Differential expression profile of genes involved in the immune response associated to progression of chronic Chagas disease.

BACKGROUND: Patients with chronic Chagas disease present marked clinical and immunological heterogeneity. During the disease, multiple immune mechanisms are activated to fight the parasite. The purpose of this study was to investigate the expression patterns of genes involved in relevant immunological processes throughout the disease in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: High-throughput RT-qPCR with QuantStudio 12K Flex real-time PCR system was used to evaluate the expression of 106 immune-related genes in PBMC from a cohort of cardiac Chagas disease patients (CCC I), asymptomatic patients (IND) and healthy donors (HD) after being stimulated with T. cruzi soluble antigens. Principal component analysis (PCA), cluster analysis and volcano plots were used to identify differentially expressed genes. In addition, gene set enrichment analysis (GSEA) was employed to identify the enriched immunological pathways in which these genes are involved. PCA revealed the existence of a statistically divergent expression profile of the 36 genes correlated with PC1 between CCC I patients and HD (p < 0.0001). Differential gene expression analysis revealed upregulation of 41 genes (expression fold-change > 1.5) and downregulation of 14 genes (expression fold-change < 0.66) (p = 8.4x10-13 to p = 0.007) in CCC I patients versus HD. Furthermore, significant differences in the expression level of specific genes have been identified between CCC I and IND patients (8 up and 1 downregulated). GSEA showed that several upregulated genes in CCC I patients participate in immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, cytokines-inflammatory response and IL-10 anti-inflammatory signaling. CONCLUSIONS: Cardiac Chagas disease patients show an antigen-specific differential gene expression profile in which several relevant immunological pathways seem to be activated. Assessment of gene expression profiles reveal unique insights into the immune response that occurs along chronic Chagas disease.

CD8+ T Cell Response Quality Is Related to Parasite Control in an Animal Model of Single and Mixed Chronic Trypanosoma cruzi Infections.

Chagas disease (ChD) is a chronic infection caused by Trypanosoma cruzi. This highly diverse intracellular parasite is classified into seven genotypes or discrete typing units (DTUs) and they overlap in geographic ranges, vectors, and clinical characteristics. Although studies have suggested that ChD progression is due to a decline in the immune response quality, a direct relationship between T cell responses and disease outcome is still unclear. To investigate the relationship between parasite control and immune T cell responses, we used two distinct infection approaches in an animal model to explore the histological and parasitological outcomes and dissect the T cell responses in T. cruzi-infected mice. First, we performed single infection experiments with DA (TcI) or Y (TcII) T. cruzi strains to compare the infection outcomes and evaluate its relationship with the T cell response. Second, because infections with diverse T. cruzi genotypes can occur in naturally infected individuals, mice were infected with the Y or DA strain and subsequently reinfected with the Y strain. We found different infection outcomes in the two infection approaches used. The single chronic infection showed differences in the inflammatory infiltrate level, while mixed chronic infection by different T. cruzi DTUs showed dissimilarities in the parasite loads. Chronically infected mice with a low inflammatory infiltrate (DA-infected mice) or low parasitemia and parasitism (Y/Y-infected mice) showed increases in early-differentiated CD8+ T cells, a multifunctional T cell response and lower expression of inhibitory receptors on CD8+ T cells. In contrast, infected mice with a high inflammatory infiltrate (Y-infected mice) or high parasitemia and parasitism (DA/Y-infected mice) showed a CD8+ T cell response distinguished by an increase in late-differentiated cells, a monofunctional response, and enhanced expression of inhibitory receptors. Overall, our results demonstrated that the infection outcomes caused by single or mixed T. cruzi infection with different genotypes induce a differential immune CD8+ T cell response quality. These findings suggest that the CD8+ T cell response might dictate differences in the infection outcomes at the chronic T. cruzi stage. This study shows that the T cell response quality is related to parasite control during chronic T. cruzi infection.

Differential Expression of Immune Response Genes in Asymptomatic Chronic Chagas Disease Patients Versus Healthy Subjects.

Infection by the Trypanosoma cruzi parasite causes Chagas disease and triggers multiple immune mechanisms in the host to combat the pathogen. Chagas disease has a variable clinical presentation and progression, producing in the chronic phase a fragile balance between the host immune response and parasite replication that keeps patients in a clinically silent asymptomatic stage for years. Since the parasite is intracellular and replicates within cells, the cell-mediated response of the host adaptive immunity plays a critical role. This function is mainly orchestrated by T lymphocytes, which recognize parasite antigens and promote specific functions to control the infection. However, little is known about the immunological markers associated with this asymptomatic stage of the disease. In this large-scale analysis, the differential expression of 106 immune system-related genes has been analyzed using high-throughput qPCR in T. cruzi antigen-stimulated PBMC from chronic Chagas disease patients with indeterminate form (IND) and healthy donors (HD) from endemic and non-endemic areas of Chagas disease. This analysis revealed that there were no differences in the expression level of most genes under study between healthy donors from endemic and non-endemic areas determined by PCA and differential gene expression analysis. Instead, PCA revealed the existence of different expression profiles between IND patients and HD (p < 0.0001), dependent on the 32 genes included in PC1. Differential gene expression analysis also revealed 23 upregulated genes (expression fold change > 2) and 11 downregulated genes (expression fold change < 0.5) in IND patients versus HD. Enrichment analysis showed that several upregulated genes in IND patients participate in relevant immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, and cytokine-inflammatory response. The antigen-specific differential gene expression profile detected in these patients and the relevant immunological pathways that seem to be activated could represent potential biomarkers of the asymptomatic form of Chagas disease, helpful to diagnosis and infection control.

Variability of the Pr77 sequence of L1Tc retrotransposon among six T. cruzi strains belonging to different discrete typing units (DTUs).

All trypanosomatid genomes are colonized by non-LTR retrotransposons which exhibit a highly conserved 77-nt sequence at their 5' ends, known as the Pr77-hallmark (Pr77). The wide distribution of Pr77 is expected to be related to the gene regulation processes in these organisms as it has promoter and HDV-like ribozyme activities at the DNA and RNA levels, respectively. The identification of Pr77 hallmark-bearing retrotransposons and the study of the associations of mobile elements with relevant genes have been analyzed in the genomes of six strains of Trypanosoma cruzi belonging to different discrete typing units (DTUs) and with different geographical origins and host/vectors. The genomes have been sequenced, assembled and annotated. BUSCO analyses indicated a good quality for the assemblies that were used in comparative analyses. The results show differences among the six genomes in the copy number of genes related to virulence processes, the abundance of retrotransposons bearing the Pr77 sequence and the presence of the Pr77 hallmarks not associated with retroelements. The analyses also show frequent associations of Pr77-bearing retrotransposons and single Pr77 hallmarks with genes coding for trans-sialidases, RHS, MASP or hypothetical proteins, showing variable proportion depending on the type of retroelement, gene class and parasite strain. These differences in the genomic distribution of active retroelements and other Pr77-containing elements have shaped the genome architecture of these six strains and might be contributing to the phenotypic variability existing among them.

TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons.

BACKGROUND: Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5' ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. METHODS: TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. RESULTS: This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein-protein interactions through other trypanosomatid nuclear proteins. CONCLUSIONS: Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite.

Serological reactivity against T. cruzi-derived antigens: Evaluation of their suitability for the assessment of response to treatment in chronic Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 6 million people worldwide. Following a mostly asymptomatic acute phase, the disease progresses to a long-lasting chronic phase throughout which life-threatening disorders to the heart and/or gastrointestinal tract will manifest in about 30% of those chronically infected. During the chronic phase, the parasitemia is low and intermittent, while a high level of anti-T. cruzi antibodies persist for years. These two features hamper post-chemotherapeutic follow-up of patients with the tools available. The lack of biomarkers for timely assessment of therapeutic response discourages a greater use of the two available anti-parasitic drugs, and complicates the evaluation of new drugs in clinical trials. Herein, we investigated in a blinded case-control study the serological reactivity over time of a group of parasite-derived antigens to potentially address follow up of T. cruzi chronically infected subjects after treatment. We tested PFR2, KMP11, HSP70, 3973, F29 and the InfYnity multiplexed antigenic array, by means of serological assays on a multi-national retrospective collection of samples. Some of the antigens exhibited promising results, underscoring the need for further studies to determine their potential role as treatment response biomarkers.

A proportion of CD4+ T cells from patients with chronic Chagas disease undergo a dysfunctional process, which is partially reversed by benznidazole treatment.

BACKGROUND: Signs of senescence and the late stages of differentiation associated with the more severe forms of Chagas disease have been described in the Trypanosoma cruzi antigen-specific CD4+ T-cell population. However, the mechanisms involved in these functions are not fully known. To date, little is known about the possible impact of benznidazole treatment on the T. cruzi-specific functional response of CD4+ T cells. METHODOLOGY/PRINCIPAL FINDINGS: The functional capacity of CD4+ T cells was analyzed by cytometric assays in chronic Chagas disease patients, with indeterminate form (IND) and cardiac alterations (CCC) (25 and 15, respectively) before and after benznidazole treatment. An increase in the multifunctional capacity (expression of IFN-γ, IL-2, TNF-α, perforin and/or granzyme B) of the antigen-specific CD4+ T cells was observed in indeterminate versus cardiac patients, which was associated with the reduced coexpression of inhibitory receptors (2B4, CD160, CTLA-4, PD-1 and/or TIM-3). The functional profile of these cells shows statistically significant differences between IND and CCC (p<0.001), with a higher proportion of CD4+ T cells coexpressing 2 and 3 molecules in IND (54.4% versus 23.1% and 4.1% versus 2.4%, respectively). A significant decrease in the frequencies of CD4+ T cells that coexpress 2, 3 and 4 inhibitory receptors was observed in IND after 24-48 months of treatment (p<0.05, p<0.01 and p<0.05, respectively), which was associated with an increase in antigen-specific multifunctional activity. The IND group showed, at 9-12 months after treatment, an increase in the CD4+ T cell subset coproducing three molecules, which were mainly granzyme B+, perforin+ and IFN-γ+ (1.4% versus 4.5%). CONCLUSIONS/SIGNIFICANCE: A CD4+ T cell dysfunctional process was detected in chronic Chagas disease patients, being more exacerbated in those patients with cardiac symptoms. After short-term benznidazole treatment (9-12 months), indeterminate patients showed a significant increase in the frequency of multifunctional antigen-specific CD4+ T cells.

New chemotherapy regimens and biomarkers for Chagas disease: the rationale and design of the TESEO study, an open-label, randomised, prospective, phase-2 clinical trial in the Plurinational State of Bolivia.

INTRODUCTION: Chagas disease (CD) affects ~7 million people worldwide. Benznidazole (BZN) and nifurtimox (NFX) are the only approved drugs for CD chemotherapy. Although both drugs are highly effective in acute and paediatric infections, their efficacy in adults with chronic CD (CCD) is lower and variable. Moreover, the high incidence of adverse events (AEs) with both drugs has hampered their widespread use. Trials in CCD adults showed that quantitative PCR (qPCR) assays remain negative for 12 months after standard-of-care (SoC) BZN treatment in ~80% patients. BZN pharmacokinetic data and the nonsynchronous nature of the proliferative mammal-dwelling parasite stage suggested that a lower BZN/NFX dosing frequency, combined with standard or extended treatment duration, might have the same or better efficacy than either drug SoC, with fewer AEs. METHODS AND ANALYSIS: New ThErapies and Biomarkers for ChagaS infEctiOn (TESEO) is an open-label, randomised, prospective, phase-2 clinical trial, with six treatment arms (75 patients/arm, 450 patients). Primary objectives are to compare the safety and efficacy of two new proposed chemotherapy regimens of BZN and NFX in adults with CCD with the current SoC for BZN and NFX, evaluated by qPCR and biomarkers for 36 months posttreatment and correlated with CD conventional serology. Recruitment of patients was initiated on 18 December 2019 and on 20 May 2021, 450 patients (study goal) were randomised among the six treatment arms. The treatment phase was finalised on 18 August 2021. Secondary objectives include evaluation of population pharmacokinetics of both drugs in all treatment arms, the incidence of AEs, and parasite genotyping. ETHICS AND DISSEMINATION: The TESEO study was approved by the National Institutes of Health (NIH), U.S. Food and Drug Administration (FDA), federal regulatory agency of the Plurinational State of Bolivia and the Ethics Committees of the participating institutions. The results will be disseminated via publications in peer-reviewed journals, conferences and reports to the NIH, FDA and participating institutions. TRIAL REGISTRATION NUMBER: NCT03981523.

Detection of Leishmania infantum DNA by real-time PCR in saliva of dogs.

This study developed a real-time quantitative PCR (qPCR) assay to detect L. infantum kinetoplast DNA (kDNA) in canine saliva. The qPCR showed an efficiency of 93.8%, a coefficient of correlation of 0.996 and a detection limit of 0.5 fg/reaction (0.005 parasites), although it detected until 0.25 fg/reaction (0.0025 parasites). When samples from 12 dogs experimentally infected with L. infantum were collected, L. infantum kDNA was detected at 16-weeks post-infection (wpi) in 41.7% and 91.7% of saliva and bone marrow samples, respectively, and at 47-wpi in 75% of both samples. L. infantum kDNA can be detected by qPCR in canine saliva, with lower sensitivity in the early stages of infection and a lower parasite load estimation compared to bone marrow. However, saliva had similar sensitivities to bone marrow in the later stages of the infection and could be used to detect L. infantum kDNA being aware of its limitations.

Trypanosoma cruzi Ikiakarora (TcIII) Draft Genome Sequence.

Trypanosoma cruzi shows a genetic diversity that has been associated with the variability of clinical manifestations, geographical distribution, and preferential parasite-vector interactions. In an effort to better understand this genetic variability, here, the draft genome of T. cruzi strain Ikiakarora (discrete typing unit TcIII), which has been associated with the sylvatic cycle, is reported.

Draft Genome Sequence of the Trypanosoma cruzi B. M. López Strain (TcIa), Isolated from a Colombian Patient.

Trypanosoma cruzi parasite strains are classified into six lineages (discrete typing units TcI to TcVI). The broad genetic diversity of T. cruzi strains has an influence on the development of the host response and pathogenesis, as well as drug susceptibility. Here, the draft genome of the T. cruzi B. M. López strain (TcIa) is reported.

Differential phenotypic and functional profile of epitope-specific cytotoxic CD8+ T cells in benznidazole-treated chronic asymptomatic Chagas disease patients.

One of the greatest challenges in Chagas disease research is the search for tools that will enable the assessment of pharmacological treatment efficacy. A recently described set of serological biomarkers composed of four parasite antigens and established criteria of treatment efficacy allowed the evaluation of the impact of benznidazole treatment a short/medium time after the treatment. In addition, cellular immunological parameters have also been described as potential indicators of the treatment response. The cytotoxic CD8+ T cells specific to five epitopes in the PFR2, PFR3, TcCA-2 and KMP11 antigens have been analysed, and these epitopes have been shown to be recognized, processed and presented in the context of a natural T. cruzi infection. In the present manuscript, we characterized these antigen-specific CD8+ T cells in indeterminate chronic Chagas disease patients both before and after (from 11 to 28 months) benznidazole treatment. The results indicate that there is a differential memory CD8+ T cell profile depending on the antigenic epitope and that the benznidazole treatment modulates the memory, differentiation and senescence phenotypes of the epitope-specific CD8+ T cells. Moreover, in these patients, the reactivity of sera against the referred set of biomarkers was evaluated. The data obtained show that the patients who met the established therapeutic efficacy criteria presented a differential phenotypic profile of the antigen-specific CD8+ T cells even prior to treatment compared to the patients who did not meet the therapeutic efficacy criteria, and this behaviour is associated with a better functionality of these CD8+ T cells.

One-year follow-up of anti-Leishmania antibody concentrations in serum and saliva from experimentally infected dogs.

The quantification of anti-Leishmania antibodies in serum and saliva by a time-resolved immunofluorometric assay is useful for the diagnosis and treatment monitoring of dogs with clinical leishmaniasis. We compared the kinetics of anti-Leishmania IgG2 and IgA antibodies in serum and saliva from 11 Beagle dogs experimentally infected with Leishmania infantum. Most dogs showed detectable concentrations of anti-Leishmania IgG2 earlier in serum (between 3 and 4 months p.i.) than in saliva (between 4 and 6 months p.i.). Overall, a high correlation between concentrations of anti-Leishmania IgG2 in serum and saliva (r = 0.853; P < 0.0001) was observed. The quantification of anti-Leishmania IgA showed less diagnostic value than IgG2, since detectable amounts of IgA were not observed in the saliva of four dogs and in the serum of one dog. In addition, a very low correlation between anti-Leishmania IgA in serum and saliva (r = 0.289; P < 0.001) was observed. Our results indicate that the antibodies against L. infantum in saliva appear approximately 1 month later than in serum, and suggest that there is a threshold for the passing of immunoglobulins from serum to saliva in dogs. These facts should be taken into consideration for a proper interpretation of saliva assays for quantification of antibodies.

A Parasite Biomarker Set for Evaluating Benznidazole Treatment Efficacy in Patients with Chronic Asymptomatic Trypanosoma cruzi Infection.

One of the current greatest challenges of Chagas disease is the establishment of biomarkers to assess the efficacy of drugs in a short period of time. In this context, the reactivity of sera from 66 adults with chronic indeterminate Chagas disease (IND) for a set of four Trypanosoma cruzi antigens (KMP11, PFR2, HSP70, and 3973d) was analyzed before and after benznidazole treatment. The results showed that the reactivity against these antigens decreased at 9, 24, and 48 months after treatment. Moreover, the 42.4% and 68.75% of IND patients met the established standard criteria of therapeutic efficacy (STEC) at 24 and 48 months posttreatment, respectively. Meeting the STEC implied that there was a continuous decrease in the reactivity of the patient sera against the four antigens after treatment and that there was a substantial decrease in the reactivity for at least two of the antigens. This important decrease in reactivity may be associated with a drastic reduction in the parasite load, but it is not necessarily associated with a parasitological cure. After treatment, a positive PCR result was only obtained in patients who did not meet the STEC. The percentage of granzyme B+/perforin+ CD8+ T cells was significantly higher in patients who met the STEC than in those who did not meet the STEC (35.2% versus 2.2%; P < 0.05). Furthermore, the patients who met the STEC exhibited an increased quality of the multifunctional response of the antigen-specific CD8+ T cells compared with that in the patients who did not meet the STEC.

T-cell exhaustion process during chronic infection caused by intracellular trypanosomatids / Proceso de agotamiento de las células T durante la infección crónica causada por tripanosomátidos intracelulares

Two of the most important neglected tropical diseases, Chagas disease and leishmaniasis, are caused by protozoan intracellular parasites of the Trypanosomatida order. These infections provoke a high social burden and lead to the death of a large number of patients. The host triggers several immune mechanisms, but in the absence of adequate treatment, the infection becomes chronic and in many cases causes the appearance of serious alterations. T lymphocytes are fundamental cells of the adaptive system and are the main immune elements that orchestrate the cell-to-cell response in the context of intracellular infections. Furthermore, it has been described that continuous and persistent stimulation in response to pathogenic antigens causes loss of antigen-specific functional capacities in the T cell subsets. This process is known as exhaustion. This review explores the results to date of the exhaustion process during chronic infections caused by the trypanosomatid parasites Leishmania spp. and Trypanosoma cruzi. A large amount of evidence shows upregulation of the markers of the exhaustion process, namely, the inhibitory receptors, during these chronic infections. This increased expression is observed in both the CD4+ and CD8+ T cell populations. In parallel, with this increased expression of inhibitory receptors, the loss of antigen-specific functional capacity of these T cells is detected, reducing the lymphoproliferative potential and the ability to produce protective molecules against these parasitic infections, such as Th1-like cytokines, among others. Additionally, a positive correlation between the high coexpression of these inhibitory molecules and the severity of the pathology is demonstrated. Furthermore, T cell populations experience a phenotypic fluctuation in the course of these infections toward the predominance of effector memory subsets with a late or terminal differentiation state. This balancing in turn affects the functional capacity of the T cells and enriches the number of cells with senescent and apoptotic characteristics. Thus, it has been demonstrated the existence of an exhaustion process that affects key populations for the parasite control. However, the role of this process in the progression of the severity of these pathologies is still unknown. The current drugs used to treat these neglected diseases seem to partially reverse this exhaustion process, denoting a reduction in the high inhibitory receptor expression observed prior to chemotherapies. An improvement in the functional capacity of these T cell populations is also observed, which could be related to the reversion of the dysfunctional process. However, the efforts made to date to evaluate blocking therapies do not lead us to a promising conclusion. It will probably be necessary to test the simultaneous blockade of several pathways and to continue advancing the knowledge to verify their possible use as immunotherapy. It is therefore necessary to continue investigating how this process is triggered and to what extent it influences the appearance of the symptomatology of patients

La enfermedad de Chagas y la leishmaniasis, causadas por parásitos protozoarios intracelulares del orden Trypanosomatida, son consideradas dos de las enfermedades tropicales desatendidas más importantes. Estas infecciones conllevan un alto desgaste social, provocando el deterioro de la salud de un gran número de pacientes e incluso su muerte. Los linfocitos T son células fundamentales del sistema adaptativo y son los principales elementos inmunitarios para el control de estas infecciones intracelulares. La presente revisión explora los estudios y resultados obtenidos hasta la fecha del proceso de agotamiento celular durante las infecciones causadas por los parásitos Leishmania spp. y Trypanosoma cruzi. Así, se recoge que la persistente estimulación celular en respuesta a antígenos de estos patógenos conduce a un proceso de pérdida de la capacidad funcional antígeno-específica en las poblaciones de células T CD4+ y CD8+. Numerosos estudios muestran la existencia de una correlación directa entre el nivel de la co-expresión de receptores inhibitorios y la gravedad de estas patologías. Paralelamente, se detecta la pérdida de la capacidad funcional específica de antígeno de estas células T, lo que reduce su potencial linfoproliferativo y su capacidad de producir moléculas protectoras contra estas infecciones. Además, durante el curso de estas infecciones se observa un incremento de la frecuencia de células T de memoria efectora con un grado de diferenciación tardía o terminal. Este balanceo fenotípico, a su vez, afecta a la capacidad funcional de las células T aumentando el número de células con características senescentes y apoptóticas. Así, los estudios realizados hasta la fecha demuestran con certeza la existencia de un proceso de agotamiento que afecta a poblaciones clave para el control parasitario. Sin embargo, actualmente se desconoce con precisión el papel que este proceso de agotamiento juega en el agravamiento de estas patologías. Los medicamentos actuales usados para tratar estas enfermedades protozoarias revierten parcialmente este proceso de agotamiento. Así, tras el tratamiento, numerosos pacientes muestra una reducción en la expresión de receptores inhibitorios y co-expresión de los mismos. También se ha observado una mejoría en la capacidad funcional de las distintas poblaciones de células T, que podría estar relacionada con la reversión del proceso disfuncional.Sin embargo, los estudios realizados hasta la fecha en la evaluación de terapias de bloqueo de los receptores inhibitorios no han conducido a resultados prometedores. Algunos autores proponen evaluar terapias de bloqueo simultáneo de varias vías de señalización, con el fin de ampliar el conocimiento sobre esta herramienta como posible inmunoterapia de control de la infección por los mencionados parásitos. Además, se considera necesario continuar investigando sobre cómo se desencadena exactamente este proceso de agotamiento celular y en qué medida influye en la aparición de la sintomatología de los pacientes y ausencia de control de la infección

Dynamics of T Cells Repertoire During Trypanosoma cruzi Infection and its Post-Treatment Modulation.

Chagas disease courses with different clinical phases and has a variable clinical presentation and progression. The acute infection phase mostly exhibits a non-specific symptomatology. In the absence of treatment, the acute phase is followed by a chronic phase, which is initially asymptomatic. This chronic asymptomatic phase of the disease is characterized by a fragile balance between the host's immune response and the parasite replication. The loss of this balance is crucial for the progression of the sickness. The virulence and tropism of the T. cruzi infecting strain together to the inflammation processes in the cardiac tissue are the main factors for the establishment and severity of the cardiomyopathy. The efficacy of treatment in chronic Chagas disease patients is controversial. However, several studies carried out in chronic patients demonstrated that antiparasitic treatment reduces parasite load in the bloodstream and leads to an improvement in the immune response against the Trypanosoma cruzi parasite. The present review is mainly focused on the cellular patterns associated to the clinical status and the evolution of the disease in chronic patients, as well as the effectiveness of the treatment related to T. cruzi infection control. Therefore, an emphasis is placed on the dynamics of specific-antigens T cell subpopulations, their memory and activation phenotypes, their functionality and their contribution to pathogenesis or disease control, as well as their association with risk of congenital transmission of the parasite.

Phenotypic and Functional Profiles of Antigen-Specific CD4+ and CD8+ T Cells Associated With Infection Control in Patients With Cutaneous Leishmaniasis.

The host immunological response is a key factor determining the pathogenesis of cutaneous leishmaniasis. It is known that a Th1 cellular response is associated with infection control and that antigen-specific memory T cells are necessary for the development of a rapid and strong protective cellular response. The present manuscript reports the analysis of the functional and phenotypic profiles of antigen-specific CD4+ and CD8+ T cells from patients cured of cutaneous leishmaniasis (CL), patients with an active process of cutaneous leishmaniasis, asymptomatic individuals with a positive Montenegro test and healthy donors (HD). Peripheral blood mononuclear cells (PBMCs) from the patients exhibited a lymphoproliferative capacity after stimulation with total soluble protein from either Leishmania panamensis (SLpA) or Leishmania infantum (SLiA) or with a recombinant paraflagellar rod protein-1 (rPFR1). Higher frequencies of antigen-specific TNAIVE cells, mainly following stimulation with rPFR1, were observed in asymptomatic and cured patients than in patients with active cutaneous leishmaniasis, while T cells from patients with active cutaneous leishmaniasis showed a higher percentage of effector memory T cells (TEM for CD4+ T cells and TEMRA for CD8+ T cells). The amount of antigen-specific CD57+/CD8+ TEMRA cells in patients with active cutaneous leishmaniasis was higher than that in cured patients and asymptomatic subjects. Regarding functionality, a more robust multifunctional CD8+ T cell response was detected in cured patients than in those with active cutaneous leishmaniasis. Moreover, cured patients showed a significant increase in the frequency of cells expressing a Th1-type cytotoxic production profile (IFN-γ+/granzyme-B/+perforin+). Patients with an active leishmaniosis process had a significantly higher frequency of CD8+ T cells expressing the inhibitory CD160 and 2B4 receptors than did cured patients. The expression profile observed in cured patients could be indicative of an imbalance toward a CD8+ Th1 response, which could be associated with infection control; consequently, the determination of this profile could be a useful tool for facilitating the clinical follow-up of patients with cutaneous leishmaniasis. The results also suggest a possible exhaustion process of CD8+ T cells associated with the evolution of Leishmania infection.

Impact of benznidazole treatment on the functional response of Trypanosoma cruzi antigen-specific CD4+CD8+ T cells in chronic Chagas disease patients.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi. The persistence of the parasite is associated with the disease chronicity and the impairment of the cellular immune response. It has been reported that the CD4+CD8+ T cell population expands in chronic Chagas disease patients. Few studies have focused on this subset of cells, and very little is known about the impact of antiparasitic treatment on this population. METHODOLOGY: Thirty-eight chronic Chagas disease patients (20 asymptomatic and 18 symptomatic) and twelve healthy controls were enrolled in this study. Peripheral blood mononuclear cells were stimulated with soluble T. cruzi antigens to analyze the production of cytokines and cytotoxic molecules by CD4+CD8+ T cells before and after benznidazole treatment. Additionally, expression and co-expression of five inhibitory receptors in these patients after treatment were studied using a multiparameter flow cytometry technique. PRINCIPAL FINDINGS: The frequency of CD4+CD8+ T cells was higher in chronic Chagas disease patients compared with healthy donors. Furthermore, a higher ratio of CD4+CD8low/CD4+CD8high subpopulations was observed in chronic Chagas disease patients than in healthy donors. Additionally, CD4+CD8+ T cells from these patients expressed and co-expressed higher levels of inhibitory receptors in direct proportion to the severity of the pathology. Benznidazole treatment reduced the frequency of CD4+CD8+ T cells and decreased the ratio of CD4+CD8low/CD4+CD8high subpopulations. The co-expression level of the inhibitory receptor was reduced after treatment simultaneously with the enhancement of the multifunctional capacity of CD4+CD8+ T cells. After treatment, an increase in the frequency of T. cruzi antigen-specific CD4+CD8+ T cells expressing IL-2 and TNF-α was also observed. CONCLUSIONS: CD4+CD8+ T cells could play an important role in the control of T. cruzi infection since they were able to produce effector molecules for parasite control. Benznidazole treatment partially reversed the exhaustion process caused by T. cruzi infection in these cells with an improvement in the functional response of the T. cruzi antigen-specific CD4+CD8+ T cells.

Biology of Trypanosoma cruzi Retrotransposons: From an Enzymatic to a Structural Point of View.

INTRODUCTION: An important portion of the Trypanosoma cruzi genome is composed of mobile genetic elements, which are interspersed with genes on all chromosomes. The L1Tc non-LTR retrotransposon and its truncated version NARTc are the most highly represented and best studied of these elements. L1Tc is actively transcribed in all three forms of the Trypanosoma parasite and encodes the proteins that enable it to autonomously mobilize. This mini review discusses the enzymatic properties of L1Tc that enable its mobilization and possibly the mobilization of other non-autonomous retrotransposons in Trypanosoma. We also briefly review the Hepatitis Delta Virus-like autocatalytic and 2A self-cleaving viral-like sequences contained in L1Tc that regulate post-transcriptional properties such as relative protein abundance and mRNA stability. Special emphasis is placed on the Pr77 dual system, which is based on the RNA pol II-dependent internal promoter of L1Tc and NARTc and the HDV-like ribozyme activity encoded by the first 77 nucleotides of the element's DNA and RNA. The high degree of conservation of the Pr77 sequence, referred to as the "Pr77-hallmark", among different trypanosomatid retroelements suggests that these mobile elements are responsible for the distribution of regulatory sequences within the genome they inhabit. CONCLUSION: We also discuss how the involvement of L1Tc and NARTc in the gene regulatory processes of these parasites could justify their domestication and long-term coexistence in these ancient organisms.