and its application to T. cruzi infection.

Comparative proteomic analysis of trypomastigotes from Trypanosoma cruzi strains with different pathogenicity.

Chagas disease, caused by the parasite Trypanosoma cruzi, is one of the most neglected diseases in Latin America, being currently a global health problem. Its immunopathogenesis is still quite unknown. Moreover, there are important differences in pathogenicity between some different T. cruzi strains. For example, in mice, Y strain produces a high acute lethality while VFRA remains in the host mostly in a chronic manner. Comparative proteomic studies between T. cruzi strains represent a complement for transcriptomics and may allow the detection of relevant factors or distinctive functions. Here for the first time, we compared the proteome of trypomastigotes from 2 strains, Y and VFRA, analyzed by mass spectrometry. Gene ontology analysis were used to display similarities or differences in cellular components, biological processes and molecular functions. Also, we performed metabolic pathways enrichment analysis to detect the most relevant pathways in each strain. Although in general they have similar profiles in the different ontology groups, there were some particular interesting differences. Moreover, there were around 10% of different proteins between Y and VFRA strains, that were shared by other T. cruzi strains or protozoan species. They displayed many common enriched metabolic pathways but some others were uniquely enriched in one strain. Thus, we detected enriched antioxidant defenses in VFRA that could correlate with its ability to induce a chronic infection in mice controlling ROS production, while the Y strain revealed a great enrichment of pathways related with nucleotides and protein production, that could fit with its high parasite replication and lethality. In summary, Y and VFRA strains displayed comparable proteomes with some particular distinctions that could contribute to understand their different biological behaviors.

Analysis of mRNA processing at whole transcriptome level, transcriptomic profile and genome sequence refinement of Trypanosoma cruzi.

The genomic sequence of Trypanosoma cruzi, the protozoan causative of Chagas disease was published more than a decade ago. However, due to their complexity, its complete haploid predicted sequence and therefore its genetic repertoire remains unconfirmed. In this work, we have used RNAseq data to improve the previous genome assembly of Sylvio X10 strain and to define the complete transcriptome at trypomastigote stage (mammalian stage). A total of 22,977 transcripts were identified, of which more than half could be considered novel as they did not match previously annotated genes. Moreover, for the first time in T. cruzi, we are providing their relative abundance levels. We have identified that Sylvio X10 trypomastigotes exhibit a predominance of surface protein genes, specifically those encoding trans-sialidase and mucin-like proteins. On the other hand, detailed analysis of the pre-mRNA processing sites revealed some similarities but also some differences in the spliced leader and different polyadenylation addition sites compared to close related kinetoplastid parasites. Our results also confirm that transcription is bidirectional as occur in other kinetoplastids and the proportion of forward-sense and reverse-sense transcripts is almost equivalent, demonstrating that a strand-specificity does not exist.

Regulatory Lymphoid and Myeloid Cells Determine the Cardiac Immunopathogenesis of Trypanosoma cruzi Infection.

Chagas disease is a multisystemic disorder caused by the protozoan parasite Trypanosoma cruzi, which affects ~8 million people in Latin America, killing 7,000 people annually. Chagas disease is one of the main causes of death in the endemic area and the leading cause of infectious myocarditis in the world. T. cruzi infection induces two phases, acute and chronic, where the infection is initially asymptomatic and the majority of patients will remain clinically indeterminate for life. However, over a period of 10-30 years, ~30% of infected individuals will develop irreversible, potentially fatal cardiac syndromes (chronic chagasic cardiomyopathy [CCC]), and/or dilatation of the gastro-intestinal tract (megacolon or megaesophagus). Myocarditis is the most serious and frequent manifestation of chronic Chagas heart disease and appears in about 30% of infected individuals several years after infection occurs. Myocarditis is characterized by a mononuclear cell infiltrate that includes different types of myeloid and lymphoid cells and it can occur also in the acute phase. T. cruzi infects and replicates in macrophages and cardiomyocytes as well as in other nucleated cells. The pathogenesis of the chronic phase is thought to be dependent on an immune-inflammatory reaction to a low-grade replicative infection. It is known that cytokines produced by type 1 helper CD4+ T cells are able to control infection. However, the role that infiltrating lymphoid and myeloid cells may play in experimental and natural Chagas disease pathogenesis has not been completely elucidated, and several reports indicate that it depends on the mouse genetic background and parasite strain and/or inoculum. Here, we review the role that T cell CD4+ subsets, myeloid subclasses including myeloid-derived suppressor cells may play in the immunopathogenesis of Chagas disease with special focus on myocarditis, by comparing results obtained with different experimental animal models.