Trypanosoma Cruzi

This paper reports the in vitro trypanocidal activity evaluation of new carbohydrazide derivatives from 3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine, substituted at C-6 position by phenyl, methyl or trifluoromethyl group. These compounds were evaluated in order to identify the antiparasitic profile against trypomastigote and amastigote forms of Trypanosoma cruzi. The 4-carbohydrazide derivatives presented different profiles of activity. In the investigation of the chemical structure influence in the trypanocidal activity, the results indicated there are large lipophilicity and volume differences among these derivatives. The complementarities of their stereoelectronic and physical-chemical aspects seem to be relevant for the biological activity against T. cruzi.

Trypanosome infections in their natural hosts are frequently di.cult to detect by microscopy\ and culture methods are unreliable and not suitable for all species of Trypanosoma[ A nested PCR strategy for detecting and identifying Trypanosoma species\ suitable for detecting both known and unknown trypanosomes\ is presented[ Thirty!two blood samples from 12 species of Australian birds and mammals were screened by a nested PCR for the presence of Trypanosoma sp[ ssrRNA[ Three infections were detected\ one in an eastern grey kangaroo "Macropus giganteus#\ one in a common wombat "Vombatus ursinus# and one in a platypus "Ornithorhynchus anatinus#[ The kangaroo and wombat are new host records for Trypanosoma sp[^the platypus parasite was Trypanosoma binneyi[ The three parasites could be distinguished by restriction fragment length polymorphisms of the ampli_ed fragment of the ssrRNA gene[ The kangaroo and wombat parasites were also isolated in a semi!solid blood agar medium[ The culture forms of the kangaroo trypanosome had an expanded~agellar sheath in which structures similar to hemidesmosomes were detected by EM[ The nested PCR was at least as sensitive as culture\ and analysis of the PCR products gave parasite!speci_c _ngerprints[ Therefore this method could be suitable for rapidly screening host animals for the presence of trypanosomes and identifying the infecting strain[ Þ 0888 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[ Keywords] Macropus giganteus^Ornithorhynchus anatinus^Ortholfersia macleayi^Trypanosoma binneyi^Trypanosoma cruzi^Vombatus ursinus^Guanidine thiocyanate

We herein describe the synthesis and characterization of nine new 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine. We evaluate their toxic effects against the different evolutive forms of Trypanosoma cruzi, and the host cell (murine macrophages). The results obtained show that mesoionic hydrochloride MI possesses the best activity profile. Compound MI may be a prototype for use in the development of a new chemotherapeutic agent with high efficiency, which may be employed in the treatment of Chagas' disease.

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The biological behaviour of 23 Trypanosoma cruzi isolates in Swiss mice was compared. Nineteen isolates were obtained from patients in the acute phase of Chagas disease (13), sylvatic reservoir hosts (Didelphis marsupialis) (3), and triatomine bugs (Rhodnius robustus) (3) from four regions of the State of Amazonas (AM). Four isolates were obtained from chronic chagasic patients in the State of Paraná (PR): three autochthones, and one allochthone from the State of Minas Gerais. Only one isolate was unable to infect the mice. The AM and PR isolates showed the largest number of significant differences from each other. The former had lower mean values in the pre-patent (5.4 days) and patent (4.6 days) periods (PP), with the parasitaemia (Pmax) reaching a peak of 9.9 Â 10 4 blood trypomastigotes (BT)/mL of blood by the 7th day following inoculation. The AM isolates also had higher positivity to fresh-blood examination (FBE) (84.1%) compared to haemoculture (HC) (58.7%) and polymerase chain reaction (PCR) (33.3%), in addition to higher mortality (2.9%). The PR isolates had higher values for PP (18.5 days) and Pmax (99.9 Â 10 4 BT/mL) as well as higher positivity to FBE (87.2%), HC (100%), and PCR (83.3%). The correlations between the biological behaviour of the T. cruzi isolates and the clinical and epidemiological characteristics of Chagas disease are discussed.

The drugs against tropical neglected diseases, especially Chagas’ Disease, were launched more than 30 years ago, and the development of resistance requires the discovery of new and more effective chemotherapeutic agents. Trypanosoma cruzi has a redox enzyme called trypanothione reductase which was successfully inhibited for peptide derivatives (McKie et al., Amino Acids, 2001, 20: 145). This work aims at studying the mechanism of inhibition of this enzyme through molecular dynamics simulations and evaluating the behavior of some derivatives when inhibiting this protein. We should affirm that any particular molecular dynamics analysis tools (Hbond pattern, 3-D root-mean-square deviation, solvent accessible surface area, etc.) cannot be used apart from the others to justify completely these peptides inhibitory patterns. Based on our results, we reproduced the experimental data and, moreover, we discriminated against a new site in enzyme aperture, which can assist the development of powerful inhibitors against trypanothione reductase enzyme.

A., ROSALES-ENCINA, J. L., . American trypanosomiasis: In situ and generalized features of parasitism and inflammation kinetics in a murine model. Experimental Parasitology 83, 267-274. Mimicking the natural conditions of mammalian infection, metacyclic trypomastigote forms of a Mexican isolate (Ninoa) of Trypanosoma cruzi were inoculated into mice in order to study inflammation kinetics and parasite clearance at the inoculation site, parasite tropism to different organs, and local inflammatory cell infiltrates. Polymorphonuclear cells were detected at the inoculation site as early as 1 hr after inoculation. Peak cell infiltrate was observed at 24 hr; at 96 hr polymorphonuclear cells had disappeared. Mononuclear cell infiltrates began at 24 hr, peaking at Day 15, and then stared disappearing by Day 30. Antigens and parasites were detected by conventional techniques up to 15 min and thereafter became undetectable. Amplification of the hypervariable region of kinetoplast minicircle DNA by polymerase chain reaction was positive from 24 hr to Day 15, and the reaction became negative on Day 30. Myositis was observed in skeletal muscle from Days 7 to 180, it progressed from slight to severe, with an inflammation process which included macrophages, plasmatic cells, and a few eosinophils, the phenotype of the infiltrating cells included LyT2 / and LyT1 / on Day 30, and both cell populations decreased in parallel on Day 180. Antigen and parasite nests were present from Days 15 to 180; in muscle the earliest time at which minicircle DNA was detected was Day 7 and it was present until Day 180. Myocarditis was also observed; it developed from slight to severe in between Days 7 and 30, then gradually decreased, and cleared up. Mononuclear cell infiltrates in the myocardium were present from Days 7 to 180. Antigen and parasite nests were detected at Days 15 and 30 and disappeared at Day 180, although minicircle DNA was detected until the last day of observation. Both skeletal and heart muscles showed inflammatory reaction foci containing T. cruzi antigen. There was twice the number of foci in heart as in skeletal muscle. This ratio was maintained by Day 30; later skeletal muscle showed persistent inflammation which was practically cleared up in the heart. Parasites or antigen were not detected by Day 180 in both skeletal and cardiac muscle; however, minicircle DNA was amplified, indicating that an small proportion of parasites evaded immune response. According to these data, Mexican Ninoa Strain has a classification as biodeme 3. ᭧

is credited with the discovery of Chagas disease because of his 1909 published findings of the Trypanosoma cruzi parasite isolated in the blood of a Brazilian patient [1]. Following his discovery, reports of Chagas disease from across Latin America were published, classifying these countries as endemic with continual transmission occurring [2]. The United States has long been considered a non-endemic country due to the opinion that transmission between local vector populations and humans is non-existent or infrequent [3]. We recently published an extensive review with published reports of routine human exposure to nocturnal Triatominae vector bites and continual reports of autochthonous transmission to Texas locals over the past 60 years [4]. In the midst of performing this historical literature review, we came across a reference to the original source of the term "kissing bug." Accessing the historical archives that were available online for five different urban newspapers, we searched for newspaper articles that included the key words "kissing bug" and were published between January 1, 1899, and December 31, 1899. What we unearthed was an unexpected "outbreak" of kissing bug assaults that were reported in newspapers across the nation. Ten years before Carlos Chagas described Chagas disease (in 1909), the US experienced a multi-city hysteria caused by the routine, nightly bites of the "kissing bug" that resulted in numerous hospitalizations and even a few deaths. On Tuesday, June 20, 1899, The Washington Post published the first article describing "the bite of a strange bug" [5]. Reporters noted "several victims.. .woke up to find both eyes nearly closed by the swelling.. .the matter is beginning to interest physicians" [5]. During the early days of the epidemic, no one had seen the insect, but only reported that it struck during the night without any initial pain. The afflicted victim would awake in the morning with swelling mostly of the eyelid and lips, and occasionally on the hand, shoulder, or arm [5-9]. Swelling typically subsided within 48 to 72 hours and was accompanied, on occasion, by fever and/or symptoms that resembled poisoning [10-12]. Sadly, several fatalities were reported throughout the continuing epidemic, with one death certificate specifically stating "chief and determining cause of death-sting of a kissing bug" [11,13-16]. Between June and July of 1899, reports of kissing bug victims were rampant across the continental US (Fig 1). More than sixty newspaper articles were published that referenced over 100 cases, with The Washington Post, The New York Times, The Atlanta Constitution, the Boston Daily Globe, and the Chicago Daily Tribune newspapers publishing the majority of the relevant articles. (Of notable interest, patients' names, ages, and residential addresses were published in the newspaper alongside their accounts.) The bug responsible for these attacks was anecdotally referred to as the "kissing bug," "Hobson bug," and "Dorsey Foultz bug" [17]. Despite the ambiguous nature of the newly emerging epidemic, early reports from local entomologists

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite. Ó

Background: Triatomines (Hemiptera, Reduviidae) are vectors of Trypanosoma cruzi, the causative agent of Chagas disease, one of the most important vector-borne diseases in Latin America. This study compares the environmental niche spaces of Triatoma brasiliensis and Triatoma melanica using ecological niche modelling and reports findings on DNA barcoding and wing geometric morphometrics as tools for the identification of these species. Methods: We compared the geographic distribution of the species using generalized linear models fitted to elevation and current data on land surface temperature, vegetation cover and rainfall recorded by earth observation satellites for northeastern Brazil. Additionally, we evaluated nucleotide sequence data from the barcode region of the mitochondrial cytochrome c oxidase subunit I (CO1) and wing geometric morphometrics as taxonomic identification tools for T. brasiliensis and T. melanica. The ecological niche models show that the environmental spaces currently occupied by T. brasiliensis and T. melanica are similar although not equivalent, and associated with the caatinga ecosystem. The CO1 sequence analyses based on pair wise genetic distance matrix calculated using Kimura 2-Parameter (K2P) evolutionary model, clearly separate the two species, supporting the barcoding gap. Wing size and shape analyses based on seven landmarks of 72 field specimens confirmed consistent differences between T. brasiliensis and T. melanica. Our results suggest that the separation of the two species should be attributed to a factor that does not include the current environmental conditions. However, as the caatinga is a biome that has existed in the area for at least the last 18,000 years, past conditions might have had an influence in the speciation process. The DNA Barcoding approach may be extended to these members of the subfamily Triatominae.

Modelos anteriores de la glico´lisis de la forma sangu´ınea de Trypanosoma brucei arrojaban valores de flujo coherentes con resultados experimentales, sin embargo asum´ıan una suma conservada de fosfatos y un balance energ´etico de cero en el interior del glicosoma. En este trabajo se construyeron modelos de las formas sangu´ınea y proc´ıclica de Trypanosoma brucei y de la forma epimastigote de Trypanosoma cruzi que predicen valores de flujo glicol´ıtico similares a los reportados experimentalmente y permiten modelar condiciones de alto consumo de ATP y G6P, como rutas de s´ıntesis y ruta de las pentosas fosfato, presentes en los par´asitos pero ignoradas hasta el momento en la los modelos existentes en la literatura. El valor del flujo glicol´ıtico del modelo de la forma sangu´ınea de T. brucei fue de 91.08 nmol de glucosa/min/mg prot., para la forma proc´ıclica de 6.99 nmol de glucosa/min/mg prot., ambos valores dentro del rango de los resultados experimentales. Ambos modelos alcanzan el estado estacionario para todas las variables, en el caso del modelo de la forma proc´ıclica es necesario introducir los t´erminos de consumo de ATP y G6P en el sistema de ecuaciones para evitar la acumulaci´on de fructosa-1,6-bisfosfato. Las proporciones de productos finales fueron similares a las experimentales tanto en el modelo de la forma sangu´ınea como en el modelo de la forma proc´ıclica. Los valores de concentracio´n de los metabolitos fueron similares a los reportados en la literatura, una mejor estimacio´n que la de modelos anteriores. Se determin´o que la produccio´n de succinato en el modelo de la forma proc´ıclica esta´ ligada al balance redox de la c´elula y a la velocidad del transporte de triosas hacia y desde la mitocondria. Se demostro´ mediante un modelo algebraico que la acumulacio´n de fructosa-1,6bisfosfato es una consecuencia directa de la configuraci´on del sistema. El que sea necesario introducir t´erminos de consumo en las ecuaciones para alcanzar el estado estacionario en todas las variables es un argumento a favor de considerar el glicosoma como una organela de s´ıntesis.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidiWed by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have diVerent size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.

Background: Usually the analysis of the various developmental stages of Trypanosoma cruzi in the experimentally infected vertebrate and invertebrate hosts is based on the morphological observations of tissue fragments from animals and insects. The development of techniques that allow the imaging of animals infected with parasites expressing luciferase open up possibilities to follow the fate of bioluminescent parasites in infected vectors. Methods: D-luciferin (60 μg) was injected into the hemocoel of the whole insect before bioluminescence acquisition. In dissected insects, the whole gut was incubated with D-luciferin in PBS (300 μg/ml) for ex vivo bioluminescence acquisition in the IVIS W Imaging System, Xenogen. Results: Herein, we describe the results obtained with the luciferase gene integrated into the genome of the Dm28c clone of T. cruzi, and the use of these parasites to follow, in real time, the infection of the insect vector Rhodnius prolixus, by a non-invasive method. The insects were evaluated by in vivo bioluminescent imaging on the feeding day, and on the 7 th, 14 th, 21 st and 28 th days after feeding. To corroborate the bioluminescent imaging made in vivo, and investigate the digestive tract region, the insects were dissected. The bioluminescence emitted was proportional to the number of protozoans in regions of the gut. The same digestive tracts were also macerated to count the parasites in distinct morphological stages with an optical microscope, and for bioluminescence acquisition in a microplate using the IVIS W Imaging System. A positive correlation of parasite numbers and bioluminescence in the microplate was obtained.

| midgut of the mosquitoes Anopheles stephensi Liston and Aedes aegypti L. (Diptera:Culicidae). Parasitol. Res. 75, 268-279 40 Huber, M. et al. (1991) Malaria parasite chitinase and penetration of the mosquito peritrophic membrane. Proc. Natl Acad. Sci. LISA 88, 2807-28111 41 Ham, P.J. et al. (1991 Effect of N-acetyI-D-glucosamine on the migration of Brugia pahangi microfilariae into the haemocoel of Aedes aegypti. Med. Vet. EntomoL 5,[485][486][487][488][489][490][491][492][493] In order to replicate, many parasites must invade host cells. Changes in the intracelhdar Ca 2÷ concentration ([Ca~-*]) of different parasites and tissue culttnv cells during their interaction have been studied. Art increar.e .61 cytosolic Ca 2 ÷ in Trypanosoma cruzi trypomastigotes occurs after association of the parasites with host cell~. Ca 2 ÷ mobilization in the host cells also takes place upon contact with T. cruzi trypomastigotes, Leishmania donovani amastigotes or Plasmodium falciparum merozoites. When Ca 2÷ transients are prevented by intracelhdar Ca 2. chelators, a decrease in parasite association to host cells is observed. This reveals the importance of [Ca'-÷]i in the process of parasitehost cell interaction, as disc,ssed here by Roberto Docampo and Silvia MoremJ.

When epimastigote forms of Trypanosoma cruzi grown in a rich medium (LIT) are transferred to a simple, chemically defined medium (TAU3AAG, containing Ca 2+, Mg 2+, K +, Na +, L-proline, L-glutamate, and L-aspartate in phosphate buffer), they transform into trypomastigote forms. Morphometric analysis of transmission electron micrographs of thin sections of parasites collected at different steps of the transformation process showed that no changes occurred in the volume density of mitochondria and cytoplasmic vacuoles. However, a significant increase in the volume density of the kinetoplast DNA network as well as the lipid inclusions and a decrease in that of the reservosome (a special type of endosome) was observed. These observations suggest that during differentiation, T. cruzi accumulates lipids and uses molecules contained in the reservosome as its main energy source.

Chagas disease is a chronic, systemic, parasitic infection caused by the protozoan Trypanosoma cruzi, and was discovered in 1909. The disease affects about 8 million people in Latin America, of whom 30-40% either have or will develop cardiomyopathy, digestive megasyndromes, or both. In the past three decades, the control and management of Chagas disease has undergone several improvements. Large-scale vector control programmes and screening of blood donors have reduced disease incidence and prevalence. Although more effective trypanocidal drugs are needed, treatment with benznidazole (or nifurtimox) is reasonably safe and effective, and is now recommended for a widened range of patients. Improved models for risk stratification are available, and certain guided treatments could halt or reverse disease progression. By contrast, some challenges remain: Chagas disease is becoming an emerging health problem in non-endemic areas because of growing population movements; early detection and ...

From the aerial parts of Cissampelos pareira L. (Menispermaceae), a chalcone-flavone dimer has been isolated which, mainly from NMR spectroscopic and MS data, was proved to be 2-(4-hydroxy-3-methoxyphenyl)-7-(4-methoxyphenyl)-6-(2-hydroxy-4,6dimethoxybenzoyl)-furano[3,2-g]benzopyran-4-one. This has been assigned the trivial name cissampeloflavone. The compound has good activity against Trypanosoma cruzi and T. brucei rhodesiense and has a low toxicity to the human KB cell line. #

El presente trabajo describe un protocolo para la obtención de poblaciones puras y estables de amastigotes extracelulares de Trypanosoma cruzi. El mismo está basado en un mecanismo de selección de estadios por efectos combinados de cambios térmicos y presencia de plasma humano en el medio de incubación. Epimastigotes (clon Bra C15C2) se incubaron a 27°C durante 96 h y subcultivados cada 96 h a 36.5°C en el medio definido F69. Luego de 3 subcultivos a 36.5°C y posterior incubación en presencia de 5% de plasma humano normal durante el mismo lapso, se obtuvo una población de parásitos constituida principalmente por amastigotes que pudo ser mantenida durante tiempo indefinido por subcultivos en medios F69 y F29. Los estudios morfológicos de dichos amastigotes mostraron estructuras similares a los obtenidos por otros métodos. La cinética de crecimiento de amastigotes dependió de la temperatura de incubación y del medio de cultivo utilizado (F29 ó infusión cerebro corazón BHI). La incubación en el medio F29 a 27°C mostró las fases pre-exponencial y logarítmica de 72-96 h y 24-48 h respectivamente. Se obtuvieron resultados similares utilizando el medio BHI. Cultivos con o sin recambio de medio, sufrieron transformación a epimastigotes al cabo de 16 días. A 36.5°C, la cinética de crecimiento en el medio F29 fue similar, aunque no se observó transformación a epimastigotes. El medio BHI no fue apto para el crecimiento de amastigotes a esta temperatura. En tanto, el F29, por subcultivos, permitió el mantenimiento de amastigotes con una pureza superior al 99% durante tiempo indefinido

Chagas disease, a chronic systemic parasitosis caused by the Kinetoplastid protozoon Trypanosoma cruzi, is the first cause of cardiac morbidity and mortality in poor rural and suburban areas of Latin America and the largest parasitic disease burden in the continent, now spreading worldwide due to international migrations. A recent change in the scientific paradigm on the pathogenesis of chronic Chagas disease has led to a consensus that all T. cruzi-seropositive patients should receive etiological treatment. This important scientific advance has spurred the rigorous evaluation of the safety and efficacy of currently available drugs (benznidazole and nifurtimox) as well as novel anti-T. cruzi drug candidates in chronic patients, who were previously excluded from such treatment. The first results indicate that benznidazole is effective in inducing a marked and sustained reduction in the circulating parasites' level in the majority of these patients, but adverse effects can lead to treatment discontinuation in 10-20% of cases. Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, are better tolerated but their efficacy at the doses and treatment duration used in the initial studies was significantly lower; such results are probably related to suboptimal exposure and/or treatment duration. Combination therapies are a promising perspective but the lack of validated biomarkers of response to etiological treatment and eventual parasitological cures in chronic patients remains a serious challenge.

Chagas disease represents a serious public health problem in South America. The first line of treatment is Nifurtimox and Benznidazole which generate toxic effects in treated patients. We have recently shown that a number of 5-nitrofuranes possess activity against Trypanosoma cruzi through oxidative stress and inhibition of parasite ergosterol biosynthesis, specifically at the level of squalene epoxidase. Here, we identify new 5-nitrofuranes and the thia-analogues with excellent effects on the viability of T. cruzi and adequate parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated, during 120 h, with the compounds showed that some of them accumulated squalene suggesting the squalene epoxidase activity inhibition of the parasite. Nifurtimox was able to accumulate squalene only at lower incubation times. Due to this fact some derivatives were also tested as antifungal agents. Quantitative structure-activity relationship studies were also performed showing relevant features for further new derivatives design. Taken together, the results obtained in the present work point to a more general effect of 5-nitrofuranes and 5-nitrothiophenes in trypanosomatids, opening potential therapeutic possibilities of them for these infectious diseases.

Ethnopharmacological relevance: In Russian traditional medicine, an extract from the mushroom Inonotus obliquus (Fr.) Pil´at is used as an anti-tumor medicine and diuretic. It has been reported that Inonotus obliquus has therapeutic effects, such as anti-inflammatory, immuno-modulatory and hepatoprotective effects. This study was designed to investigate the chemical composition and biological properties of aqueous and ethanolic extracts of Inonotus obliquus from Finland, Russia, and Thailand. Their antioxidative, antimicrobial, and antiquorum properties were tested as well as the cytotoxicity on various tumor cell lines.

Anti-native DNA antibodies can be detected by indirect immunofluorescence assay with Crithidia luciliae, displaying an annular image due to a kinetoplast containing double stranded DNA. Other structures such as membrane, flagellum and basal corpuscle can be stained as well, showing what is called atypical fluorescent images. As C. luciliae belongs to the Trypanosomatidae family, which include the human pathogens Trypanosoma cruzi and Leishmania spp., it was considered that these atypical images could be caused by cross-reactions. Serological studies for Chagas' disease were performed in 105 serum samples displaying atypical images. Sixty four percent of the samples from non endemic and 78.3% from endemic areas for Chagas' disease showed fluorescence in both, membrane and flagellum (joint image). Fifty samples from normal blood donors and 57 samples from patients with conective tissue diseases were tested with C. luciliae. None of them presented the joint image except for two...

Carreira, J. C. A., Jansen, A. M., Meirelles, M. de N., Costa e Silva, F., Lenzi, H. L. 2001. Trypanosoma cruzi in the scent glands of Didelphis marsupialis: The kinetics of colonization. Experimental Parasitology97, 129–140. This study examined the dynamics of colonization of Trypanosoma cruzi in the scent glands of the opossum Didelphis marsupialis following direct inoculation with 105 epimastigotes of isolate G-49 (an opossum-derived strain). One, three, and five days, 1 month, and 1 year after inoculation, scent glands were fixed for analysis using brightfield and electron microscopies. One day after inoculation the parasites, mainly as epimastigotes, were randomly distributed into the lumen. From the third day on, the parasites still in the form of epimastigotes tended to concentrate closer to the epithelium. The flagellates reached the definitive distribution pattern on the fifth day, when they formed huge clusters deep into the foveae. In samples collected 1 month and 1 year after inoculation, the ratio of epimastigotes:trypomastigotes was 1:1, with epimastigotes predominating near the epithelium and trypomastigotes far from it. Our observations suggest that T. cruzi grows continuously in the scent glands and does not depend on adhesion to promote metacyclogenesis. Metacyclogenesis far from the epithelium seems to be an important selective advantage to both host and parasite, since it assures the elimination of the infective forms of the parasite when the host expels the glands' contents, which occurs in frightening situations or at times of stress. The morphological characteristics of infected and noninfected scent glands using transmission and scanning electron microscopies were also described.

A partir del caso de una niña de seis años con diagnóstico confirmado de enfermedad de Chagas aguda, procedente de la localidad de Pozuzo (Oxapampa-Junín) en al selva central del Perú, un área no señalada como endémica en el país, se realizó un estudio ...

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtelomeric in location while one is chromosomeinternal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a public health problem in South America affecting millions of people, and more recently several thousands in countries where the disease is not endemic. Due to the magnitude of the problem, finding a cure for this disease remains a major challenge. The aim of this study is to evaluate the trypanocidal activity of ergosterol peroxide (5a, 8a-epidioxy-22E-ergosta-6, 22-dien-3b-ol) isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida. The ergosterol peroxide showed strong trypanocidal activity on the intracellular form of T. cruzi. Ergosterol peroxide had an inhibitory concentration (IC 50) of 6.74 mg/mL on T. cruzi, but showed no lytic action on erythrocytes and no cytotoxic effect on mammalian cells at concentrations higher than 1600 mg/mL. The interaction of Trypanosoma cruzi with ergosterol peroxide in vitro resulted in a strong lytic activity possibly due to the disruption of the parasite membrane. This is the first report of trypanocidal activity, a new biological property of ergosterol peroxide isolated from Pleurotus ostreatus (Jacq.) P. Kumm. f. sp. Florida.

Molecular biology techniques were used to identify 218 fungi from soil samples collected from four islands of Antarctica. These consisted of 22 taxa of 15 different genera belonging to the Zygomycota, Ascomycota, and Basidiomycota. Mortierella, Antarctomyces, Pseudogymnoascus, and Penicillium were the most frequently isolated genera and Penicillium tardochrysogenum, Penicillium verrucosus, Goffeauzyma gilvescens, and Mortierella sp. 2 the most abundant taxa. All fungal isolates were cultivated using solid-state fermentation to obtain their crude extracts. Pseudogymnoascus destructans, Mortierella parvispora, and Penicillium chrysogenum displayed antiparasitic activities, whilst extracts of P. destructans, Mortierella amoeboidea, Mortierella sp. 3, and P. tardochrysogenum showed herbicidal activities. Reported as pathogenic for bats, different isolates of P. destructans exhibited trypanocidal activities and herbicidal activity, and may be a source of bioactive molecules to be conside...

The aim of this review is to gather the current knowledge of Trypanosoma cruzi's virulence factors described to date in an integrative way, relating these with the parasite's life cycle and trying to elucidate their importance in each process. Several aspects relevant for the parasite's survival, such as invasion, resistance to oxidative damage, escape from the phagolysosomal vacuole and differentiation, among others, will be discussed. However, there is still a lot to learn about what virulence really means in T. cruzi and which parasite molecules are absolutely required to make T. cruzi one of the most successful pathogens to invade, survive and persist in a mammalian host.

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly speci¢c for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae di¡er in sequence, conformation and substrate speci¢city suggests that a di¡erent family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly speci¢c for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae di¡er in sequence, conformation and substrate speci¢city suggests that a di¡erent family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases.

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Four compounds were isolated from Brazilian propolis. They are identified as: (1) 3-prenyl-4-hydroxycinnamic acid (PHCA), (2) 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyrane (DCBEN), (3) 3,5-diprenyl-4-hydroxycinnamic acid (DHCA), and (4) 2,2-dimethyl-6-carboxyethenyl-8-prenyl-2H-1-benzopyran (DPB). The structures of the compounds were determined by MS and NMR techniques. All compounds were assayed against Trypanosoma cruzi and the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis. Compounds (1) to (4) were active against T. cruzi. Except (1), all compounds presented activity against the bacteria tested. When compounds (1)-(3) were tested in the guinea pig isolated trachea, all induced a relaxant effect similar to propolis extract.

As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3 Å and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6 Å over 482 C atoms). Kinetically , the K m for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The K m for NADPH for the T. brucei enzyme was found to be 0.77 M using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S = K m values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thiori-dazine and citalopram. The relative IC 50 values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.

This manuscript describes the evaluation of anti-infective potential in vitro of organic extracts from nine sponges, one ascidian, two octocorals, one bryozoan, and 27 seaweed species collected along the Brazilian coast. Antimicrobial activity was tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) by the disk diffusion method. Antiprotozoal activity was evaluated against Leishmania braziliensis (MHOM/BR/96/LSC96-H3) promastigotes and Trypanosoma cruzi (MHOM/BR/00/Y) epimastigotes by MTT assay. Activity against intracellular amastigotes of T. cruzi and L. brasiliensis in murine macrophages was also evaluated. Antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the plaque number reduction assay (IC 50). Cytotoxicity on VERO cells was evaluated by the MTT assay (CC 50). The results were expressed as SI = CC 50 /IC 50. The most promising antimicrobial results were obtained against S. aureus and C. albicans with Dragmacidon reticulatum. Among the seaweeds, only Osmundaria obtusiloba showed moderate activity against P. aeruginosa. Concerning antiprotozoal activity, Bugula neritina, Carijoa riseii, Dragmaxia anomala and Haliclona (Halichoclona) sp. showed the most interesting results, mainly against extracellular promastigote forms of L. braziliensis (66, 35.9, 97.2, and 43.6% inhibition, respectively). Moreover, six species of seaweeds Anadyomene saldanhae, Caulerpa cupressoides, Canistrocarpus cervicornis, Dictyota sp., Ochtodes secundiramea, and Padina sp. showed promising results against L. braziliensis (87.9, 51.7, 85.9, 93.3, 99.7, and 80.9% inhibition, respectively), and only Dictyota sp. was effective against T. cruzi (60.4% inhibition). Finally, the antiherpes activity was also evaluated, with Haliclona (Halichoclona) sp. and Petromica citrina showing the best results (SI = 11.9 and SI > 5, respectively). All the active extracts deserve special attention in further studies to chemically characterize the bioactive compounds, and to perform more refined biological assays.

Many studies have shed light on the mechanisms underlying both immunoprotection and immune dysregulation arising after Trypanosoma cruzi infection. However, little is known about the impact of benznidazole (N-benzyl-2-nitroimidazole acetamide), the drug available for clinical treatment of the infection, on the immune system in the infected host. In the present study we investigated the effect of benznidazole therapy on the lymphoid compartment during the course of experimental T. cruzi infection. Although amelioration of a variety of clinical and parasitological signs was observed in treated mice, amelioration of splenocyte expansion was not detected. Interestingly, this sustained splenomegaly observed in benznidazole-treated mice showed a preferential expansion of CD8 ؉ T lymphocytes. Moreover, although benznidazole treatment blocked the expansion of recently activated CD4 ؉ and CD8 ؉ T cells seen in infected hosts, benznidazole treatment led to a selective expansion of effector and memory CD8 ؉ T lymphocytes in association with a lower rate of apoptosis. In addition, the surviving treated animals were protected from reinfection. Together, these data suggest that, in addition to its well-known direct role in blocking parasite replication in vivo, benznidazole appears to directly affect immune regulation in T. cruzi-infected hosts.