False positive serology of prepandemic chagasic samples with SARS-CoV-2 antigen.

OBJECTIVE: To determine whether prepandemic sera from patients with Chagas disease recognise SARS-CoV-2 antigens. MATERIALS AND METHODS: Forty sera from patients with Chagas disease were tested for the presence of IgG cross-reactivity against the nucleocapsid protein (NP) and spike (S) SARS-CoV-2 proteins by ELISA. Positive samples were tested again using a different ELISA and CLIA, both against NP. RESULTS: None of the sera from patients with Chagas disease, previously confirmed as positive for the presence of anti-Trypanosoma cruzi antibodies reacted against the SARS-CoV-2 S protein, and six samples tested positive for the NP antigen (15%). The six positive samples were re-tested, five remained positive by ELISA and all were negative by CLIA. CONCLUSION: According to our data, false-positive results might be a concern in the detection of SARS-CoV-2 antibodies in patients with Chagas disease.

A terpenoid-rich extract from Clethra fimbriata exhibits anti-Trypanosoma cru zi activity and induces T cell cytokine production.

Chagas disease, a worldwide public health concern, is a chronic infection caused by Trypanosoma cruzi. Considering T. cruzi chronic persistence correlates with CD4+ and CD8+ T cell dysfunction and the safety and efficacy profiles of Benznidazol and Nifurtimox, the two drugs currently used for its etiological treatment, are far from ideal, the search of new trypanocidal treatment options is a highly relevant issue. Therefore, the objective of this work was to evaluate the trypanocidal effect and cytokine production induction of three extracts (hexane, dichloromethane and hydroalcoholic) obtained from Clethra fimbriata, a plant traditionally used as a febrifuge in Colombia. Additionally, the extracts' major components with the highest trypanocidal activity were determined. It was evidenced C. fimbriata hexane extract exhibited the highest activity capable of inhibiting the three parasite developmental stages with an IC50/EC50 of 153.9 ± 29.5 (epimastigotes), 39.3 ± 7.2 (trypomastigotes), and 45.6 ± 10.5 (amastigotes) µg/mL, presenting a low cytotoxicity in VERO cells with a selectivity index ranging from 6.49 to 25.4. Moreover, this extract induced trypomastigote apoptotic death and inhibited parasite cell infection. The extract also induced IFN-γ and TNF production in CD4+ and CD8+ T cells, as well as de novo production of the cytotoxic molecules granzyme B and perforin in CD8+ T cells from healthy donors. Fatty acids and terpenes represented C. fimbriata key compounds. Thus, the trypanocidal activity and cytokine production induction of the hexane extract may be associated with terpene presence, particularly, triterpenes.

Trypanosoma cruzi-specific CD8+ T cells and other immunological hallmarks in chronic Chagas cardiomyopathy: Two decades of research.

Trypanosoma cruzi, the causal agent of Chagas disease, has coexisted with humans for thousands of years. Therefore, the parasite has developed several mechanisms of antigenic variability that has allowed it to live inside the cells and evade the host immune response. Since T. cruzi displays an intracellular cycle-stage, our research team focused on providing insights into the CD8+ T cells immune response in chronic Chagas cardiomyopathy. We began our work in the 2000s studying parasite antigens that induce natural immune responses such as the KMP11 protein and TcTLE, its N-terminal derived peptide. Different approaches allowed us to reveal TcTLE peptide as a promiscuous CD8+ T cell epitope, able of inducing multifunctional cellular immune responses and eliciting a humoral response capable of decreasing parasite movement and infective capacity. Next, we demonstrated that as the disease progresses, total CD8+ T cells display a dysfunctional state characterized by a prolonged hyper-activation state along with an increase of inhibitory receptors (2B4, CD160, PD-1, TIM-3, CTLA-4) expression, an increase of specific terminal effector T cells (TTE), a decrease of proliferative capacity, a decrease of stem cell memory (TSCM) frequency, and a decrease of CD28 and CD3ζ expression. Thus, parasite-specific CD8+ T cells undergo clonal exhaustion, distinguished by an increase in late-differentiated cells, a mono-functional response, and enhanced expression of inhibitory receptors. Finally, it was found that anti-parasitic treatment induces an improved CD8+ T cell response in asymptomatic individuals, and a mouse animal model led us to establish a correlation between the quality of the CD8+ T cell responses and the outcome of chronic infection. In the future, using OMICs strategies, the identification of the specific cellular signals involved in disease progression will provide an invaluable resource for discovering new biomarkers of progression or new vaccine and immunotherapy strategies. Also, the inclusion of the TcTLE peptide in the rational design of epitope-based vaccines, the development of immunotherapy strategies using TSCM or the blocking of inhibitory receptors, and the use of the CD8+ T cell response quality to follow treatments, immunotherapies or vaccines, all are alternatives than could be explored in the fight against 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.

Seroprevalence and seroconversion rates to SARS-CoV-2 in interns, residents, and medical doctors in a University Hospital in Bogotá, Colombia / Tasas de seroprevalencia y seroconversión al SARS-CoV-2 en internos, residentes y médicos en un Hospital Universitario de Bogotá, Colombia

Abstract Objectives: To determine the prevalence of antibodies to SARS-CoV-2 and the incidence of seroconversion in the first month of follow-up among interns, residents, and medical doctors attending patients at a University Hospital in Bogota (Colombia). Design or methods: A cross-sectional and a prospective study were performed during June, July, and August 2020 to assess seroprevalence and seroconversion rates using CLIA IgG for SARS-CoV-2. LFA IgG and IgM and ELFA IgM were also determined to explore concordance with CLIA IgG. Results: At baseline, 8 (2.28% 95%CI 1.16-4.43%) participants were IgG positive for SARS-CoV-2 by CLIA. At the end of the study, 21 (5.98% 95%CI 3.94-8.97%) individuals seroconverted by CLIA IgG. In all, 29 individuals had IgG by CLIA and of these 11 (3.13% 95%CI 1.76-5.52%) were asymptomatic. No associations with risk factors for infection were identified. CLIA IgG had moderate concordance (>962 samples) with LFA IgG and ELFA IgM, but minimal with LFA IgM. Conclusions: Our report is the first in Latina America on seroprevalence and seroconversion rates in medical healthcare workers. The relatively high rate (>3%) of asymptomatic health care workers with evidence of previous SARS-CoV-2 infection underscores the need to screen this population for infection to prevent infection/disease spread.

Resumen Objetivos: Determinar la prevalencia de anticuerpos frente al SARS-CoV-2 y la incidencia de seroconversión en el primer mes de seguimiento en internos, residentes y médicos que atienden pacientes en un Hospital Universitario de Bogotá (Colombia). Diseño y métodos: Se realizó un estudio transversal y prospectivo durante junio, julio y agosto de 2020 para evaluar las tasas de seroprevalencia y seroconversión utilizando CLIA IgG para SARS-CoV-2. También se determinaron LFA IgG e IgM y ELFA IgM para explorar la concordancia con CLIA IgG. Resultados: Al inicio del estudio, 8 (2,28% IC del 95% 1,16-4,43%) participantes fueron IgG positivos para SARS-CoV-2 por CLIA. Al final del estudio, 21 (5,98% IC 95% 3,94-8,97%) individuos seroconvirtieron por CLIA IgG. En total, 29 individuos tenían IgG por CLIA y de estos 11 (3,13% 95% IC 1,76-5,52%) eran asintomáticos. No se identificaron asociaciones con factores de riesgo de infección. El CLIA IgG tuvo una concordancia moderada (> 962 muestras) con LFA IgG y ELFA IgM, pero mínima con el LFA IgM. Conclusiones: Nuestro informe es el primero en América Latina sobre tasas de seroprevalencia y seroconversión en trabajadores médicos de la salud. La tasa relativamente alta (> 3%) de trabajadores de la salud asintomáticos con evidencia de infección previa por SARS-CoV-2 resalta la necesidad de realizar pruebas de detección de infección en esta población para prevenir la propagación de la infección.

Preliminary chemical characterization of ethanolic extracts from Colombian plants with promising anti - Trypanosoma cruzi activity.

Chagas disease is caused by Trypanosoma cruzi, and it is an important cause of morbidity and mortality in Latin America. There are no vaccines, and the chemotherapy available to treat this infection has serious side effects. In a search for alternative treatments, we determined the in vitro susceptibility of epimastigote and trypomastigote forms of T. cruzi and the cytotoxic effects on peripheral blood mononuclear cells (PBMCs) of ethanolic extracts obtained from six different plant species. The ethanolic extracts of Ageratina vacciniaefolia, Clethra fimbriata and Siparuna sessiliflora showed antiprotozoal activity against epimastigotes and low cytotoxicity in mammalian cells. However, only the ethanolic extract of C. fimbriata showed activity against T. cruzi trypomastigotes, and it had low cytotoxicity in PBMCs. An analysis on the phytochemical composition of C. fimbriata extract showed that its metabolites were primarily represented by two families of compounds: flavonoids and terpenoids. Lastly, we analyzed whether the A. vacciniaefolia, C. fimbriata, or S. sessiliflora ethanolic extracts induced IFN-γ or TNF-α production. Significantly, ethanolic extracts of C. fimbriata induced TNF-α production and S. sessiliflora induced both cytokines. In addition, C. fimbriata and S. sessiliflora induced the simultaneous secretion of IFN-γ and TNF-α in CD8+ T cells. The antiprotozoal and immunomodulatory activity of C. fimbriata may be related to the presence of flavonoid and triterpene compounds in the extract. Thus, these findings suggest that C. fimbriata may represent a valuable source of new bioactive compounds for the therapeutic treatment of Chagas disease that combines trypanocidal activity with the capacity to boost the immune response.

Diminished mitogen-induced T cell proliferation by Trypanosoma cruzi antigens associated with antigen-presenting cell modulation and CD3 signaling.

Chronic infection by Trypanosoma cruzi decreases T cell proliferation and it is most likely accompanied by changes in signals required for activation. We assessed the effect of T. cruzi antigens on mitogen-induced proliferation of T cells from uninfected individuals and the association with the expression of molecules involved in antigen presentation, T cell costimulation and activation, and cytokine production. T. cruzi antigen exposure reduced mitogen-induced proliferation of CD4+ and CD8+ T cells in PBMC cultures, but only reduced mitogen-induced proliferation in the CD4+ T cells from sorted cell cultures cocultured with antigen-pulsed CD3- cells. CD40/CD80 and CD86 expression were reduced in antigen-pulsed DCs and monocytes, respectively. TNF-α, IL-10 and CCL17 levels were increased in cultures with antigen-pulsed CD3- cells, while CD3ζ chain expression was reduced in T cells from cultures with antigen. Our findings suggest that T. cruzi could alter T cell proliferation indirectly by downregulating costimulatory molecules and inducing the secretion of IL-10 and directly by decreasing TCR signaling.

Intermediate Monocytes and Cytokine Production Associated With Severe Forms of Chagas Disease.

Monocytes are classified according to their CD14 and CD16 expression into classical (reparative), intermediate (inflammatory), and non-classical. This study assessed the frequency of monocyte and the relationship between monocyte subset percentages and the levels of blood cytokines in Colombian chagasic patients with different clinical forms. This study included chagasic patients in different clinical stages: indeterminate (IND) n = 14, chronic chagasic cardiomyopathy (CCC) n = 14, and heart transplant chagasic (HTCC) n = 9; controls with non-chagasic cardiopathy (NCC) n = 15, and healthy individuals (HI) n = 15. Peripheral blood mononuclear cells (PBMCs) were isolated, labeled for CD14, CD16, and HLA-DR, and analyzed by flow cytometry. Cytokines were measured with a bead-based immunoassay. Percentages of total CD14+ CD16+ and CD14+ HLA-DR+ monocytes were higher in patients with heart involvement (CCC, HTCC, and NCC) than controls. Percentages of intermediate monocytes increased in symptomatic chagasic patients (CCC and HTCC) compared to asymptomatic chagasic patients (IND) and controls (HI). Asymptomatic chagasic patients (IND) had higher percentages of classical monocytes, an increased production of CCL17 chemokine compared to chagasic symptomatic patients (CCC), and their levels of CCL17 was positively correlated with the percentage of classical monocyte subset. In CCC, the percentages of intermediate and classical monocytes were positively correlated with IL-6 levels, which were higher in this group compared to HI, and negatively with IL-12p40 concentration, respectively. Remarkably, there also was an important increased of classical monocytes frequency in three chronic chagasic patients who underwent cardiac transplant, of which one received anti-parasitic treatment. Our findings suggest that cardiac chagasic patients have an increased percentage of inflammatory monocytes and produce more IL-6, a biomarker of heart failure and left ventricular dysfunction, whereas asymptomatic chagasic individuals present a higher percentage of reparative monocytes and CCL17.

An Animal Model of Acute and Chronic Chagas Disease With the Reticulotropic Y Strain of Trypanosoma cruzi That Depicts the Multifunctionality and Dysfunctionality of T Cells.

Chagas disease (ChD), a complex and persistent parasitosis caused by Trypanosoma cruzi, represents a natural model of chronic infection, in which some people exhibit cardiac or digestive complications that can result in death 20-40 years after the initial infection. Nonetheless, due to unknown mechanisms, some T. cruzi-infected individuals remain asymptomatic throughout their lives. Actually, no vaccine is available to prevent ChD, and treatments for chronic ChD patients are controversial. Chronically T. cruzi-infected individuals exhibit a deterioration of T cell function, an exhaustion state characterized by poor cytokine production and increased inhibitory receptor co-expression, suggesting that these changes are potentially related to ChD progression. Moreover, an effective anti-parasitic treatment appears to reverse this state and improve the T cell response. Taking into account these findings, the functionality state of T cells might provide a potential correlate of protection to detect individuals who will or will not develop the severe forms of ChD. Consequently, we investigated the T cell response, analyzed by flow cytometry with two multicolor immunofluorescence panels, to assess cytokines/cytotoxic molecules and the expression of inhibitory receptors, in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi-infected mice. Acute ChD induced a high antigen-specific multifunctional T cell response by producing IFN-γ, TNF-α, IL-2, granzyme B, and perforin; and a high frequency of T cells co-expressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional antigen-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) co-expressed on T cells. Taken together, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression. Thus, these results constitute a model that will facilitate an in-depth search for immune markers and correlates of protection, as well as long-term studies of new immunotherapy strategies for ChD.

CD8+ T Cell Response to Trypanosoma cruzi Antigens during Chronic Chagas Disease.

Flow cytometry is a valuable technique in cellular immunology that allows evaluating effective parameters of the immune response associated with CD8+ T cells. During Chagas disease, infection caused by Trypanosoma cruzi parasite, similar to other intracellular infectious agents, antigen-specific CD8+ T cells are essential for controlling the infection. However, CD8+ T cell response is only partially effective in some chronic Chagas disease patients. Thus, characterization and phenotyping of T. cruzi-specific CD8+ T cells are of great importance during chronic Chagas disease.

Trypanosoma cruzi Detection in Colombian Patients with a Diagnosis of Esophageal Achalasia.

Achalasia is a motility disorder of the esophagus that might be secondary to a chronic Trypanosoma cruzi infection. Several studies have investigated esophageal achalasia in patients with Chagas disease (CD) in Latin America, but no related studies have been performed in Colombia. The goals of the present study were to determine the presence of anti-T. cruzi antibodies in patients with esophageal achalasia who visited a referral hospital in Bogotá, Colombia, and to detect the presence of the parasite and its discrete typing units (DTUs). This cross-sectional study was conducted in adult patients (18-65 years old) who were previously diagnosed with esophageal achalasia and from whom blood was drawn to assess antibodies against T. cruzi using four different serological tests. Trypanosoma cruzi DNA was detected by conventional polymerase chain reaction (cPCR) and quantitative polymerase chain reaction (qPCR). In total, 38 patients, with an average age of 46.6 years (standard deviation of ±16.2) and comprising 16 men and 22 women, were enrolled. Five (13.15%) patients were found to be positive for anti-T. cruzi antibodies by indirect immunofluorescence assay (IFA), and two patients who were negative according to IFA were reactive by both enzyme-linked immunosorbent assay and immunoblot (5.3%). Parasite DNA was detected in two of these seven patients by cPCR and in one of these by qPCR. The parasite DTU obtained was TcI. In summary, this study identified T. cruzi in Colombian patients with esophageal achalasia, indicating that digestive compromise could also be present in patients with chronic CD.

T cells responding to Trypanosoma cruzi detected by membrane TNF-α and CD154 in chagasic patients.

INTRODUCTION: Chagas disease is a parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. Variability in cytokine secretion among chronic Trypanosoma cruzi-infected patients might preclude the identification of the pool of antigen specific T cells. The goal of this study was to determine the fraction of T cells responding to T. cruzi antigen measured by the expression of membrane TNF-α and CD154. METHODS: A total of 21 chagasic patients, 11 healthy and 5 non-chagasic cardiomyopathy controls were analyzed. PBMCs were short-term cultured in the presence of anti-CD28, anti-CD49d, anti-TNF-α, and TACE (TNF-α converting enzyme) inhibitor either under T. cruzi-lysate or polyclonal stimuli. Cells were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD154, and analyzed with flow cytometry. RESULTS: CD4+ and CD8+ T cells in chagasic patients displayed higher percentages of membrane-bound TNF-α+ and CD154+ compared with controls after T. cruzi-antigen stimulation. Both markers displayed a positive correlation in the T cell subpopulations analyzed. Symptomatic chagasic patients were differentiated from asymptomatic patients based on the expression of CD154 and membrane TNF-α in TCD4+ and TCD8+ compartments, respectively. CONCLUSIONS: These results show that both markers could be useful for assessing the pool of antigen-specific T cells in chronic chagasic patients.

Antiparasitic Treatment Induces an Improved CD8+ T Cell Response in Chronic Chagasic Patients.

Chagas disease is a chronic infection caused by Trypanosoma cruzi, an intracellular protozoan parasite. Chronic chagasic patients (CCPs) have dysfunctional CD8+ T cells that are characterized by impaired cytokine production, high coexpression of inhibitory receptors, and advanced cellular differentiation. Most patients diagnosed in the chronic phase of Chagas disease already exhibit heart involvement, and there is no vaccination that protects against the disease. Antiparasitic treatment is controversial as to its indication for this stage of the disease. There is a lack of biological markers to evaluate the effectiveness of antiparasitic treatment, and little is known about the effect of the treatment on CD8+ T cells. Thus, the aim of the current study was to analyze the early effects of antiparasitic treatment on CD8+ T cells from CCPs with asymptomatic clinical forms of disease. To evaluate the CD8+ T cell subsets, expression of inhibitory receptors, and functionality of T cells in CCPs, PBMCs were isolated. The results showed that treatment of CCPs with the asymptomatic form of the disease induces an increase in the frequency of CD8+ central memory T cells and terminal effector T cells, a decrease in the coexpression of inhibitory receptors, an improved Ag-specific CD8+ T cell response exhibited by the individual production of IFN-γ or IL-2, and a multifunctional CD8+ T cell profile of up to four functions (IFN-γ+IL-2+Perforin+Granzyme B+). These findings suggest that, in CCPs, antiparasitic treatment improved the quality of Ag-specific CD8+ T cell responses associated with a decrease in inhibitory receptor coexpression, which could serve as biomarkers for monitoring the effectiveness of antiparasitic treatment.

Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients.

BACKGROUND: TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. METHODOLOGY/PRINCIPAL FINDINGS: From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07-0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. CONCLUSION/SIGNIFICANCE: These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy.

Altering the motility of Trypanosoma cruzi with rabbit polyclonal anti-peptide antibodies reduces infection to susceptible mammalian cells.

Trypanosoma cruzi's trypomastigotes are highly active and their incessant motility seems to be important for mammalian host cell infection. The kinetoplastid membrane protein-11 (KMP-11) is a protein expressed in all parasite stages, which induces a cellular and humoral immune response in the infected host, and is hypothesized to participate in the parasite's motility. An N-terminal peptide from KMP-11, termed K1 or TcTLE, induced polyclonal antibodies that inhibit parasitic invasion of Vero cells. The goal of this study was to evaluate the motility and infectivity of T. cruzi when exposed to polyclonal anti-TcTLE antibodies. Rabbits were immunized with TcTLE peptide along with FIS peptide as an immunomodulator. ELISA assay results showed that post-immunization sera contained high titers of polyclonal anti-TcTLE antibodies, which were also reactive against the native KMP-11 protein and live parasites as detected by immunofluorescence and flow cytometry assays. Trypomastigotes of T. cruzi were incubated with pre- or post-immunization sera, and infectivity to human astrocytes was assessed by Giemsa staining/light microscope and flow cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled parasites. T. cruzi infection in astrocytes decreased approximately by 30% upon incubation with post-immunization sera compared with pre-immunization sera. Furthermore, trypomastigotes were recorded by video microscopy and the parasite's flagellar speed was calculated by tracking the flagella. Trypomastigotes exposed to post-immunization sera had qualitative alterations in motility and significantly slower flagella (45.5 µm/s), compared with those exposed to pre-immunization sera (69.2 µm/s). In summary, polyclonal anti-TcTLE serum significantly reduced the parasite's flagellar speed and cell infectivity. These findings support that KMP-11 could be important for parasite motility, and that by targeting its N-terminal peptide infectivity can be reduced.

Low frequency of circulating CD8+ T stem cell memory cells in chronic chagasic patients with severe forms of the disease.

BACKGROUND: CD8+ T cells have been shown to play a crucial role in Trypanosoma cruzi infection. Memory CD8+ T cells can be categorised based on their distinct differentiation stages and functional activities as follows: stem cell memory (TSCM), central memory (TCM), transitional memory (TTM), effector memory (TEM) and terminal effector (TTE) cells. Currently, the immune mechanisms that control T. cruzi in the chronic phase of the infection are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To characterise the CD8+ T cell subsets that could be participating in the control of T. cruzi infection, in this study, we compared total and T. cruzi-specific circulating CD8+ T cells with distinctive phenotypic and functional features in chronic chagasic patients (CCPs) with different degrees of cardiac dysfunction. We observed a decreased frequency of total TSCM along with an increased frequency of TTE in CCPs with severe disease. Antigen-specific TSCM cells were not detectable in CCPs with severe forms of the disease. A functional profile of CD8+ T cell subsets among CCPs revealed a high frequency of monofunctional CD8+ T cells in the most severe patients with IFN-γ+- or TNF-α+-producing cells. CONCLUSIONS/SIGNIFICANCE: These findings suggest that CD8+ TSCM cells may be associated with the immune response to T. cruzi and outcome of Chagas disease, given that these cells may be involved in repopulating the T cell pool that controls infection.

Mammalian cellular culture models of Trypanosoma cruzi infection: a review of the published literature.

Cellular culture infection with Trypanosoma cruzi is a tool used to dissect the biological mechanisms behind Chagas disease as well as to screen potential trypanocidal compounds. Data on these models are highly heterogeneous, which represents a challenge when attempting to compare different studies. The purpose of this review is to provide an overview of the cell culture infectivity assays performed to date. Scientific journal databases were searched for articles in which cultured cells were infected with any Trypanosoma cruzi strain or isolate regardless of the study's goal. From these articles the cell type, parasite genotype, culture conditions and infectivity results were extracted. This review represents an initial step toward the unification of infectivity model data. Important differences were detected when comparing the pathophysiology of Chagas disease with the experimental conditions used in the analyzed studies. While Trypanosoma cruzi preferentially infects stromal cells in vivo, most of the assays employ epithelial cell lines. Furthermore, the most commonly used parasite strain (Tulahuen-TcVI) is associated with chagasic cardiomyopathy only in the Southern Cone of South America. Suggestions to overcome these discrepancies include the use of stromal cell lines and parasite genotypes associated with the known characteristics of the natural history of Chagas disease.

[Preliminary evaluation of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® in Colombian individuals]. / Evaluación preliminar de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® en individuos colombianos.

INTRODUCTION: The diagnosis of Chagas' disease is essential to provide early treatment and improve patients' prognosis . The discriminatory efficiency of the serological tests varies according to the disease prevalence and the test- antigen used . OBJECTIVE: To evaluate the discriminatory efficiency of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (Nova Tec Immunodiagnostica GmbBH) in a group of Colombian individuals, using indirect immunofluorescence antibody testing (IFAT) and enzyme immunoassay (ELISA) tests as references. MATERIALS AND METHODS: Seventy-eight samples from chronic chagasic patients (36 asymptomatic and 42 symptomatic) and 21 healthy controls were included. Seventeen samples from non-infected people with Chagas' disease epidemiological risk, seven with leishmaniasis and nine with non-chagasic cardiomyopathy were also analyzed. Real time PCR was performed on four individuals whose results differed among tests. RESULTS: Significant differences at 450 nm optical absorbance were found (p<0.0001) when the median absorbance values of healthy controls (0.143), asymptomatic (2.401) and symptomatic (2.776) chagasic patients were compared, as well as when asymptomatic and symptomatic patients (p=0.0408) and seronegative people with epidemiological risk (0.232), cardiomyopathy (0.367) or leishmaniasis (0.337) were compared with chagasic patients (p<0.0001). Finally, there were differences among healthy controls and non-infected people with epidemiological risk (p=0.0264), patients with non-chagasic cardiomyopathy (p=0.0015) and patients with leishmaniasis (p=0.002). Real-time PCR was positive in three out of four analyzed cases. CONCLUSIONS: The commercial ELISA test allowed us to discriminate the chagasic patients from the controls. A phase II study of diagnostic tests for determining field reliability of this test is required.

Evaluación preliminar de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® en individuos colombianos / Preliminary evaluation of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® in Colombian individuals

Introducción. El diagnóstico de la enfermedad de Chagas es fundamental para brindar un tratamiento oportuno y mejorar el pronóstico del paciente. La capacidad discriminatoria de las pruebas serológicas para el diagnóstico varía de acuerdo con la prevalencia de la enfermedad y el antígeno utilizado en la prueba. Objetivo. Evaluar la capacidad discriminatoria de la prueba comercial Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (NovaTec Immunodiagnostica GmbH) en un grupo de individuos colombianos utilizando la prueba de inmunofluorescencia indirecta (IFI) y el ensayo de inmunoabsorción enzimática (ELISA) como referencia. Materiales y métodos. Se incluyeron 78 muestras de pacientes crónicos (36 asintomáticos y 42 sintomáticos) y 21 de controles sanos. También se analizaron 17 individuos no infectados con riesgo epidemiológico para la enfermedad de Chagas, siete con leishmaniasis y nueve con enfermedad cardiaca. Se evaluaron por PCR en tiempo real cuatro individuos cuyos resultados variaron entre pruebas. Resultados. Se encontraron diferencias significativas a una densidad óptica de 450 nm (p<0,0001) al comparar la mediana de la absorbancia entre los controles sanos (0,143) y los asintomáticos (2,401) o sintomáticos (2,776), entre los asintomáticos y sintomáticos (p=0,0408), entre los seronegativos con riesgo (0,232), individuos con enfermedades cardiacas (0,367) o con leishmaniasis (0,337) y los pacientes con enfermedad de Chagas (p<0,0001), y entre los controles sanos y los pacientes seronegativos con riesgo (p=0,0264), con enfermedades cardiacas (p=0,0015) o con leishmaniasis (p=0,002). La PCR en tiempo real fue positiva en tres de los cuatro casos. Conclusiones. Esta prueba comercial de ELISA permitió discriminar a los pacientes con Chagas de los controles. Se requieren estudios de fase II para determinar las características operativas de la prueba.

Introduction: The diagnosis of Chagas´ disease is essential to provide early treatment and improve patients´ prognosis . The discriminatory efficiency of the serological tests varies according to the disease prevalence and the test- antigen used . Objective: To evaluate the discriminatory efficiency of the commercial kit Chagas ( Trypanosoma cruzi ) IgG-ELISA ® (Nova Tec Immunodiagnostica GmbBH) in a group of Colombian individuals, using indirect immunofluorescence antibody testing (IFAT) and enzyme immunoassay (ELISA) tests as references. Materials and methods: Seventy-eight samples from chronic chagasic patients (36 asymptomatic and 42 symptomatic) and 21 healthy controls were included. Seventeen samples from non-infected people with Chagas´ disease epidemiological risk, seven with leishmaniasis and nine with non-chagasic cardiomyopathy were also analyzed. Real time PCR was performed on four individuals whose results differed among tests. Results: Significant differences at 450 nm optical absorbance were found (p<0.0001) when the median absorbance values of healthy controls (0.143), asymptomatic (2.401) and symptomatic (2.776) chagasic patients were compared, as well as when asymptomatic and symptomatic patients (p=0.0408) and seronegative people with epidemiological risk (0.232), cardiomyopathy (0.367) or leishmaniasis (0.337) were compared with chagasic patients (p<0.0001). Finally, there were differences among healthy controls and non-infected people with epidemiological risk (p=0.0264), patients with non-chagasic cardiomyopathy (p=0.0015) and patients with leishmaniasis (p=0.002). Real-time PCR was positive in three out of four analyzed cases. Conclusions: The commercial ELISA test allowed us to discriminate the chagasic patients from the controls. A phase II study of diagnostic tests for determining field reliability of this test is required.

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo / Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFN ? , IL-2 y TNFa frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi . Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFN ? , IL-2 y TNFa fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi . Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica.

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN ? , IL-2 and TNF a using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes producing IFN ? , IL-2 and TNF a was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8 + T lymphocyte subpopulations corresponding to those reported in the literature.