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Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.

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BACKGROUND: Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES: Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS: We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS: Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in ß-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION: The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

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BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

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The activation of signaling pathways involving protein tyrosine kinases (PTKs) has been demonstrated during Trypanosoma cruzi invasion. Herein, we describe the participation of FAK/Src in the invasion of cardiomyocytes by T. cruzi. The treatment of cardiomyocytes with genistein, a PTK inhibitor, significantly reduced T. cruzi invasion. Also, PP1, a potent Src-family protein inhibitor, and PF573228, a specific FAK inhibitor, also inhibited T. cruzi entry; maximal inhibition was achieved at concentrations of 25µM PP1 (53% inhibition) and 40µM PF573228 (50% inhibition). The suppression of FAK expression in siRNA-treated cells and tetracycline-uninduced Tet-FAK(WT)-46 cells significantly reduced T. cruzi invasion. The entry of T. cruzi is accompanied by changes in FAK and c-Src expression and phosphorylation. An enhancement of FAK activation occurs during the initial stages of T. cruzi-cardiomyocyte interaction (30 and 60min), with a concomitant increase in the level of c-Src expression and phosphorylation, suggesting that FAK/Src act as an integrated signaling pathway that coordinates parasite entry. These data provide novel insights into the signaling pathways that are involved in cardiomyocyte invasion by T. cruzi. A better understanding of the signal transduction networks involved in T. cruzi invasion may contribute to the development of more effective therapies for the treatment of Chagas' disease.

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Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.

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We present the results of the first detailed study of the antiproliferative and ultrastructural effects of amiodarone on Trypanosoma cruzi, the causative agent of Chagas' disease. Moreover, we report the effects of this compound on the recovery of F-actin fibrils, connexin43, and contractility in T. cruzi-infected cardiac myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the symptomatic treatment of Chagas' disease patients with cardiac compromise. In addition, recent studies identified its antifungal and antiprotozoal activities, which take place through Ca(2+) homeostasis disruption and ergosterol biosynthesis blockade. We tested different concentrations of amiodarone (2.5 to 10 µM) on infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the clinically relevant intracellular amastigote form of T. cruzi. Ultrastructural analyses revealed that amiodarone had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distribution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular parasites. Amiodarone is, therefore, a promising compound for the development of new drugs against T. cruzi.

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We have previously demonstrated selection favoring the JG strain of Trypanosoma cruzi in hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

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We have previously demonstrated selection favoring the JG strain of Trypanosoma cruziin hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

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We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in cultured L(6)E(9) myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains. Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains, and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed proportional overall gene-expression alterations. These results indicate that infection with different parasite strains modulates similar but not identical pathways in the host cells.

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Caveolae are motile, membrane-bound compartments that contain a number of molecules that participate in cell signaling. Caveolins are protein markers of caveolae and function in a variety of biological processes. Caveolin-3 (Cav-3) is expressed in muscle cells and Cav-3 null mice display a cardiomyopathic phenotype. Ultrastructural cytochemistry, confocal microscopy and immunoblotting revealed a reduction in Cav-3 expression and an activation of ERK (extracellular-signal-regulated kinase) 48 hours after Trypanosoma cruzi infection of cultured cardiac myocytes. CD-1 mice infected with the Brazil strain of T. cruzi displayed reduced expression of Cav-3 and activation of ERK 66 days post infection (dpi).   By 180 dpi there was a normalization of these values. These data suggest that the reduction in Cav-3 expression and the activation of ERK during the early phase of infection may contribute to the pathogenesis of chagasic cardiomyopathy.

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In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.

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We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.

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Leishmania (Leishmania) chagasi infection activates macrophages, which release several microbicidal agents, including peptidases, to eliminate the parasite. Leishmanicidal mediators released in large amounts may cause morphological and/or functional injuries to the liver. In order to investigate the involvement of peptidases in this phenomenon, an in vitro co-culture model of peritoneal macrophages infected with L. chagasi and hepatocytes was used. High levels of released hepatic transaminases were found in supernatants from infected co-cultures at the same time point in which alterations in hepatocyte morphology and maximum proteolytic activity were observed. The largest proteolytic activity being at pH 10 as well as the greatest efficiency of treatment with 1,10-phenantroline observed in supernatants from the infected co-cultures suggests the presence of metallopeptidases during the leishmanicidal activity by infected macrophages. Furthermore, TNF-alpha levels and high levels of TGF-beta were increased at this time point, and this can be related to the synthesis of metallopeptidases and the conversion of the latent form to the active form. Metallopeptidase activities were detected by gelatin SDS-PAGE in higher amounts in infected macrophages and co-culture supernatant; moreover, one metallopeptidase migrating at 85 kDa produced in excess (41% more) by infected macrophages was identified as MMP-9. This metallopeptidase may be participating in this phenomenon together with other leishmanicidal factors released by these host cells.

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Cardiac damages caused by in vivo infection with Trypanosoma cruzi are still not fully clarified. Here we describe for the first time an in vitro model of fibrosis, hypertrophy, and remodeling induced by T. cruzi in cardiomyocyte spheroids (cardiac microtissues). In this new 3-dimensional system, cardiac spheroids showed spontaneous contractility, with typical cardiac morphology and production of extracellular matrix components. There were 4- and 6-fold increases, respectively, in the area and the volume of T. cruzi-infected cardiomyocytes and whole microtissues, together with a 50% reduction of the cell population. Immunofluorescence showed increased expression of fibronectin, collagen IV, and laminin in the microtissues 144 h after infection. T. cruzi infection induced an increase in both the cellular area and the extracellular matrix components in cardiac spheroids, which contributed to an increase in total microtissue volume, making this a powerful 3-dimensional in vitro model for the study of cardiac-tissue hypertrophy, fibrosis, and remodeling.

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Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)-binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S(35)]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction.

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In this study we assessed the susceptibility of primary fibroblast culture of chicken embryo to infection of P. gallinaceum sporozoites as well as the initial development of exoerithrocytic stages. Fibroblasts were obtained from the chest muscles of chicken embryos and sporozoites were obtained from experimentally infected Aedes fluviatilis salivary glands. After 1h, 3h, 24h, 48h and 72h periods pos-infection, cell cultures were fixed and analyzed both by indirect immunofluorescent-antibody test with anti-circumsporozoite protein monoclonal antibodies and by transmission electron microscopy. Circumsporozoite protein was detected in all parasitic forms. The mean percentage of fibroblasts with adhered or penetrated sporozoites did not significantly increase proportionately to the concentration of parasites in the inoculum, and independently if fetal calf or normal chicken sera were used in the culture medium. It was noted that the longer the incubation time, higher the possibility of the sporozoites to adhere and penetrate to fibroblats. Spozoites were observed penetrating in the fibroblast after 3h incubation when 0.68% of the cells had adhered parasites. Differentiation and development of the exoerythrocytic forms was observed after 24h incubation, when an average of 0,14% of the parasites have already invaded the cells. Developing parasites were found until 72h, when only 0.04% of fibroblasts were infected. Fibroblast cell culture seems to be a valuable experimental tool for in vitro investigation of the exoerytrocytic cycle of P. gallinaceum.

No presente estudo, avaliamos a susceptibilidade de cultura primária de fibroblastos de embrião de galinha à infecção por esporozoítas de P. gallinaceum, assim como o desenvolvimento de estágios do ciclo exoeritrocítico. Fibroblastos foram obtidos a partir da musculatura do peito de embriões de galinha e esporozoítas foram obtidos de glândulas salivares de Aedes fluviatilis experimentalmente infectados. Após períodos de 1h, 3h, 24h, 48h e 72h após a infecção, culturas de células foram fixadas e analisadas através de imunofluorescência indireta empregando-se anticorpos monoclonais contra a proteína circum-esporozoíta e microscopia eletrônica de transmissão. Proteína circum-esporozoíta foi detectada em todas as formas parasitárias. O percentual médio de fibroblastos com esporozoítas aderidos ou já penetrados não aumentou proporcionalmente com a concentração de parasitos no inóculo e independeu se o soro utilizado no cultivo celular era soro bovino fetal ou soro de galinha normal. Foi observado que, quando maior é o período de incubação, maior é a possibilidade dos esporozoítas aderirem e penetrarem nos fibroblastos. Esporozoítas foram observados penetrando em fibroblastos depois de 3h de incubação, quando 0,68% das células tinham parasitos aderidos. A diferenciação e o desenvolvimento das formas exoeritrocíticas foram observados após 24h de incubação, quando somente 0.04% dos fibroblastos achavam-se infectados. A cultura primária de fibroblastos de galinha parece ser um valioso modelo experimental para a investigação in vitro do ciclo exoeritrocítico do P. gallinaceum.

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The cytokine transforming growth factor-beta (TGF-beta) plays various functions in the control of Trypanosoma cruzi infectivity and in the progression of Chagas' disease. When we immunostained T. cruzi-infected cardiomyocytes (after either in vivo or in vitro infections) for TGF-beta, we observed stronger immunoreactivity in parasites than in host cells. TGF-beta immunoreactivity evolved during parasite cycle progression, with intense staining in amastigotes versus very faint staining in trypomastigotes. TGF-beta was present on the surface of amastigotes, in the flagellar pocket, and in intraparasitic vesicles as revealed by electron microscopy. However, no ortholog TGF-beta gene could be identified in the genome of T. cruzi by in silico analysis or by extensive polymerase chain reaction and reverse transcriptase-polymerase chain reaction studies. Immunoreactive TGF-beta was most probably taken up by the parasite from the host cell cytoplasm because such an internalization process of biotinylated TGF-beta could be observed in axenic amastigotes in vitro. These observations represent the first example of a novel mechanism by which a primitive unicellular protozoan can use host cell TGF-beta to control its own intracellular life cycle.

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Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.

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The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.

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We present the results of the first detailed study of the molecular and cellular basis of the antiproliferative effects of the bisphosphonate risedronate (Ris) on Trypanosoma cruzi, the causative agent of Chagas' disease. Ris and related compounds, which block poly-isoprenoid biosynthesis at the level of farnesyl pyrophosphate synthase, are currently used for the treatment of bone resorption disorders, but also display selective activity against trypanosomatid and apicomplexan parasites. Ris induced a dose-dependent effect on growth of the extracellular epimastigote form of T. cruzi; complete growth arrest and cell lysis ensued at 150 microM. Growth inhibition was associated with depletion of the parasite's endogenous sterols, but complete growth arrest and loss of cell viability took place before full depletion of these compounds, suggesting that disappearance of other essential poly-isoprenoids is involved in its anti-parasitic action. Ris had a variety of effects on cellular ultrastructure, including mitochondrial swelling, disorganisation of other organelles, such as reservosomes and the kinetoplast, together with the appearance of autophagic vesicles and progressive vacuolization of the cytoplasm. Ris had selective antiproliferative effects against the clinically relevant amastigote form of T. cruzi, and at 100 microM, was able to prevent completely the development of T. cruzi infection of murine muscle heart or Vero cells, and to cure cultures which were already infected. Ris induced drastic ultrastructural alterations in the intracellular parasites and blocked amastigote to trypomastigote differentiation, with no biochemical or ultrastructural effects on the host cells, which fully recovered their normal structure and activity after treatment. Ris is, therefore, a promising lead compound for the development of new drugs against T. cruzi.

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