Trypanosoma Cruzi Infection Research Articles

Expression patterns of Galectin-3 and NLRP3 in Chagas reactivation and graft damage in heart transplants.

This study aimed to assess the expression patterns of galectin-3 (Gal-3) and NLRP3 in heart transplant recipients according to the presence of reactivated Trypanosoma cruzi infection or allograft rejection in Chagas and non-Chagas heart transplant recipients. Gal-3 and NLRP3 expression levels were analyzed in endomyocardial biopsies from 31 heart transplant recipients, including 16 patients with chronic Chagas disease (ChD) and 15 without ChD. Samples were evaluated during periods of graft rejection or ChD reactivation, characterized by intense myocardial cellular infiltrate, and after remission of the infiltrate, classified by histopathological severity. The transcriptional levels of genes encoding Gal-3, NLRP3, Asc, caspase-1, and IL-1β were identified using the GEO2T tool across different experimental conditions. Gal-3 expression was lower in the myocardial infiltrate of ChD patients compared to non-ChD patients (p < 0.0001), whereas NLRP3 positivity was higher in ChD patients (p < 0.05). In a murine model of T. cruzi infection, elevated Gal-3 mRNA and NLRP3 inflammasome levels were observed in myocardial interstitial cells (p < 0.05). Peripheral blood mononuclear cells and cells from rodent cardiac allografts showed increased Gal-3 mRNA and NLRP3 levels compared to non-transplanted and rodent cardiac isografts (p < 0.001). Our findings suggest that Gal-3 and NLRP3 may be important biomarkers for differentiating heart transplant recipients with and without ChD regarding the myocardial immunological processes.

Zileuton, a 5-Lypoxigenase Inhibitor, is Antiparasitic and Prevents Inflammation in the Chronic Stage of Heart Chagas Disease.

Chronic Chagas cardiomyopathy is associated with an unbalanced immune response and impaired heart function, and available drugs do not prevent its development. Zileuton (Zi), a 5-lypoxigenase inhibitor, affects inflammatory/pro-resolution mediators. Herein, Zi treatment in the early phase of infection reduced parasitemia associated mainly with the direct effect of Zi on the parasite, and the enzyme epoxide hydrolase was the potential molecular target behind the trypanocidal effect. In the intermediate acute phase of infection, Zi reduced the number of innate and adaptive inflammatory cells, increased the level of SOCS2 expression in the heart associated with lower inflammation, and improved cardiac function. Zi treatment initiated in the chronic stage increased the level of SOCS2 expression in the heart, reduced inflammation, and improved cardiac function. Our data suggest that Zi protects against Trypanosoma cruzi infection by acting directly on the parasite and reducing heart damage and is a promising option for the treatment of Chagas disease.

Effects of fasting on the interplay between temperature and Trypanosoma cruzi infection on the life cycle of the Chagas disease vector Rhodnius prolixus.

Rhodnius prolixus, a triatomine insect, is one of the most important vectors of Chagas disease in South America. Its interaction with the parasite Trypanosoma cruzi, the causative agent of this disease, is known to be deeply affected by ambient temperature and the nutritional status of the insect vector. In this study, we investigated how starvation affects the life cycle of R. prolixus and the population dynamics of the parasite inside the intestine of the vector at four temperatures ranging from 24°C to 30°C. The weights and molting times of chronically infected and uninfected insects were monitored through repeated 30-day fasting periods from first instar to adult stage, assessing their capacity to retain blood meal weight between developmental stages and tracking parasite concentrations in their urine. Our results demonstrate that ambient temperature is a crucial factor affecting the resistance of R. prolixus to starvation, as survival, body weight, and weight retention greatly decreased in high temperature treatments. Furthermore, we showed that temperature significantly influenced whether T. cruzi established an infection in early instars, with few insects developing infections at the lowest and highest temperature treatments. Additionally, we discovered that a fasting period of 30 days induces a steady decrease in parasite populations in the vector over its lifetime. Infection by T. cruzi had no effect on the survival, molting time, and nutritional factors monitored in our protocol. Our results highlight the importance of nutrition as a determining factor for the development of the vector and the parasite, providing valuable insights for elucidating the complex interplay between temperature and nutrition in shaping the epidemiology of Chagas disease in a changing climate.

Changes in lipid abundance are associated with disease progression and treatment response in chronic Trypanosoma cruzi infection

BackgroundChagas disease, caused by the parasite Trypanosoma cruzi, is a zoonosis that affects more than seven million people. Current limitations on the diagnosis of the disease hinder the prognosis of patients and the evaluation of treatment efficacy, slowing the development of new therapeutic options. The infection is known to disrupt several host metabolic pathways, providing an opportunity for the identification of biomarkers.MethodsThe metabolomic and lipidomic profiles of a cohort of symptomatic and asymptomatic patients with T. cruzi infection and a group of uninfected controls were analysed using liquid chromatography/mass spectrometry. Differences among all groups and changes before and after receiving anti-parasitic treatment across those with T. cruzi infection were explored.ResultsThree lipids were found to differentiate between symptomatic and asymptomatic participants: 10-hydroxydecanoic acid and phosphatidylethanolamines PE(18:0/20:4) and PE(18:1/20:4). Additionally, sphinganine, 4-hydroxysphinganine, hexadecasphinganine, and other sphingolipids showed post-treatment abundance similar to that in non-infected controls.ConclusionsThese molecules hold promise as potentially useful biomarkers for monitoring disease progression and treatment response in patients with chronic T. cruzi infection.Graphical

Evaluation of the Vitros® Anti-T. cruzi Assay for its incorporation into the Trypanosoma cruzi Infection Diagnostic Algorithm

Background and objectiveSerological screening for Chagas disease (CD) in Latin American adults living in Europe is a cost-effective strategy for transmission prevention. The World Health Organization recommends two different serological tests including native and recombinant antigens for IgG detection. In Spain, most commercialized native tests require manual processing. We aimed to evaluate the sensitivity and specificity of the automated Vitros® Anti-T. cruzi native antigen-based test. MethodsA total of 556 serum samples were tested using two different tests: 1) our reference assay, a chemiluminescence immunoassay employing a recombinant multi-antigen protein (Liaison® XL Murex Chagas); 2) a chemiluminescence immunoassay with native antigen (Vitros® Anti-T. cruzi assay). Additionally, 180 samples were also processed by a manual indirect immunofluorescent assay (Chagas IFA). Sensibility, specificity and kappa index were calculated. ResultsVitros® showed a kappa index of 0.94 (IC 95%: 0.86-1.03) compared to Liaison® XL with a sensitivity of 93.6% and specificity of 99.5%. Compared to IFA, Vitros® showed a kappa index of 0.61 (IC 95%: 0.47-0.76), sensitivity of 97.5% and specificity of 70.37%. Discrepant results were obtained mainly in treated patients. ConclusionsThe Vitros® Anti-T. cruzi assay showed potential for implementation as an automated serological screening test, enhancing the diagnostic process in high-throughput microbiology laboratories.

Reverse remodelling and clinical outcomes in patients with chagas cardiomyopathy with reduced ejection fraction

Abstract Introduction Chagas disease affects around 8 million individuals globally and about 30% develop cardiac manifestations and, in time, heart failure with reduced ejection fraction (HFrEF). Chagas cardiomyopathy (CC) patients often have a poorer prognosis compared to other causes of HFrEF, owing to a higher rate of ventricular arrhythmias and thromboembolic conditions. Reverse remodelling (RR) has been linked to enhanced mortality and morbidity outcomes in HFrEF due to non-Chagas causes, but the effect of RR on CC prognosis is still unclear. Methods This retrospective study assessed 1043 patients diagnosed with CC via positive serology for Trypanosoma cruzi infection coupled with cardiopathy and ventricular dysfunction [left ventricular ejection fraction (LVEF) &amp;lt; 40%] during the follow-up period from January 2006 to September 2021. The primary outcome measured included overall mortality and heart transplantation in relation to RR status. Propensity score matching was utilised to adjust for confounding factors. Positive reverse remodelling (PRR) was characterised as patients displaying improved LVEF, and negative reverse remodelling (NRR) as those with persistent ventricular dysfunction during follow-up. Results Initially, 221 (21.2%) were categorised as PRR and 822 (78.8%) as NRR. Participants in the PRR group were predominantly female (51.6% versus 41.4%; p &amp;lt; 0.001), older [59 years (53 – 66) versus 56 years (47 – 64); p = 0.007], more prone to hypertension (43.4% versus 35.8%; p = 0.036), and presented with higher initial heart rates [70 bpm (60 – 80) versus 65 bpm (60 – 75); p = 0.002], systolic blood pressure [120 mmHg (110 – 130) versus 110 mmHg (100 – 120); p &amp;lt; 0.001], diastolic blood pressure [80 mmHg (69 – 82) versus 70 mmHg (60 – 80); p &amp;lt; 0.001], and LVEF [30.0% (26.0 – 35.0) versus 29% (25.0 – 34.0); p &amp;lt; 0.001] compared to the NRR group. Moreover, the PRR group had a lower intake of beta-blockers (BB) (76.6% versus 87.0%; p = 0.004), spironolactone (38.5% versus 59.5%; p &amp;lt; 0.001), and triple therapy (30.9% versus 50.4%; p &amp;lt; 0.001). Following propensity score matching, 200 patients remained for the final analysis, with 100 in each group. Cox proportional hazards model analysis revealed PRR as an independent predictor [hazard ratio (HR): 0.367; 95% confidence interval (CI) 0.218 – 0.616; p &amp;lt; 0.001] of mortality from all causes or heart transplantation. The PRR group exhibited a longer event-free survival duration than the NRR group (10.332 years; 95% CI 9.557 – 11.107 versus 7.348 years; 95% CI 6.606 – 8.089; p &amp;lt; 0.001). Discussion This study suggests a positive association between the PRR and increased survival in patients with CHD and HFrEF. Previously published studies didn’t demonstrate this association, possibly due to the small number of patients included. Conclusion PRR predicts a better long-term survival free from all-cause mortality and cardiac transplantation in Chagas cardiomyopathy with LVEF &amp;lt; 40%.Kaplan-Meier event-free survival curve

Effects of Free and Nanoencapsulated Benznidazole in Acute Trypanosoma cruzi Infection: Role of Cholinergic Pathway and Redox Status

Background/Objectives: The Trypanosoma cruzi infection promotes an intense inflammatory process that affects several tissues. The cholinergic system may exert a regulatory immune response and control the inflammatory process. This study aimed to evaluate the comparative effect of free and nanoencapsulated benznidazole in acute T. cruzi infection to assess hematological, biochemical, and oxidative status triggered by the cholinergic system. Methods: For this, fifty female Swiss mice were distributed in eight groups, i.e., uninfected and infected animals under four treatment protocols: untreated (control—CT); vehicle treatment (Eudragit L 100—EL-100); benznidazole treatment (BNZ); and nanoencapsulated benznidazole treatment (NBNZ). After eight treatment days, the animals were euthanized for sample collection. Results: The peak of parasitemia was at day 7 p.i., and the BNZ and NBNZ controlled and reduced the parasite rate but showed no efficacy in terms of total elimination of parasites analyzed by RT-PCR in both infected groups. The infection promotes significant anemia, leukopenia, and thrombocytopenia, which the BNZ improves. There was an increase in AChE activity during infection, leading to a pro-inflammatory response and an increase in M1 and M2 mACh receptors in the BNZ group, showing that the treatment interacted with the cholinergic pathway. In addition, a pro-oxidative response was characterized in the infection and mainly in the infected BNZ and NBNZ groups. The histopathological analysis showed significative splenomegaly and inflammatory infiltrate in the heart, liver, and spleen. Conclusions: The administration of the BNZ or NBNZ reverses hematological, hepatic, and renal alterations through cholinergic signaling and stimulates a pro-inflammatory response during acute T. cruzi infection.

Trypanosoma cruzi infection in American black bears (Ursus americanus): A case report in a cub from California and serologic survey for exposure in wild black bears from several states

Trypanosoma cruzi is an important cause of disease and death in humans and dogs, and although wildlife infections are common, less is known about disease manifestations. A 12-week-old male American black bear (Ursus americanus) cub with mild lethargy and anorexia presented to a wildlife rehabilitation center in Lake Tahoe, California. The cub continued to become increasingly weak and showed decreasing interest in play and other activities. The cub was anemic and had increased γ-glutamyltransferase (GGT) liver enzymes. A large number of trypanosomes were noted on a thin blood smear. Trypanosoma cruzi was isolated in culture from a subsequent blood collection. Proliferative bony lesions were noted on radiographs, but this finding was considered unrelated to the T. cruzi infection. The number of parasites observed in thin blood smears dramatically dropped over time, but it remained PCR positive until at least nine months. The cub continued to gain weight and became increasingly active. Serum samples from the cub were positive with three different serologic assays (IFA, ELISA, and ICT). The bear was not treated because of the decreasing parasitemia and the improvement in activity and appetite. Although the bear could not be released due to issues unrelated to T. cruzi, it remains healthy in a captive facility. Sequence analysis of the DHFR-TS and COII-ND1 gene sequences confirmed the bear was infected with DTC TcIV. Following the detection of this clinical case, a serologic survey was conducted to determine the prevalence of T. cruzi exposure of black bears in California, North Carolina, and Pennsylvania. Because no serologic assay has been validated for use in bears, three different assays were used. Marked differences in apparent seroprevalence range from 1% (requiring all three assays to be positive) to ∼20.7% (requiring only one assay to be positive). Black bears are naturally exposed to T. cruzi across the United States. Future studies using PCR testing of tissues or blood would be needed to better understand the prevalence of T. cruzi in wild black bears, lineages most commonly associated with infection, and if T. cruzi represents a health threat to bears.

Copaifera spp. oleoresins control Trypanosoma cruzi infection in human trophoblast cells (BeWo) and placental explants

Congenital Chagas disease (CCD) is a worldwide neglected problem with significant treatment limitations. This study aimed to evaluate the potential of Copaifera spp. oleoresins (ORs) against Trypanosoma cruzi infection in trophoblast cells (BeWo lineage) and human chorionic villous explants (HCVE). The cytotoxicity of ORs was investigated using LDH and MTT assays. T. cruzi (Y strain) proliferation, invasion and reversibility were assessed in OR-treated BeWo cells, and proliferation was evaluated in OR-treated HCVE. The ultrastructure of T. cruzi trypomastigotes and amastigotes treated with ORs were analyzed by scanning and transmission electronic microscopy. ROS production in infected and treated BeWo cells and cytokines in BeWo and HCVE were measured. The ORs irreversibly decreased T. cruzi invasion, proliferation and release in BeWo cells by up to 70 %, 82 % and 80 %, respectively, and reduced parasite load in HCVE by up to 80 %. Significant structural changes in treated parasites were observed. ORs showed antioxidant capacity in BeWo cells, reducing ROS production induced by T. cruzi infection. Also, T. cruzi infection modulated the cytokine profile in both BeWo cells and HCVE; however, treatment with ORs upregulated cytokines decreased by T. cruzi infection in BeWo cells, while downregulated cytokines increased by the T. cruzi infection in HCVE. In conclusion, non-cytotoxic concentrations of Copaifera ORs demonstrated promising potential for controlling T. cruzi infection in models of the human maternal-fetal interface.

Interlaboratory Harmonization Study and Prospective Evaluation of the PURE–Trypanosoma cruzi–Loop-Mediated Isothermal Amplification Assay for Detecting Parasite DNA in Newborn's Dried Blood Spots

Timely diagnosis of vertical Trypanosoma cruzi infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi-infected mothers are required to undergo serological testing after nine months, which risks loss to follow-up. Alternatively, the Loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA offers high analytical and clinical performance and easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultra-rapid DNA extraction method combined with the LAMP in dried blood spots (DBS) on FTA cards. The procedure has been implemented in ten public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruzi at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBS from 222 newborns at birth and/or two months, detecting T. cruzi microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with Real Time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.

Electrocardiographic abnormalities are associated with seropositive Trypanosoma cruzi infection status using a simplified cardiac diagnostic evaluation in dogs.

To describe associations between cardiac abnormalities and Trypanosoma cruzi serostatus by use of a simplified diagnostic evaluation in dogs at risk for T cruzi infection. A prospective, cross-sectional study was performed using a simplified diagnostic evaluation including high-sensitivity cardiac troponin I, 30-second ECG, and echocardiogram with 7 variables in 46 client-owned dogs from high-risk environments. Dogs were categorized as serologically positive (SP), negative (SN), or discordant (SD) by use of 2 antibody tests. Functional evaluation of cardiac health scores and blood PCR were obtained. Dogs were SP (n = 19), SN (17), and SD (10), with 9 PCR positive (7 SP, 1 SN, 1 SD). Troponin was above reference range in 6 of 46 (4 SP, 1 SN, 1 SD), and functional evaluation of cardiac health scores were 0 in all dogs. Conduction system abnormalities (prolonged interval durations, second-degree atrioventricular block, splintered QRS complex) and ventricular arrhythmias were documented in 8 (7 SP, 0 SN, 1 SD). Twenty-six (12 SP, 8 SN, 6 SD) had echocardiographic abnormalities, most often myxomatous mitral valve disease (MMVD) and left ventricular enlargement. Seropositive dogs were significantly older and had a higher likelihood of MMVD. Conduction system abnormalities were associated with positive serostatus. Echocardiographic abnormalities were complicated by MMVD and did not distinguish between serostatus. An ECG with assessment and detailed measurement of complexes and cardiac troponin I are simple tests to perform with abnormalities detected in seroreactive dogs. Electrocardiographic abnormalities in high-risk or seroreactive dogs should prompt further evaluation and monitoring of T cruzi infection.

Testosterone leads to Trypanosoma cruzi glycoprotein synthesis and increased of inflammatory mediators in bone marrow-derived macrophages

Despite all the scientific progress in recent decades to unravel the immune processes and the way the parasite bypasses the immune system, Chagas disease is still a major public health problem, affecting an estimated 3.5 million people. Among the components that may participate in the response against the parasite, testosterone has been gaining more and more visibility. Studies indicate that the parasite itself seems to carry out steroidogenesis, in which, in co-culture with androgen precursors, T. cruzi has been shown to produce TS, but the purpose of the TS synthesized by the parasite and how this can influence its invasion glycoproteins is still unclear unknown. The aim of this study was to evaluate the influence of testosterone in Trypanosoma cruzi infection on the immune response of bone marrow-derived macrophages. Bone marrow from male rats was extracted and cultured with RMPI medium containing 30% L929 cell supernatant for macrophage differentiation. The cells were incubated for 10 days and, after this period, they were seeded in 96 wells in the amount of 1 x 105 cells per well. TS was added at different concentrations of 20 μM, 10 μM, 5 μM and 1 μM and then infected with the Y strain of T. cruzi, at a rate of 10 parasites per cell, with the culture remaining for six, 12 and 24 h. The supernatant was collected and the production of nitric oxide (NO), tumor necrosis factor (TNF) and the number of cell parasites was assessed by staining with 4′-6′-diamino-2-phenylindole (DAPI) and ranked by high Content Screening (HSC). The parasite was then cultured with the addition of TS, at the mentioned concentrations, leaving it for six and 12 h and then performing the RT-PCR of the mucins. DAPI staining revealed a significant increase in the number of parasites in cells containing TS. The exception was observed when 1 μM of hormone/well was used. A reduction in TNF production was found with 20 and 10 μM of TS for 6 h stimulation, although increased levels were observed with 5 and 1 μM, similar to the infected control. However, there was an increase in TNF production and not after 12 h. The relative expression of parasite glycoprotein 82 was increased with the presence of TS in the medium, regardless of time. Our data suggest that TS may contribute to cellular immunosuppression, increasing parasite infection in the cell, as well as inflammatory mediators that lead to cell and tissue damage in infected individuals, as well as the possible use of TS to allow their invasion into the cell hosts.

Efficacy of a Zn-based metalorganic framework doped with benznidazole on acute experimental Trypanosoma cruzi infection.

Metal-Organic Frameworks (MOFs) have been shown to enhance the activity of encapsulated compounds by facilitating their passage across cell membranes, thereby enabling controlled and selective release. This study investigates the efficacy of BNZ@Zn-MOFs against the acute phase of Trypanosoma cruzi infection in a mouse model. The particles were synthesized by electroelution (EL), doped with BZN via mechanochemistry, and characterized using scanning electron microscopy (SEM), infrared spectroscopy (FTIR), and X-ray diffraction (XRD). BNZ@Zn-MOFs released 80% of the encapsulated BZN within 3h, demonstrating no cytotoxicity in NIH-3T3 and HeLa cells. Furthermore, in a model of acute experimental T. cruzi-infection in BALB/c mice, the delivery system exhibited antiparasitic activity at a significantly lower BZN concentration compared to free BZN treatment. PCR analysis of treated mice revealed no parasite DNA in their tissues, and hematoxylin-eosin staining showed no apparent damage to tissue architecture. Additionally, serum levels of liver function enzymes remained unchanged, indicating no adverse effects on liver function. This delivery system, utilizing suboptimal BZN doses, enables the preservation of drug activity while potentially facilitating a substantial decrease in side effects associated with Chagas disease treatment.

Trypanosoma cruzi Infection in Pregnancies without Congenital Transmission Is Associated with Reduced Fetal Growth: A Cross-Sectional Study in Argentina, Honduras, and Mexico.

We aimed to measure the association between Trypanosoma cruzi infection in pregnancy and reduced fetal growth in the absence of T. cruzi congenital transmission. We conducted a cross-sectional study of secondary data of all singleton live births between 2011 and 2013 in five hospitals from Argentina, Honduras, and Mexico. We excluded newborns with T. cruzi infection. Noninfected pregnant people were those without any positive rapid tests. The main study outcomes were birth weight, head circumference, and length for gestational age and sex. Logistic regression models were adjusted for country, age, education level, and obstetric history. Of the 26,544 deliveries, 459 (1.7%) pregnant people were found by rapid tests to be positive for T. cruzi. Of these, 320 were positive by enzyme-linked immunosorbent assay and 231 had a positive polymerase chain reaction (PCR) test. Uninfected newborns from T. cruzi-infected pregnant people were more likely to have birth weights below the 5th and 10th percentiles and head circumferences below the 3rd and 10th percentiles. Among T. cruzi-infected pregnant people diagnosed by PCR, the odds ratios were 1.58 for birth weight below the 10th percentile (95% CI, 1.12-2.23) and 1.57 for birth weight below the 5th percentile (95% CI, 1.02-2.42). Higher T. cruzi parasitic loads in pregnancy had a stronger association with reduced fetal growth (both in birth weight and head circumference), with an odds ratio of 2.31 (95% CI, 1.36-3.91) for a birth weight below the 5th percentile. The association shows, irrespective of causality, that newborns of pregnancies with T. cruzi have an increased risk of reduced fetal growth. We recommend further studies to assess other potential confounders and the causality of these associations.

Dynamics of Trypanosoma cruzi infection in hamsters and novel association with progressive motor dysfunction.

Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.

Protective Efficacy of the Epitope-Conjugated Antigen N-Tc52/TSkb20 in Mitigating Trypanosoma cruzi Infection through CD8+ T-Cells and IFNγ Responses.

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.

Enteric nervous system regeneration and functional cure of experimental digestive Chagas disease with trypanocidal chemotherapy

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a female C3H/HeN mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology and the impact of treatment with the front-line, anti-parasitic drug benznidazole. Curative treatment 6 weeks post-infection resulted in sustained recovery of gastrointestinal transit function, whereas treatment failure led to infection relapse and gradual return of DCD symptoms. Neuro/immune gene expression patterns shifted from chronic inflammation to a tissue repair profile after cure, accompanied by increased cellular proliferation, glial cell marker expression and recovery of neuronal density in the myenteric plexus. Delaying treatment until 24 weeks post-infection led to partial reversal of DCD, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk of irreversible enteric neuromuscular tissue damage and dysfunction developing highlights the importance of prompt diagnosis and treatment. These findings support the concept of treating asymptomatic, T. cruzi-infected individuals with benznidazole to prevent DCD development.

Preclinical evaluation of combined therapy with amiodarone and low-dose benznidazole in a mouse model of chronic Trypanosoma cruzi infection

Chagasic chronic cardiomyopathy (CCC) is the primary clinical manifestation of Chagas disease (CD), caused by Trypanosoma cruzi. Current therapeutic options for CD are limited to benznidazole (Bz) and nifurtimox. Amiodarone (AMD) has emerged as most effective drug for treating the arrhythmic form of CCC. To address the effects of Bz and AMD we used a preclinical model of CCC. Female C57BL/6 mice were infected with T. cruzi and subjected to oral treatment for 30 consecutive days, either as monotherapy or in combination. AMD in monotherapy decreased the prolonged QTc interval, the incidence of atrioventricular conduction disorders and cardiac hypertrophy. However, AMD monotherapy did not impact parasitemia, parasite load, TNF concentration and production of reactive oxygen species (ROS) in cardiac tissue. Alike Bz therapy, the combination of Bz and AMD (Bz/AMD), improved cardiac electric abnormalities detected T. cruzi-infected mice such as decrease in heart rates, enlargement of PR and QTc intervals and increased incidence of atrioventricular block and sinus arrhythmia. Further, Bz/AMD therapy ameliorated the ventricular function and reduced parasite burden in the cardiac tissue and parasitemia to a degree comparable to Bz monotherapy. Importantly, Bz/AMD treatment efficiently reduced TNF concentration in the cardiac tissue and plasma and had beneficial effects on immunological abnormalities. Moreover, in the cardiac tissue Bz/AMD therapy reduced fibronectin and collagen deposition, mitochondrial damage and production of ROS, and improved sarcomeric and gap junction integrity. Our study underlines the potential of the Bz/AMD therapy, as we have shown that combination increased efficacy in the treatment of CCC.

CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection.

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model, we found that Treg cells play a role during the initial stages after T. cruzi infection, restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation, exhaustion and functional markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model.

Systemic inflammatory Th1 cytokines during Trypanosoma cruzi infection disrupt the typical anatomical cell distribution and phenotypic/functional characteristics of various cell subsets within the thymus

The thymus plays a crucial role in T cell differentiation, a complex process influenced by various factors such as antigens, the microenvironment and thymic architecture. The way the thymus resolves infections is critical, as chronic persistence of microbes or inflammatory mediators can obstruct the differentiation.Here, we illustrate that following inflammatory T helper 1 infectious processes like those caused by Candida albicans or Trypanosoma cruzi, single positive thymocytes adopt a mature phenotype. Further investigations focused on T. cruzi infection, reveal a substantial existence of CD44+ cells in both the cortical and medullary areas of the thymus at the onset of infection. This disturbance coincides with heightened interferon gamma (IFNγ) production by thymocytes and an increased cytotoxic capacity against T. cruzi-infected macrophages. Additionally, we observe a reduced exportation capacity in T. cruzi-infected mice. Some alterations can be reversed in IFNγ knockout mice (KO). Notably, the majority of these effects can be replicated by systemic expression of interleukin (IL)-12+IL-18, underlining the predominantly inflammatory rather than pathogen-specific nature of these phenomena.Understanding the mechanisms through which systemic inflammation disrupts normal T cell development, as well as subsequent T cell exportation to secondary lymphoid organs (SLO) is pivotal for comprehending susceptibility to diseases in different pathological scenarios.