Valorizing Constituents of Cashew Nut Shell Liquid toward the Sustainable Development of New Drugs against Chagas Disease.

Six new ether phospholipid analogues encompassing constituents from cashew nut shell liquid as the lipid portion were synthesized in an effort to valorize byproducts of the cashew industry toward the generation of potent compounds against Chagas disease. Anacardic acids, cardanols, and cardols were used as the lipid portions and choline as the polar headgroup. The compounds were evaluated for their in vitro antiparasitic activity against different developmental stages of Trypanosoma cruzi. Compounds 16 and 17 were found to be the most potent against T. cruzi epimastigotes, trypomastigotes, and intracellular amastigotes exhibiting selectivity indices against the latter 32-fold and 7-fold higher than current drug benznidazole, respectively. Hence, four out of six analogues can be considered as hit-compounds toward the sustainable development of new treatments for Chagas disease, based on inexpensive agro-waste material.

Design, synthesis and biological evaluation of antiparasitic dinitroaniline-ether phospholipid hybrids.

A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.

Silver and copper-benznidazole derivatives as potential antiparasitic metallodrugs: Synthesis, characterization, and biological evaluation.

Currently the only drug available to treat Chagas disease in Brazil is benznidazole (BZN). Therefore, there is an urgent need to discover and develop new anti- Trypanosoma cruzi candidates. In our continuous effort to enhance clinical antiparasitic drugs using synergistic strategy, BZN was coordinated to silver and copper ions to enhance its effectiveness to treat that illness. In this work, the syntheses of four novel metal-BZN complexes, [Ag(BZN)2]NO3·H2O (1), [CuCl2(BZN)(H2O)]·1/2CH3CN (2), [Ag(PPh3)2(BZN)2]NO3·H2O (3), and [Cu(PPh3)2(BNZ)2]NO3·2H2O (4), and their characterization using multiple analytical and spectroscopic techniques such as Infrared (FTIR), Nuclear Magnetic Resonance (1H, 13C, 31P), UV-Visible (UV-Vis), Electron Paramagnetic Resonance (EPR), conductivity and elemental analysis are described. IC50 (Half-maximal inhibitory concentration) values of Ag-BZN compounds are about five to ten times lower than benznidazole itself in both proliferation stages of the parasite (epimastigotes and amastigotes). The cytotoxicity of both compounds in human cells (fibroblasts and hepatocytes) are comparable to BZN, indicating that Ag-BZN complexes can be more selective than BZN.

Effects of the natural antimicrobial peptide aureocin A53 on cells of Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis in the excised teat model.

Aureocin A53 is an N-formylated antimicrobial peptide (AMP) produced by Staphylococcus aureus. Aureocin A53 has a broad spectrum of antimicrobial activity against human and animal pathogens. In the present study, its antagonistic activity was investigated towards 30 strains of S. aureus and 30 strains of Streptococcus spp. isolated from bovine mastitis cases in Brazil. Bovine mastitis is a disease that causes a major economic impact worldwide. Aureocin A53 inhibited the growth of all 60 strains tested, including multidrug-resistant streptococcal isolates and strains of S. aureus belonging to different pulsotypes. This AMP proved to be bactericidal against the six target strains randomly selected among staphylococci and streptococci, also exhibiting a lytic mode of action against the staphylococcal cells. Furthermore, it was determined that 2,048 AU/mL of the AMP were required to inhibit 99.99% of the cell growth of the strain less sensitive to aureocin A53. Aureocin A53 was not toxic to bovine mammary gland epithelial cells after a 24-h exposure and maintained its antimicrobial activity when tested in the excised-teat model against strains of S. aureus and Streptococcus agalactiae, the species responsible for most intramammary infections, not only in Brazil but in other countries as well. Therefore, the use of aureocin A53 in the development of new pharmacological products for the prophylaxis and/or treatment of bovine mastitis was considered promising.

Promising fluconazole based zinc(II) and copper(II) coordination polymers against Chagas disease.

A series of new transition metal coordination polymers, [Zn(Ac)2(FLZ)2]n (1), [Zn(FLZ)2(Cl)2]n (2), {[Zn(FLZ)2](NO3)2}n (3), [Cu(FLZ)2(CH3COO)4]n (4), {[Cu(FLZ)2Cl2]}n (5) and {[Cu(FLZ)2](NO3)2}n (6), were synthesized by the reaction of fluconazole (FLZ) with the respective zinc or copper salts under mild conditions. The molecular structure of these compounds was elucidated by several analytical and spectroscopy techniques such as elemental analyses, 1H and 13C{1H} nuclear magnetic resonance, electronic paramagnetic resonance, and infrared spectroscopy. Single-crystal X-ray diffraction confirmed the structure of the compounds 2, 4, 5 and 6 in solid state. The antichagasic activity of these compounds was evaluated against different forms of Trypanosoma cruzi. Compound 2 exhibited the highest activity against intracellular amastigotes. The ultrastructural changes in epimastigotes and intracellular amastigotes were investigated. These promising biological results demonstrated that the zinc or copper coordination polymers can form very active anti-parasitic compounds. The resulting compounds are more effective than the free azole drug and, consequently, great candidates for the treatment of Chagas disease.

Design, Synthesis and Antiparasitic Evaluation of Click Phospholipids.

A library of seventeen novel ether phospholipid analogues, containing 5-membered heterocyclic rings (1,2,3-triazolyl, isoxazolyl, 1,3,4-oxadiazolyl and 1,2,4-oxadiazolyl) in the lipid portion were designed and synthesized aiming to identify optimised miltefosine analogues. The compounds were evaluated for their in vitro antiparasitic activity against Leishmania infantum and Leishmania donovani intracellular amastigotes, against Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the substituents of the heterocyclic ring (tail) and the oligomethylene spacer between the head group and the heterocyclic ring was found to affect the activity and toxicity of these compounds leading to a significantly improved understanding of their structure-activity relationships. The early ADMET profile of the new derivatives did not reveal major liabilities for the potent compounds. The 1,2,3-triazole derivative 27 substituted by a decyl tail, an undecyl spacer and a choline head group exhibited broad spectrum antiparasitic activity. It possessed low micromolar activity against the intracellular amastigotes of two L. infantum strains and T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes, while its cytotoxicity concentration (CC50) against THP-1 macrophages ranged between 50 and 100 µM. Altogether, our work paves the way for the development of improved ether phospholipid derivatives to control neglected tropical diseases.

Basic Biology of Trypanosoma cruzi.

The present review addresses basic aspects of the biology of the pathogenic protozoa Trypanosoma cruzi and some comparative information of Trypanosoma brucei. Like eukaryotic cells, their cellular organization is similar to that of mammalian hosts. However, these parasites present structural particularities. That is why the following topics are emphasized in this paper: developmental stages of the life cycle in the vertebrate and invertebrate hosts; the cytoskeleton of the protozoa, especially the sub-pellicular microtubules; the flagellum and its attachment to the protozoan body through specialized junctions; the kinetoplast-mitochondrion complex, including its structural organization and DNA replication; glycosome and its role in the metabolism of the cell; acidocalcisome, describing its morphology, biochemistry, and functional role; cytostome and the endocytic pathway; the organization of the endoplasmic reticulum and Golgi complex; the nucleus, describing its structural organization during interphase and division; and the process of interaction of the parasite with host cells. The unique characteristics of these structures also make them interesting chemotherapeutic targets. Therefore, further understanding of cell biology aspects contributes to the development of drugs for chemotherapy.

May the epimastigote form of Trypanosoma cruzi be infective?

For many years it has been considered that there are three basic developmental stages of Trypanosoma cruzi: Epimastigote (Epi), Amastigote (Ama) and Trypomastigote (Typo). Epi and Ama are able to divide while Trypo does not divide. Epi are not infective while Ama and Trypo are able to infect host cells. Here we review the available data for the epimastigote form. Taken together the data show that (a) there are intermediate forms between epimastigotes and trypomastigotes in axenic cultures as well as between amastigote and trypomastigote forms within the cells (both in vitro and in vivo), and (c) that the intermediate forms, here designated as "Transitional Epimastigote", most of the time considered as epimastigotes, are able to infect cells. The recognition of the existence of this stage is of practical importance for those work with T. cruzi. Many laboratories working only with T. cruzi in axenic cultures usually consider to work with nonpathogenic cultures. This attitude needs to be changed requiring special care by those working with this protozoan to avoid accidental infections in the laboratory. In view of these observation a new scheme for the life cycle of T. cruzi is proposed.

Toxoplasma gondii Mechanisms of Entry Into Host Cells.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite. Toxoplasma can invade and multiply inside any nucleated cell of a wide range of homeothermic hosts. The canonical process of internalization involves several steps: an initial recognition of the host cell surface and a sequential secretion of proteins from micronemes followed by rhoptries that assemble a macromolecular complex constituting a specialized and transient moving junction. The parasite is then internalized via an endocytic process with the establishment of a parasitophorous vacuole (PV), that does not fuse with lysosomes, where the parasites survive and multiply. This process of host cell invasion is usually referred to active penetration. Using different cell types and inhibitors of distinct endocytic pathways, we show that treatment of host cells with compounds that interfere with clathrin-mediated endocytosis (hypertonic sucrose medium, chlorpromazine hydrochloride, and pitstop 2 inhibited the internalization of tachyzoites). In addition, treatments that interfere with macropinocytosis, such as incubation with amiloride or IPA-3, increased parasite attachment to the host cell surface but significantly blocked parasite internalization. Immunofluorescence microscopy showed that markers of macropinocytosis, such as the Rab5 effector rabankyrin 5 and Pak1, are associated with parasite-containing cytoplasmic vacuoles. These results indicate that entrance of T. gondii into mammalian cells can take place both by the well-characterized interaction of parasite and host cell endocytic machinery and other processes, such as the clathrin-mediated endocytosis, and macropinocytosis.

Membrane-bound extracellular vesicles secreted by parasitic protozoa: cellular structures involved in the communication between cells.

The focus of this review is a group of structures/organelles collectively known as extracellular vesicles (EVs) that are secreted by most, if not all, cells, varying from mammalian cells to protozoa and even bacteria. They vary in size: some are small (100-200 nm) and others are larger (> 200 nm). In protozoa, however, most of them are small or medium in size (200-400 nm). These include vesicles from different origins. We briefly review the biogenesis of this distinct group that includes (a) exosome, which originates from the multivesicular bodies, an important component of the endocytic pathway; (b) ectosome, formed from a budding process that takes place in the plasma membrane of the cells; (c) vesicles released from the cell surface following a process of patching and capping of ligand/receptor complexes; (d) other processes where tubules secreted by the parasite subsequently originate exosome-like structures. Here, special emphasis is given to EVs secreted by parasitic protozoa such as Leishmania, Trypanosoma, Plasmodium, Toxoplasma, Cryptosporidium, Trichomonas, and Giardia. Most of them have been characterized as exosomes that were isolated using several approaches and characterized by electron microscopy, proteomic analysis, and RNA sequencing. The results obtained show clearly that they present several proteins and different types of RNAs. From the functional point of view, it is now clear that the secreted exosomes can be incorporated by the parasite itself as well as by mammalian cells with which they interact. As a consequence, there is interference both with the parasite (induction of differentiation, changes in infectivity, etc.) and with the host cell. Therefore, the EVs constitute a new system of transference of signals among cells. On the other hand, there are suggestions that exosomes may constitute potential biotechnology tools and are important players of what has been designated as nanobiotechnology. They may constitute an important delivery system for gene therapy and molecular-displaying cell regulation capabilities when incorporated into other cells and even by interfering with the exosomal membrane during its biogenesis, targeting the vesicles via specific ligands to different cell types. These vesicles may reach the bloodstream, overflow through intercellular junctions, and even pass through the central nervous system blood barrier. There is evidence that it is possible to interfere with the composition of the exosomes by interfering with multivesicular body biogenesis.

Synthesis and Biological Activity of Novel Zinc-Itraconazole Complexes in Protozoan Parasites and Sporothrix spp.

The new complexes Zn(ITZ)2Cl2 (1) and Zn(ITZ)2(OH)2 (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, 1H and 13C{1H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.

In vitro activities of adamantylidene-substituted alkylphosphocholine TCAN26 against Trypanosoma cruzi: Antiproliferative and ultrastructural effects.

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.

Clathrin coated pit dependent pathway for Trypanosoma cruzi internalization into host cells.

A number of intracellular pathogens are internalized by host cells via multiple endocytic pathways, including Trypanosoma cruzi, the etiological agent of Chagas disease. Clathrin-mediated endocytosis is the most characterized endocytic pathway in mammalian cells. Its machinery was described as being required in mammalian cells for the internalization of large particles, including pathogenic bacteria, fungi, and large virus. To investigate whether T. cruzi entry into host cells can also take advantage of the clathrin-coated vesicle-dependent process, we utilized well-known inhibitors of clathrin-coated vesicle formation (sucrose hypertonic medium, chlorpromazine hydrochloride and pitstop 2) and small interference RNA (siRNA). All treatments drastically reduced the internalization of infective trypomastigotes and amastigotes of T. cruzi by phagocytic (macrophages) and epithelial cells. Clathrin labeling, as observed by fluorescence and electron microscopy, was also observed around the parasites from the initial stages of infection until the complete formation of the parasitophorous vacuole. These unexpected observations suggest the participation of the clathrin pathway in the T. cruzi entry process.

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis.

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.

Hyicin 4244, the first sactibiotic described in staphylococci, exhibits an anti-staphylococcal biofilm activity.

Hyicin 4244 is a small antimicrobial peptide with a broad spectrum of activity that was found in the culture supernatant of Staphylococcus hyicus 4244, the genome of which was then sequenced. The bacteriocin gene cluster (hyiSABCDEFG) was mined from its single chromosome and exhibited a genetic organization similar to that of subtilosin A. All genes involved in hyicin 4244 biosynthesis proved to be transcribed and encode proteins that share at least 42% similarity to proteins encoded by the subtilosin A gene cluster. Due to its resemblance to subtilosin A and the presence of three thioether bonds in its structure, hyicin 4244 is assumed to be a 35-amino acid circular sactibiotic, the first to be described in staphylococci. Hyicin 4244 inhibited 14 staphylococcal isolates from either human infections or bovine mastitis, all biofilm formers. Hyicin 4244 significantly reduced the number of colony-forming units (CFU) and the biofilm formation by two strong biofilm-forming strains randomly chosen as representatives of the strains involved in human infections and bovine mastitis. It also reduced the proliferation and viability of sessile cells in established biofilms. Therefore, hyicin 4244 proved not only to prevent biofilm formation by planktonic cells, but also to penetrate the biofilm matrix in vitro, exerting bactericidal activity against staphylococcal sessile cells. This bacteriocin has the potential to become an alternative antimicrobial for either prevention or treatment of biofilm-related infections caused by different staphylococcal species.

Macropinocytosis: a pathway to protozoan infection.

Among the various endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large amounts of plasma membrane together with extracellular medium, leading to macropinosome formation. These structures are formed when plasma membrane ruffles are assembled after actin filament rearrangement. In dendritic cells, macropinocytosis has been reported to play a role in antigen presentation. Several intracellular pathogens are internalized by host cells via multiple endocytic pathways and macropinocytosis has been described as an important entry site for various organisms. Some bacteria, such as Legionella pneumophila, as well as various viruses, use this pathway to penetrate and subvert host cells. Some protozoa, which are larger than bacteria and virus, can also use this pathway to invade host cells. As macropinocytosis is characterized by the formation of large uncoated vacuoles and is triggered by various signaling pathways, which is similar to what occurs during the formation of the majority of parasitophorous vacuoles, it is believed that this phenomenon may be more widely used by parasites than is currently appreciated. Here we review protozoa host cell invasion via macropinocytosis.

Structures containing galectin-3 are recruited to the parasitophorous vacuole containing Trypanosoma cruzi in mouse peritoneal macrophages.

Trypanosoma cruzi has a complex life cycle where the infective forms for the vertebrate host are trypomastigotes and amastigotes. Both forms invade and lyse their parasitophorous vacuole (PV) membrane, entering into the cytoplasm of its host cells. Galectin-3 (Gal-3) is a protein abundantly distributed in macrophages and epithelial cells. Previous studies demonstrated that Gal-3 binds to a 45KDa mucin of trypomastigotes surface, enhancing its adhesion to the extracellular matrix and even its entry into cells. Gal-3 has another novel cytoplasmic function recently described: a vacuole lyses marker in intracellular bacteria. Considering (1) the importance of Gal-3 during T. cruzi early infection and (2) the importance of T. cruzi PV lyses for parasite differentiation and replication, this study intended to explore a possible recruitment of structures containing Gal-3 (G3CSs) to T. cruzi PVs. Microscopy analyses showed these G3CSs around PVs after 30 and 90 min of amastigotes and trypomastigotes infection, respectively. This recruitment was specific for T. cruzi PVs since we did not observe the same distribution at macrophages vacuoles containing fluorescent microspheres (FM). Concomitantly, this study intended to analyze the participation of actin cytoskeleton in T. cruzi PV maturation. We observed that actin filaments form a "belt-like" structure around trypomastigotes and amastigotes PVs, also labeled for Gal-3. At the time proposed for PV lysis, we observed an actin disassembling while LAMP-1 was recruited to PVs membrane. However, this pattern was maintained in macrophages derived from Gal-3 knockout mice, revealing that the actin belt structure forms independently from Gal-3. Taken together, these data suggest that G3CSs are recruited to vicinity of T. cruzi PV and that actin filaments localize and remain around T. cruzi PVs until the time of its lysis.

Trypanosoma cruzi: Entry into Mammalian Host Cells and Parasitophorous Vacuole Formation.

Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are the metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. The recognition between the parasite and mammalian host cell, involves numerous molecules present in both cell types, and similar to several intracellular pathogens, T. cruzi is internalized by host cells via multiple endocytic pathways. Morphological studies demonstrated that after the interaction of the infective forms of T. cruzi with phagocytic or non-phagocytic cell types, plasma membrane (PM) protrusions can form, showing similarity with those observed during canonical phagocytosis or macropinocytic events. Additionally, several molecules known to be molecular markers of membrane rafts, macropinocytosis, and phagocytosis have been demonstrated to be present at the invasion site. These events may or may not depend on the host cell lysosomes and cytoskeleton. In addition, after penetration, components of the host endosomal-lysosomal system, such as early endosomes, late endosomes, and lysosomes, participate in the formation of the nascent parasitophorous vacuole (PV). Dynamin, a molecule involved in vesicle formation, has been shown to be involved in the PV release from the host cell PM. This review focuses on the multiple pathways that T. cruzi can use to enter the host cells until complete PV formation. We will describe different endocytic processes, such as phagocytosis, macropinocytosis, and endocytosis using membrane microdomains and clathrin-dependent endocytosis and show results that are consistent with their use by this smart parasite. We will also discuss others mechanisms that have been described, such as active penetration and the process that takes advantage of cell membrane wound repair.

Effects of amiodarone and posaconazole on the growth and ultrastructure of Trypanosoma cruzi.

The antifungal posaconazole (PCZ) is the most advanced candidate for the treatment of Chagas disease, having potent anti-Trypanosoma cruzi activity in vitro and in animal models of the disease as well as an excellent safety profile in humans. Amiodarone (AMD) is the antiarrhythmic drug most frequently used for the symptomatic treatment of chronic Chagas disease patients, but it also has specific anti-T. cruzi activity. When used in combination, these drugs exhibit potent synergistic activity against the parasite. In the present work, electron microscopy was used to analyse the effects of both compounds, acting individually or in combination, against T. cruzi. The 50% inhibitory concentration (IC(50)) against epimastigote and amastigote forms was 25 nM and 1.0 nM for PCZ and 8 µM and 5.6 µM for AMD, respectively. The antiproliferative synergism of the drugs (fractional inhibitory concentration<0.5) was confirmed and the ultrastructural alterations in the parasite induced by them, leading to cell death, were characterised using electron microscopy. These alterations include intense wrinkling of the protozoan surface, swelling of the mitochondrion, shedding of plasma membrane vesicles, the appearance of vesicles in the flagellar pocket, alterations in the kinetoplast, disorganisation of the Golgi complex, accumulation of lipid inclusions in the cytoplasm, and the formation of autophagic vacuoles, the latter confirmed by immunofluorescence microscopy. These findings indicate that the association of PCZ and AMD may constitute an effective anti-T. cruzi therapy with low side effects.

Review on Trypanosoma cruzi: Host Cell Interaction.

Trypanosoma cruzi, the causative agent of Chagas' disease, which affects a large number of individuals in Central and South America, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are metacyclic and bloodstream trypomastigote and amastigote. Metacyclic trypomastigotes are released with the feces of the insect while amastigotes and bloodstream trypomastigotes are released from the infected host cells of the vertebrate host after a complex intracellular life cycle. The recognition between parasite and mammalian host cell involves numerous molecules present in both cell types. Here, we present a brief review of the interaction between Trypanosoma cruzi and its host cells, mainly emphasizing the mechanisms and molecules that participate in the T. cruzi invasion process of the mammalian cells.