Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.

In Vitro Identification of Phosphorylation Sites on TcPolß by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolß in Trypanosoma cruzi Epimastigotes.

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolß) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolß by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolß. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolß. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolß phosphorylation and enzymatic activity in T. cruzi epimastigotes.

From molecules to ecosystems: Insights into a network of interactions for a Chagas disease outbreak using Triatoma brasiliensis as natural samplers.

Exploring the dynamics of disease transmission involves an understanding of complex interactions within the eco-epidemiologic framework. In the context of Chagas disease (CD), elements are mainly represented by the interactions among the pathogen, insect vector, host, humans and the environment. We performed quantitative and qualitative analyses on a dataset derived from 98 Triatoma brasiliensis infected by trypanosomatids, which were linked to a CD outbreak in the semi-arid region of northeastern Brazil. We extracted invertebrate-derived DNA (iDNA) from these insects, comprising 18 populations around the outbreak area, each indicative of various strata of anthropogenic influence. Food source (FS) diversity, representing potential parasite reservoirs, was determined through mitochondrial gene (cyt b) sequencing of vertebrates, and parasite genotyping was accessed using fluorescent amplified fragment barcodes (FFLB) of trypanosomatids. We also assessed the residents' awareness of breeding sites for CD vectors in the inspected houses. The quantification of Trypanosoma cruzi was estimated via real-time PCR and is denominated here as the average parasite load (PL) per insect (T. cruzi/intestinal unit). We aimed to address vector-parasite-host-environment interactions that were discussed based on their significance among the components. Notably, among the significant interactions, we observed that the PL in the insects was significantly influenced by FS. Infected insects that fed on the classic reservoir, Didelphis albiventris, and Galea spixii exhibited higher PLs, compared to those that fed on Kerodon rupestris (p < 0.04)-a primary host. While D. albiventris is already recognized as a synanthropic species, we propose that G. spixii may also be undergoing a synanthropic process. Conversely, domestic cats are frequently identified as FS in infected insects from the sylvatic environment, suggesting a possible change in their behavior towards a wild state. Therefore, we propose that neglected anthropogenic actions have facilitated the reciprocal (sylvatic-peridomestic) circulation of T. cruzi-especially noted for TcI because it was predominant in insects found in peridomestic environments. Residents are often unaware of the existence of insect breeding grounds near their homes, particularly when it involves the storage of materials without planning for use, such as piles of tiles, bricks and wood. Although indirect inferences about the interaction among vector-parasite-host-environment are still incipient, we highlight the potential use of vectors as natural samplers of biological and ecological components in transmitting the disease.

Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review

ABSTRACT Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Anti-Trypanosoma cruzi Potential of Vestitol Isolated from Lyophilized Red Propolis.

Chagas disease (CD) is a worldwide public health problem, and the drugs available for its treatment have severe limitations. Red propolis is a natural extract known for its high content of phenolic compounds and for having activity against T. cruzi. The aim of this study was to investigate the trypanocidal potential of red propolis to isolate, identify, and indicate the mode of action of the bioactive compounds. The results revealed that the total phenolic content was 15.4 mg GAE/g, and flavonoids were 7.2 mg QE/g. The extract was fractionated through liquid-liquid partitioning, and the trypanocidal potential of the samples was evaluated using the epimastigote forms of the Y strain of T. cruzi. In this process, one compound was characterized by MS, 1H, and 13C NMR and identified as vestitol. Cytotoxicity was evaluated employing MRC-5 fibroblasts and H9C2 cardiomyocytes, showing cytotoxic concentrations above 15.62 µg/mL and 31.25 µg/mL, respectively. In silico analyses were applied, and the data suggested that the substance had a membrane-permeation-enhancing effect, which was confirmed through an in vitro assay. Finally, a molecular docking analysis revealed a higher affinity of vestitol with farnesyl diphosphate synthase (FPPS). The identified isoflavan appears to be a promising lead compound for further development to treat Chagas disease.

Blimp-1 signaling pathways in T lymphocytes is essential to control the Trypanosoma cruzi infection-induced inflammation.

In many infectious diseases, the pathogen-induced inflammatory response could result in protective immunity that should be regulated to prevent tissue damage and death. In fact, in Trypanosoma cruzi infection, the innate immune and the inflammatory response should be perfectly controlled to avoid significant lesions and death. Here, we investigate the role of Blimp-1 expression in T cells in resistance to T. cruzi infection. Therefore, using mice with Blimp-1 deficiency in T cells (CKO) we determined its role in the controlling parasites growth and lesions during the acute phase of infection. Infection of mice with Blimp-1 ablation in T cells resulted failure the cytotoxic CD8+ T cells and in marked Th1-mediated inflammation, high IFN-γ and TNF production, and activation of inflammatory monocyte. Interestingly, despite high nitric-oxide synthase activation (NOS-2), parasitemia and mortality in CKO mice were increased compared with infected WT mice. Furthermore, infected-CKO mice exhibited hepatic lesions characteristic of steatosis, with significant AST and ALT activity. Mechanistically, Blimp-1 signaling in T cells induces cytotoxic CD8+ T cell activation and restricts parasite replication. In contrast, Blimp-1 represses the Th1 response, leading to a decreased monocyte activation, less NOS-2 activation, and, consequently preventing hepatic damage and dysfunction. These data demonstrate that T. cruzi-induced disease is multifactorial and that the increased IFN-γ, NO production, and dysfunction of CD8+ T cells contribute to host death. These findings have important implications for the design of potential vaccines against Chagas disease.

Discovery of Novel Inhibitors of Cruzain Cysteine Protease of Trypanosoma cruz.

Chagas disease (CD) is a parasitic disease endemic in several developing countries. According to the World Health Organization, approximately 6-8 million people worldwide are inflicted by CD. The scarcity of new drugs, mainly for the chronic phase, is the main reason for treatment limitation in CD. Therefore, there is an urgent need to discover new targets for which new therapeutical agents could be developed. Cruzain cysteine protease (CCP) is a promising alternative because this enzyme exhibits pleiotropic effects by acting as a virulence factor, modulating host immune cells, and interacting with host cells. This systematic review was conducted to discover new compounds that act as cruzain inhibitors, and their effects in vitro were studied through enzymatic assays and molecular docking. Additionally, the advances and perspectives of these inhibitors are discussed. These findings are expected to contribute to medicinal chemistry in view of the design of new, safe, and efficacious inhibitors against Trypanosoma cruzi CCP detected in the last decade (2013-2022) to provide scaffolds for further optimization, aiming toward the discovery of new drugs.

Cafeteria diet-induced obesity remodels immune response in acute Trypanosoma cruzi infection.

BACKGROUND: Obesity is a global problem associated with several conditions, including hypertension, diabetes, arthritis and cardiovascular diseases. With the increase in the prevalence of obesity in recent years, mostly in developing countries, it is important to study its impact on various diseases, including infectious illnesses, such as Chagas disease, caused by the protozoan Trypanosoma cruzi. Considering that a diet rich in salt, sugar, and fat is associated with obesity, this study aimed to evaluate the influence of cafeteria diet (CAF)-induced obesity on immune responses in T. cruzi-infected rats. METHODS: Male Wistar Hannover rats were provided with water and food ad libitum (chow group). The CAF-fed groups received a normal rodent diet or CAF. The animals were intraperitoneally infected with 105 trypomastigote forms of the Y strain of T. cruzi present in the whole blood from a previously infected mouse. RESULTS: CAF-fed rats showed a significant increase in visceral adipose tissue weight compared to chow-fed rats. A significant reduction in CD3+ CD4+ helper splenic T cells was observed in obese-infected rats compared to non-obese-infected rats, as well as CD11b and macrophages. In addition, macrophages from obese animals displayed reduced RT1b levels compared to those from control animals. Moreover, INF-γ, an important factor in macrophage activation, was reduced in obese-infected rats compared with their counterparts. CONCLUSIONS: These results indicate that a CAF can impair the cell-mediated immune response against T. cruzi.

Pro-inflammatory cytokines are modified during the multiplication of Trypanosoma cruzi within the placental chorionic villi and are associated with the level of infection via the signaling pathway NF-κB.

PROBLEM: Congenital Trypanosoma cruzi (T. cruzi) infection has been associated with changes in the levels of TNF-α and IFN-γ during the pregnancy. Therefore, we propose to study the participation and dynamics of proinflammatory cytokines in the infection process of placental explants infected by T. cruzi in vitro. METHOD OF STUDY: Chorionic villous explants (CVE) obtained of human term placentas (n = 8) from normal pregnancies were cultured with 105 trypomastigotes/mL of Tulahuen strain DTU VI for 0, 2, 4, 16, 24, 48 and 72 h. Explants were treated with sulfasalazine (SULF) (5 mM) and N-acetyl-cysteine (NAC) (15 mM), as inhibitors molecules of NF-κB pathway, or LPS (1 µg/mL) for 24 and 72 h p.i. Motile trypomastigotes were counted in culture supernatants. Immunohistochemistry and ELISA for TNF-α, IFN-γ, IL-1ß, IL-4, and IL-10 were performed in CVE and culture supernatants respectively. The parasite load was measured by RT-qPCR. RESULTS: T. cruzi invades the chorionic villi from 4 h p.i. increasing significantly its DNA at 48 and 72 h p.i. of culture (parasite multiplication phase). They were detected in stromal cells, which was related to elevation of TNF-α, IL-1ß, IFN-γ, and IL-10. The inhibition of NF-κB activity in the explants decreased the production of the analyzed cytokines, showing elevated levels of T. cruzi DNA during the multiplication phase of the parasite. CONCLUSIONS: Placental tissue modifies the secretion of pro-inflammatory cytokines during the phase of parasite multiplication, but not during the invasion phase, which in turns modifies the level of infection via the signaling pathway NF-κB.

A novel palladium complex with a coumarin-thiosemicarbazone hybrid ligand inhibits Trypanosoma cruzi release from host cells and lowers the parasitemia in vivo.

In this work, two analogous coumarin-thio and semicarbazone hybrid compounds were prepared and evaluated as a potential antichagasic agents. Furthermore, palladium and platinum complexes with the thiosemicarbazone derivative as ligand (L1) were obtained in order to establish the effect of metal complexation on the antiparasitic activity. All compounds were fully characterized both in solution and in solid state including the resolution of the crystal structure of the palladium complex by X-ray diffraction methods. Unexpectedly, all experimental and theoretical characterizations in the solid state, demonstrated that the obtained palladium and platinum complexes are structurally different: [PdCl(L1)] and [PtCl2(HL1)]. All the studied compounds lower the proliferation of the amastigote form of Trypanosoma cruzi while some of them also have an effect on the trypomastigote stage. Additionally, the compounds inhibit T. cruzi release from host cells in variable extents. The Pd compound presented a remarkable profile in all the in vitro experiments, and it showed no toxicity for mammalian cells in the assayed concentrations. In this sense, in vivo experiments were performed for this compound using an acute model of Chagas disease. Results showed that the complex significantly lowered the parasite count in the mice blood with no significant toxicity.

Nitazoxanide: A Drug Repositioning Compound with Potential Use in Chagas Disease in a Murine Model.

Chagas disease (ChD), caused by Trypanosoma cruzi, is the most serious parasitosis in the western hemisphere. Benznidazole and nifurtimox, the only two trypanocidal drugs, are expensive, difficult to obtain, and have severe side effects. Nitazoxanide has shown to be effective against protozoa, bacteria, and viruses. This study aimed to evaluate the nitazoxanide efficacy against the Mexican T. cruzi Ninoa strain in mice. Infected animals were orally treated for 30 days with nitazoxanide (100 mg/kg) or benznidazole (10 mg/kg). The clinical, immunological, and histopathological conditions of the mice were evaluated. Nitazoxanide- or benznidazole-treated mice had longer survival and less parasitemia than those without treatment. Antibody production in the nitazoxanide-treated mice was of the IgG1-type and not of the IgG2-type as in the benznidazole-treated mice. Nitazoxanide-treated mice had significantly high IFN-γ levels compared to the other infected groups. Serious histological damage could be prevented with nitazoxanide treatment compared to without treatment. In conclusion, nitazoxanide decreased parasitemia levels, indirectly induced the production of IgG antibodies, and partially prevented histopathological damage; however, it did not show therapeutic superiority compared to benznidazole in any of the evaluated aspects. Therefore, the repositioning of nitazoxanide as an alternative treatment against ChD could be considered, since it did not trigger adverse effects that worsened the pathological condition of the infected mice.

Identification of inhibitors for the transmembrane Trypanosoma cruzi eIF2α kinase relevant for parasite proliferation.

The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.

Severe Chagas disease in Ecuador: a countrywide geodemographic epidemiological analysis from 2011 to 2021.

Background: Chagas disease is a neglected and often forgotten tropical disease caused by the Trypanosoma cruzi. This parasite can be transmitted through the direct contact of human skin with feces and urine of the triatomine insect. According to the World Health Organization (WHO), an estimated 6-7 million people are infected worldwide, killing at least 14,000 every year. The disease has been reported in 20 of the 24 provinces of Ecuador, with El Oro, Guayas, and Loja being the most affected. Methodology: We analyzed the morbidity and mortality rates of severe Chagas disease in Ecuador on a nationwide, population-based level. Hospitalization cases and deaths were also examined based on altitude, including low (< 2,500 m) and high (> 2,500 m) altitudes, according to the International Society. Data was retrieved from the National Institute of Statistics and Census hospital admissions and in-hospital mortality databases from 2011 to 2021. Results: A total of 118 patients have been hospitalized in Ecuador since 2011 due to Chagas disease. The overall in-hospital mortality rate was 69.4% (N = 82). Men have a higher incidence rate (4.8/1,000,000) than women, although women have a significantly higher mortality rate than men (6.9/1,000,000). Conclusion: Chagas disease is a severe parasitic condition that primarily affects rural and poorer areas of Ecuador. Men are more likely to be infected due to differences in work and sociocultural activities. Using average elevation data, we conducted a geodemographic analysis to assess incidence rates by altitude. Our findings indicate that the disease is more common at low and moderate altitudes, but recent increases in cases at higher altitudes suggest that environmental changes, such as global warming, could be driving the proliferation of disease-carrying vectors in previously unaffected areas.

Prevalence of Trypanosoma cruzi infection in a cohort of people living with HIV/AIDS from an urban area.

The prevalence rate of coinfection Chagas disease (CD) and HIV in Brazil is between 1.3 and 5%. Serological tests for detecting CD use total antigen, which present cross reactivity with other endemic diseases, such as leishmaniasis. It is urge the use of a specific test to determinate the real prevalence of T. cruzi infection in people living with HIV AIDS (PLWHA). Here, we evaluated the prevalence of T. cruzi infection in a cohort of 240 PLWHA living in urban area from São Paulo, Brazil. Enzyme Linked Immunosorbent Assay, using epimastigote alkaline extract antigen from T. cruzi (ELISA EAE), returned a 2.0% prevalence. However by Immunoblotting, using trypomastigote excreted-secreted antigen (TESA Blot) from T. cruzi, we detected a prevalence of 0.83%. We consider that the real prevalence of T. cruzi-infection in PLWHA is 0.83%, lower than reported in literature; this is due to TESA Blot specificity, probably excluding false positives for CD immunodiagnosis. Our results demonstrate a real need to apply diagnostic tests with high sensitivity and specificity that can help assess the current status of CD/HIV coinfection in Brazil in order to stratify the effective risk of reactivation and consequently decreasing mortality.

Identification of inhibitors as drug candidates against Chagas disease.

Chagas disease, after more than a century after its discovery, is still a major public health problem. It is estimated that approximately 10 million people worldwide are infected with T. cruzi. However, the situation is more critical in Latin America and other regions where the disease is endemic. The largest number of cases occurs in Brazil, Argentina, and Mexico as more than 100 million people in these regions are located in areas with a high risk of contamination by the vector. The need for new therapeutic alternatives is urgent, as the available drugs have severe limitations such as low efficacy and high toxicity. From this scenario, in this work, we employed the virtual screening technique using cruzain and BDF2 as key biological targets for the survival of the parasite. Our objective was to identify potential inhibitors of T. cruzi trypomastigotes, which could be considered drug candidates against Chagas disease. For this, we employed different in silico methodologies and the obtained results were corroborated using in vitro biological assays. For the VS studies, a database containing synthetic compounds was simulated at the binding site of cruzain and BDF2. In addition, pharmacophoric models were constructed in the initial phases of VS, as well as other advanced analyses (molecular dynamics simulations, calculations of binding free energy, and ADME prediction) were carried out and the results allowed the selection of potential inhibitors of T. cruzi. Based on the obtained data, 32 different compounds commercially available were subjected to biological tests against the trypomastigote form of T. cruzi. As result, 11 of those compounds displayed significant activity against T. cruzi and can be considered potential candidates for the treatment of Chagas disease.

Chagas cardiomyopathy is associated with a high susceptibility to T. cruzi infection in monocyte-derived macrophages and a predominance of CD4+CD45RO+ T-cells with immunoregulatory patterns.

The pathogenesis of Chronic Chagas Cardiomyopathy (CCC) is still not fully understood, and the persistence of the parasite in tissues seems to be essential for the onset and progression of heart disease, tissue destruction, and chronic inflammation. It is clear that the polarity found between the asymptomatic (IND) and cardiac clinical forms refers mainly to the mechanisms involved in the regulation of the host's immune response. Thus, to elucidate aspects of the susceptibility of host phagocytes to T. cruzi infection, the present study explored novel aspects of innate immune response, integrating data on susceptibility to infection and intracellular replication, using monocyte-derived macrophages from CCC patients, together with memory CD4+ T-cells (CD45RO+). The isolation of PBMC was conducted by means of in vitro infection assay with T. cruzi trypomastigotes and flow cytometry analysis of the intracytoplasmic cytokine production by CD4+T-cells. Our findings indicated that monocytes derived from individuals with CCC are more susceptible to the infection and replication of intracellular amastigotes. Moreover, the stimulation of CD4+ T-cells from CCC patients, together with T. cruzi trypomastigotes, induces a predominance of a regulatory response over a type 1 response, demonstrated by an increase in IL-10 production and a reduction in the IFN-γ and IFN-γ/IL-10. Suppression of the function of monocyte-derived macrophages, from CCC patients, to control trypomastigote infection and intracellular replication sheds light on a potential susceptibility of these cells isolated from peripheral blood, which may reflect the ineffectiveness of parasite control by phagocytes in cardiac tissues, which can subsequently result in serious heart disease.

Antichagasic evaluation, molecular docking and ADMET properties of the chalcone (2E)-3-(2-fluorophenyl)-1-(2-hydroxy- 3,4,6-trimethoxyphenyl)prop-2-en-1-one against Trypanosoma cruzi.

Characterized as a neglected disease, Chagas disease is an infection that, in the current scenario, affects about 8 million people per year, with a higher incidence in underdeveloped countries, Chagas is responsible for physiological disabilities that result in impacts that are slightly reflected in world socioeconomic stability. Although treatments are based on drugs such as Benznidazole, the pathology lacks a continuous treatment method with low toxicological incidence. The present study estimates the anti-chagasic activity of the synthetic chalcone CPN2F based on the alignment between in vitro tests and structural classification in silico studies, molecular docking and ADMET studies. The in vitro tests showed a reduction in the protozoan metabolism in host cells (LLC-MK2). At the same time, the molecular docking models evaluate this growth inhibition through the synergistic effect associated with Benznida- zole against validated therapeutic target key stages (Cruzaine TcGAPDH and Trypanothione reductase) of the Trypanosoma cruzi development cycle. The in silico prediction results reveal an alignment between pharmacokinetic attributes, such as renal absorption and release, which allow the preparation of CPN2F as an antichagasic drug with a low incidence of organic toxicity.Communicated by Ramaswamy H. Sarma.

Transcriptional changes during metacyclogenesis of a Colombian Trypanosoma cruzi strain.

During its life cycle, Trypanosoma cruzi undergoes physiological modifications in order to adapt to insect vector and mammalian host conditions. Metacyclogenesis is essential, as the parasite acquires the ability to infect a variety of mammalian species, including humans, in which pathology is caused. In this work, the transcriptomes of metacyclic trypomastigotes and epimastigotes were analyzed in order to identify differentially expressed genes that may be involved in metacyclogenesis. Toward this end, in vitro induction of metacyclogenesis was performed and metacyclic trypomastigotes obtained. RNA-Seq was performed on triplicate samples of epimastigotes and metacyclic trypomastigotes. Differential gene expression analysis showed 513 genes, of which 221 were upregulated and 292 downregulated in metacyclic trypomastigotes. The analysis showed that these genes are related to biological processes relevant in metacyclogenesis. Within these processes, we found that most of the genes associated with infectivity and gene expression regulation were upregulated in metacyclic trypomastigotes, while genes involved in cell division, DNA replication, differentiation, cytoskeleton, and metabolism were mainly downregulated. The participation of some of these genes in T. cruzi metacyclogenesis is of interest, as they may be used as potential therapeutic targets in the design of new drugs for Chagas disease.

Activation-induced marker assays for identification of Trypanosoma cruzi-specific CD4 or CD8 T cells in chronic Chagas disease patients.

Antigen-specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on-going vaccine strategies. In this context, the present study focuses on T-cell assays that define the parasite-specificity on the basis of upregulation of TCR stimulation-induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD-L1, CD11a, CD49d, HLA-DR, CD38) to reliably identify activated CD4+ and CD8+ T-cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation-induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD-L1 surface markers are efficient at detecting T. cruzi-specific CD4+ T cells in CCD patients, in comparison to non-infected donors, after both stimulation times. For CD8+ T cells, only PD-L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite-specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi-specific CD4+ and CD8+ T cells.