Trypanosoma Cruzi Trypomastigotes Research Articles

Anti-Trypanosomal Bufadienolides from the Oocytes of the Toad Rhinella alata (Anura, Bufonidae).

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1-7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3-7 resulted in a good anti-trypanosomal activity profile. Among these, 16β-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease.

Structural design, synthesis, and anti-Trypanosomatidae profile of new Pyridyl-thiazolidinones

The present work reports the synthesis of a novel series of pyridine-thiazolidinones with anti-Trypanosoma cruzi and leishmanicidal activities (compounds 10–27), derived from 2 or 4-pyridine thiosemicarbazones (1–9). The in vitro assays were performed with Trypanosoma cruzi trypomastigotes and amastigotes, as well as with Leishmania amazonensis promastigotes and amastigotes. The cytotoxicity profile was evaluated using the cell line RAW 264.7. From the 18 pyridine-thiazolidinones, 5 were able to inhibit trypomastigotes. Overall, all compounds inhibited amastigotes, highlighting compounds 15 (0.60 μM), 18 (0.64 μM), 17 (0.81 μM), and 27 (0.89 μM). Compounds 15 and 18 were able to induce parasite cell death through necrosis induction. Analysis by scanning electron microscopy showed that T. cruzi trypomastigotes treated with compounds 15 and 18 induced morphological changes such as shortening, retraction and curvature of the parasite body and leakage of internal content. Regarding the antiparasitic evaluation against Leishmania amazonensis, only compound 27 had a higher selectivity compared to Miltefosine against the amastigote form (IC50 = 5.70 μM). Our results showed that compound 27 presented an antiparasitic activity for both Trypanosoma cruzi and Leishmania amazonensis. After in silico evaluation, it was suggested that the new pyridine-thiazolidinones had an appropriate drug-likeness profile. Our results pointed out a new chemical frame with an anti-Trypanosomatidae profile. The pyridine-thiazolidinones presented here for the first time could be used as a starting point for the development of new antiparasitic agents.

Optimizing XCMS parameters for GC-MS metabolomics data processing: a case study.

Optimizing metabolomics data processing parameters is a challenging and fundamental task to obtain reliable results. Automated tools have been developed to assist this optimization for LC-MS data. GC-MS data require substantial modifications in processing parameters, as the chromatographic profiles are more robust, with more symmetrical and Gaussian peaks. This work compared an automated XCMS parameter optimization using the Isotopologue Parameter Optimization (IPO) software with manual optimization of GC-MS metabolomics data. Additionally, the results were compared to online XCMS platform. GC-MS data from control and test groups of intracellular metabolites from Trypanosoma cruzi trypomastigotes were used. Optimizations were performed on the quality control (QC) samples. The results in terms of the number of molecular features extracted, repeatability, missing values, and the search for significant metabolites showed the importance of optimizing the parameters for peak detection, alignment, and grouping, especially those related to peak width (fwhm, bw) and noise ratio (snthresh). This is the first time that a systematic optimization using IPO has been performed on GC-MS data. The results demonstrate that there is no universal approach for optimization but automated tools are valuable at this stage of the metabolomics workflow. The online XCMS proves to be an interesting processing tool, helping, above all, in the choice of parameters as a starting point for adjustments and optimizations. Although the tools are easy to use, there is still a need for technical knowledge about the analytical methods and instruments used.

A monoclonal antibody against a Leishmania mexicana COX-like enzymatic activity also recognizes similar proteins in different protozoa of clinical importance.

In Leishmania mexicana, the protease gp63 has been documented as the protein responsible for cyclooxygenase (COX) activity. The present work aimed to obtain a monoclonal antibody capable of recognizing this protein without blocking the COX-like enzymatic activity. The antibody produced by the selected hybridoma was named D12 mAb. The antigen recognized by the D12 mAb was characterized by the determination of COX activity associated with immune complexes in the presence of exogenous arachidonic acid (AA) using the commercial Activity Assay Abcam kit. LSM-SMS analysis validated the identity of the antigen associated with the D12 mAb as the L. mexicana protease gp63. Confocal microscopy assays with the D12 mAb detected, by cross-recognition, similar proteins in other protozoan parasites. COX-like molecules are located in vesicular structures, homogeneously distributed throughout the cytoplasm in amastigotes (intracellular infectious phase) and promastigotes of L. mexicana, and trophozoites of Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. However, in Giardia duodenalis trophozoites, the distribution of the COX-like molecule was also in perinuclear areas. In comparison, in Trypanosoma cruzi trypomastigotes, the distribution was mainly observed in the plasma membrane. Structural analyses of COX-2-like antigens revealed continuous and discontinuous epitopes for B cells, which could be relevant in the cross-reaction of D12 mAb with the analyzed parasites. These results indicate that the D12 mAb against the L. mexicana gp63 also recognizes a COX-like molecule in several protozoan parasites, suggesting that this D12 mAb could potentially be used in combined therapies against infectious diseases.

Immunological Modulation and Control of Parasitaemia by Ayahuasca Compounds: Therapeutic Potential for Chagas's Disease.

Ayahuasca is a psychoactive and psychedelic decoct composed mainly of Banisteriopsis caapi and Psychotria viridis plant species. The beverage is rich in alkaloids and it is ritualistically used by several indigenous communities of South America as a natural medicine. There are also reports in the literature indicating the prophylaxis potential of Ayahuasca alkaloids against internal parasites. In the present study, Ayahuasca exhibited moderate in vitro activity against Trypanosoma cruzi trypomastigotes (IC50 95.78 μg/mL) compared to the reference drug benznidazole (IC50 2.03 μg/mL). The β-carboline alkaloid harmine (HRE), isolated from B. caapi, was considered active against the trypomastigotes forms (IC50 6.37), and the tryptamine N, N-dimethyltryptamine (DMT), isolated from P. viridis was also moderately active with IC50 of 21.02 μg/mL. Regarding the in vivo evaluations, no collateral effects were observed. The HRE alone demonstrated the highest trypanocidal activity in a dose-responsive manner (10 and 100 mg/kg). The Ayahuasca and the association between HRE and DMT worsened the parasitaemia, suggesting a modulation of the immunological response during the T. cruzi infection, especially by increasing total Immunoglobulin (IgG) and IgG1 antibody levels. The in silico molecular docking revealed HRE binding with low energy at two sites of the Trypanothione reductase enzyme (TR), which are absent in humans, and thus considered a promissory target for drug discovery. In conclusion, Ayahuasca compounds seem to not be toxic at the concentrations of the in vivo evaluations and can promote trypanocidal effect in multi targets, including control of parasitaemia, immunological modulation and TR enzymatic inhibition, which might benefit the treatments of patients with Chagas' disease. Moreover, the present study also provides scientific information to support the prophylactic potential of Ayahuasca against internal parasites.

Front Cover: Trypanocidal Activity of Dysphania ambrosioides, Lippia alba, and Tetradenia riparia Essential Oils against Trypanosoma cruzi (Chem. Biodiversity 12/2021)

Front Cover. The essential oils from Dysphania ambrosioides (L.) Mosyakin & Clemants (Amaranthaceae), and Lippia alba (Mill.) N.E. Brown (Verbenaceae) leaves, grown in Brazil Southeast showed high activities against Trypanosoma cruzi trypomastigote and amastigote forms. These essential oils displayed moderate toxicity against LLCMK2 cells, but the essential oil of D. ambrosioides showed a high selectivity index (SI) for trypomastigote and amastigote forms. On the other hand, the essential oil of Tetradenia riparia (Hochst.) Codd (Lamiaceae) grown in Brazil Southeast displayed the best in vivo trypanocidal effects by reducing the parasitemia after intraperitoneal administration as reported by Pagotti et al. in their full paper at 10.1002/cbdv.202100678.

Psilostachyins as trypanocidal compounds: Bioguided fractionation of Ambrosia tenuifolia chemically modified extract

This work focusses on the chemical diversification of an Ambrosia tenuifolia extract and its bioguided fractionation, aiming to unveil the chemical entity responsible for the trypanocidal activity. Besides, a revision of the phytochemical study of this species, based on previous reports of the antiparasitic psilostachyins A and C as main compounds, was conducted. To improve the biological properties of a plant extract through a simple chemical reaction, the oxidative diversification of the dichloromethane extract of this plant species was carried out. A bioguided fractionation of a chemically modified extract was performed by evaluating the inhibitory activity against Trypanosoma cruzi trypomastigotes. This experiment led to the isolation of one of the most active compounds. In general terms, epoxidized metabolites were obtained as a result of the oxidation of the major metabolite of the species. The trypanocidal activity of some tested metabolites overperformed the reference drug, benznidazole, displaying no cytotoxicity at trypanocidal concentrations. Key structure-activity relationships were obtained for designing previously undescribed antiparasitic sesquiterpene lactones.

Stress Induces Release of Extracellular Vesicles by Trypanosoma cruzi Trypomastigotes.

All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes.

Ready-to-use qPCR for detection of Cyclospora cayetanensis or Trypanosoma cruzi in food matrices

Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2–8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions.(170 words)

Proteome-wide modulation of S-nitrosylation in Trypanosoma cruzi trypomastigotes upon interaction with the host extracellular matrix

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SignificanceProtein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Purification of Glycosylphosphatidylinositol-Anchored Mucins from Trypanosoma cruzi Trypomastigotes and Synthesis of α-Gal-Containing Neoglycoproteins: Application as Biomarkers for Reliable Diagnosis and Early Assessment of Chemotherapeutic Outcomes of Chagas Disease.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

Antitrypanosomal activity of epi-polygodial from Drimys brasiliensis and its effects in cellular membrane models at the air-water interface

Epi-polygodial, a drimane sesquiterpene was isolated from Drimys brasiliensis (Winteraceae). This compound demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (IC50 = 5.01 μM) with a selectivity index higher than 40. These results were correlated with the effects observed when this compound was incorporated in cellular membrane models of protozoans, represented by Langmuir monolayers of dipalmitoylphosphoethanolamine (DPPE). Surface pressure-area isotherms showed that epi-polygodial expands DPPE monolayers at higher areas and condenses them at lower areas, which was attributed to the preferential interaction with the polar heads of the lipid. This mechanism of action could be corroborated with Polarization-Modulation Reflection-Absorption Spectroscopy and Brewster Angle Microscopy. These results pointed to the fact that the interaction of epi-polygodial with DPPE monolayers at the air-water interface affects the physical chemical properties of the mixed film, which may be important to comprehend the interaction of this drug with cellular membranes at the molecular level.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study

BackgroundDrug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease.MethodsIn this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches.ResultsSertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 μM, and activity against bloodstream trypomastigotes, with IC50 of 14 μM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline.ConclusionsThe present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.

Primary evidence of the mechanisms of action of HIV aspartyl peptidase inhibitors on Trypanosoma cruzi trypomastigote forms

The development of HIV aspartyl peptidase inhibitors (HIV-PIs) and their introduction into AIDS therapy preceded a significant decrease in the incidence, morbidity and mortality of relevant protozoan co-infections. However, few data are available about how HIV-PIs act on pathogenic parasites, such as Trypanosoma cruzi, the etiological agent of Chagas disease. Therefore, the aim of the present work was to evaluate different physiological aspects of the treatment of the infective trypomastigote forms of T. cruzi with the HIV-PIs, nelfinavir and lopinavir. At the LD50/4 h doses, both HIV-PIs significantly reduced the trypomastigote size and markedly increased the granularity/complexity. Transmission electron microscopy analysis associated to biochemical assays permitted definition of the main HIV-PIs targets in the parasite. Lopinavir and nelfinavir induced (i) plasma membrane shedding, particularly in the flagellar region, which drastically affected parasite integrity; (ii) strong mitochondrial swelling with rare matrix fragmentation, which were linked to severely reduced hydrolytic activity of dehydrogenases and organelle membrane depolarization; (iii) increased generation of reactive oxygen species (ROS); (iv) dilation of both nuclear envelope (without DNA disruption) and endoplasmic reticulum (with formation of autophagosomes), and (v) accumulation of intracellular lipid droplets, revealing a typical lipid metabolism disorder. Collectively, our study demonstrated that nelfinavir and lopinavir target vital cellular structures of trypomastigotes, culminating in irreversible metabolic injuries that lead to T. cruzi death.

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

BackgroundMajor drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds.MethodsFruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS).ResultsThe most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite.ConclusionsThe selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Synthesis and biological evaluation of potential inhibitors of the cysteine proteases cruzain and rhodesain designed by molecular simplification

Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.

Epitope Capsid-Incorporation: New Effective Approach for Vaccine Development for Chagas Disease

Background:Previously we reported that a hexon-modified adenovirus (Ad) vector containing the invasive neutralizing epitope of Trypanosoma cruzi (T. cruzi) trypomastigote gp83 (Ad5-gp83) provided immunoprotection against T. cruzi infection. The purpose of this work was to design an improved vaccine for T. cruzi using a novel epitope capsid incorporation strategy. Thus, we evaluated the immunoprotection raised by co-immunization with Ad5-gp83 and an Ad vector containing an epitope (ASP-M) of the T. cruzi amastigote surface protein 2.Methods:Protein IX (pIX)-modified Ad vector (Ad5-pIX-ASP-M) was generated, characterized, and validated. C3H/He mice were immunized with Ad5-pIX-ASP-M and Ad5-gp83 and the cell-mediated responses were evaluated by enzyme-linked immunospot (ELISPOT) assay and intracellular staining. Immunized mice were challenged with T. cruzi to evaluate the vaccine efficacy.Results:Our findings indicate that Ad5-pIX-ASP-M was viable. Specific CD8+ T-cell mediated responses prior to the challenge show an increase in IFNγ and TNFα production. A single immunization with Ad5-pIX-ASP-M provided protection from T. cruzi infection, but co-immunizations with Ad5-pIX-ASP-M and Ad5-gp83 provided a higher immunoprotection and increased survival rate of mice.Conclusions:Overall, these results suggest that the combination of gp83 and ASP-M specific epitopes onto the capsid-incorporated adenoviruses would provide superior protection against Chagas disease as compared with Ad5-gp83 alone.

Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole Analogues of Neolignans Veraguensin, Grandisin and Machilin G

Sixteen 1,4-diaryl-1,2,3-triazole compounds derived from the natural products veraguensin, grandisin and machilin G were synthesized, with yields of 78-92%. Biological activity tests against Leishmania amazonensis promastigotes showed that three of these compounds were the most active, with maximum inhibitory concentration (IC50) values of 1.1, 3.71 and 7.23 µM. One compound was highly active against Leishmania infantum, with an IC50 value of 5.2 µM, and one derivative showed an IC50 value of 28.6 µM against Trypanosoma cruzi trypomastigotes. Regarding structure-activity relationship (SAR), hybrid 1,2,3-triazolic compounds containing a methylenedioxy group, showed the best antileishmanial and antitrypanosomal activities.

Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection?

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x104 culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-β and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI versus OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.

Synthesis and biological evaluation of quinones derived from natural product komaroviquinone as anti-Trypanosoma cruzi agents.

Current chemotherapy drugs for Chagas' disease are insufficient due to their limited efficacy; however, anti-trypanosomal agents have recently shown promise. As such, synthetic intermediates of komaroviquinone were evaluated for anti-trypanosomal activity. Based on the results, a series of novel quinone derivatives were screened for anti-trypanosomal activity and mammalian cytotoxicity. Several quinone derivatives displayed higher antiprotozoal activity against Trypanosoma cruzi trypomastigotes than the reference drug benznidazole, without concomitant toxicity toward the host cell.