Identificaci��n de genotipos de aislamientos de <i>Trypanosoma cruzi</i> mediante an��lisis de ADN

El estudio mediante zimogramas electrofor��ticos de extractos de <i>Trypanozoma cruzi /i> aislados de humanos, animales dom��sticos y selv��ticos y de vectores procedentes de toda el ��rea end��mica de Argentina, permiti�� reconocer la existencia de 12 zimodemos (z) o "cepas isoenzim��ticas". De los zimodemos aislados de hospederos humanos, s��lo dos Z1 y Z12 se encuentran con gran frecuencia y tienen amplia distribuci��n geogr��fica. Se ha observado una correlaci��n significativa entre el zimodemo del par��sito infectante y el cuadro cl��nico. El compromiso card��aco es significativamente mayor en pacientes con Z12 que en aquellos con Z1. Por esta raz��n, la tipificaci��n de la cepa infectante puede ser ��til con fines pron��sticos. Sin embargo, la metodolog��a utilizada para la determinaci��n del zimodemo es muy laboriosa e insume mucho tiempo, lo que reduce las posibilidades de aplicaci��n cl��nica. Dada la similitud del agrupamiento obtenido por el an��lisis de los zimodemos y del ADNk, los problemas de la caracterizaci��n isoenzim��tica de aislamientos pueden ser evitados mediante estudio del ADN del par��sito. (...) Objetivo general Identificaci��n de zimodemos de <i>Trypanosoma cruzi</i> mediante an��lisis de ADN. Objetivos espec��ficos a) Aislamientos de <i>T. cruzi</i> de pacientes chag��sicos cr��nicos con diferentes cuadros cl��nicos ser��n caracterizados utilizando isoenzimas como marcadores gen��ticos. b) Se establecer�� la relaci��n entre la sintomatolog��a observada en cada caso y el zimodemo al cual pertenecen los par��sitos aislados. c) A partir del ADN aislado se proceder�� a: 1. Amplificaci��n de segmentos de ADN al azar dando lugar a fragmentos de ADN que permitan identificar los diferentes zimodemos. 2. Hibridizaci��n con sondas de ADN, lo que permitir��a identificar el zimodemo al que pertenecen los par��sitos de dicho aislamiento. 3. Amplificar, mediante PCR, la regi��n HVRm de <i>T. cruzi</i> directamente en muestras biol��gicas y proceder a su tipificaci��n con sondas espec��ficas.

VARIABILIDAD CL��NICA DE LA MIOCARDIOPAT��A CHAG��SICA: INFLUENCIA DE LA COMPOSICI��N GEN��TICA DEL TRYPANOSOMA CRUZI

El descubrimiento de t��cnicas m��s sensibles para la detecci��n del T. cruzi en el enfermo chag��sico rescat�� el rol primordial del par��sito en la patogenia y actualmente se considera a la enfermedad como el producto de la interacci��n de los genomas del par��sito y el humano. Sin embargo a��n queda por responder por qu�� el 30% de las personas infectadas evolucionan hacia una enfermedad card��aca y el 70% permanece asintom��tico aunque con serolog��a persistente; as�� como tambi��n la amplia variabilidad cl��nica, que puede resultar desde una cardiopat��a sin consecuencias hasta producir muerte s��bita. En este sentido, se ha descripto que la variabilidad gen��tica del par��sito debe estar relacionada con el tropismo del mismo a los diferentes ��rganos del hu��sped y, por lo tanto, con la forma cl��nica de la enfermedad y con las diferencias observadas luego del tratamiento espec��fico de la enfermedad. Es por ello que proponemos determinar la importancia que tiene la composici��n gen��tica del aislamiento de T. cruzi que infect�� al hu��sped y/o la de los clones diferentes que pueden aparecer en sangre para explicar la amplia variabilidad de s��ntomas y signos que manifiestan los pacientes con cardiopat��a chag��sica cr��nica. Estos resultados contribuir��n al entendimiento de la fisiopatogenia de la miocardiopat��a chag��sica y sus variabilidades cl��nicas y facilitar��n establecer el pron��stico y tratamiento de la enfermedad. Pacientes que concurran al Hospital Materno Infantil de la Provincia de C��rdoba, al Hospital Nacional de Cl��nicas y a la Cl��nica Sucre ser��n tratados de acuerdo con la declaraci��n de Helsinki y firmar��n consentimiento informado. Se seguir�� la evoluci��n cl��nico-cardiol��gica por radiograf��a, electrocardiograf��a y ecocardiograf��a. La serolog��a para Chagas se determinar�� por HAI-ELISA. Se obtendr��n muestras de sangre de estos pacientes que se clasificar��n con serolog��a positiva para Chagas sin cardiopat��a, con cardiopat��a leve y con cardiopat��a severa. Extracci��n del ADN: las muestras de sangre perif��rica de cada paciente se mezclar��n con igual volumen de guanidina 6M/EDTA 0,5M. El ADN se extraer�� por t��cnicas convencionales con fenol:cloroformo:alcohol isoam��lico y luego se precipitar�� con etanol. Finalmente la soluci��n se resuspender�� en agua est��ril libre de nucleasas. Se conservar�� a -4�� C hasta su uso para la amplificaci��n del contenido de ADN del par��sito por la reacci��n en cadena de la polimerasa (PCR). PCR: la detecci��n de los par��sitos en cada muestra se determinar�� mediante la amplificaci��n por PCR de un fragmento de la regi��n variable correspondiente al minic��rculo del ADN del kinetoplasto (kADN), utilizando primers espec��ficos para dicha regi��n. An��lisis de la regi��n variable del kADN por enzimas de restricci��n: la caracterizaci��n de los par��sitos de cada muestra se realizar�� adem��s mediante el an��lisis de los fragmentos producidos luego de la digesti��n con enzimas de restricci��n (RFLP). El amplificado producto de la PCR se utilizar�� para la digesti��n con las enzimas de restricci��n y los fragmentos obtenidos ser��n separados por electroforesis en geles de agarosa 2% te��idos con bromuro de etidio. An��lisis de los resultados: Los perfiles de bandas obtenidos luego de la digesti��n con las enzimas de restricci��n de las muestras de sangre de los pacientes se correlacionar��n con la sintomatolog��a cl��nica de cada uno de ellos para determinar si existe relaci��n entre la variabilidad gen��tica del par��sito infectante y la variedad cl��nica presentada. Los perfiles de bandas obtenidos luego de la RFLP de las muestras de sangre se analizar��n cualitativamente por observaci��n de los geles.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (�����m ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10����M Ca(2+) was significantly decreased by 50����g/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125��mM glucose. In RLM suspended in a medium containing 10����M Ca(2+) this lectin, at 50����g/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and �����m disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Trypanosoma Cruzi Strains From Triatomine Collected In Bahia And Rio Grande Do Sul, Brazil.

Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship.

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 ��M. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.

A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 �� 2.47 ��mol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 &#956;g/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA % > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Trypanosoma cruzi benznidazole susceptibility in vitro does not predict the therapeutic outcome of human Chagas disease

Therapeutic failure of benznidazole (BZ) is widely documented in Chagas disease and has been primarily associated with variations in the drug susceptibility of Trypanosoma cruzi strains. In humans, therapeutic success has been assessed by the negativation of anti-T. cruzi antibodies, a process that may take up to 10 years. A protocol for early screening of the drug resistance of infective strains would be valuable for orienting physicians towards alternative therapies, with a combination of existing drugs or new anti-T. cruzi agents. We developed a procedure that couples the isolation of parasites by haemoculture with quantification of BZ susceptibility in the resultant epimastigote forms. BZ activity was standardized with reference strains, which showed IC50 to BZ between 7.6-32 ��M. The assay was then applied to isolates from seven chronic patients prior to administration of BZ therapy. The IC50 of the strains varied from 15.6 �� 3-51.4 �� 1 ��M. Comparison of BZ susceptibility of the pre-treatment isolates of patients considered cured by several criteria and of non-cured patients indicates that the assay does not predict therapeutic outcome. A two-fold increase in BZ resistance in the post-treatment isolates of two patients was verified. Based on the profile of nine microsatellite loci, sub-population selection in non-cured patients was ruled out.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Chemometric Studies on Natural Products as Potential Inhibitors of the NADH Oxidase from Trypanosoma cruzi Using the VolSurf Approach

Natural products have widespread biological activities, including inhibition of mitochondrial enzyme systems. Some of these activities, for example cytotoxicity, may be the result of alteration of cellular bioenergetics. Based on previous computer-aided drug design (CADD) studies and considering reported data on structure-activity relationships (SAR), an assumption regarding the mechanism of action of natural products against parasitic infections involves the NADH-oxidase inhibition. In this study, chemometric tools, such as: Principal Component Analysis (PCA), Consensus PCA (CPCA), and partial least squares regression (PLS), were applied to a set of forty natural compounds, acting as NADH-oxidase inhibitors. The calculations were performed using the VolSurf+ program. The formalisms employed generated good exploratory and predictive results. The independent variables or descriptors having a hydrophobic profile were strongly correlated to the biological data.

Cyclooligomerisation of azido-alkyne-functionalised sugars: synthesis of 1,6-linked cyclic pseudo-galactooligosaccharides and assessment of their sialylation by Trypanosoma cruzi trans-sialidase

Cyclic pseudo-galactooligosaccharides were synthesized by cyclooligomerisation of isomeric azido-alkyne derivatives of beta-D-galactopyranose under Cu(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition reaction conditions. The principal products isolated were cyclic dimers and trimers, with lower amounts of cyclic tetramer and pentamer also evident in some cases. Molecular mechanics calculations suggest very compact but flexible structures for the cyclic trimers, with secondary OH groups exposed outside the macrocycle and available for enzymatic glycosylation. The cyclic dimers and trimers represent a new type of acceptor substrate for Trypanosoma cruzi trans-sialidase, giving rise to doubly and triply sialylated glycomacrocycles, respectively.

Histometry of the sublingual gland in male and female mice (Mus musculus) infected with the RAL strain of the Chagas parasite, Trypanosoma cruzi

Trypanosoma cruzi. The aim of this work was to analyze histologically and histometrically the sublingual gland of mice infected with the RAL strain of T cruzi, according to the sex. Swiss mice (Mus musculus) were inoculated with 2 x 10(4) blood trypomastigotes of the RAL strain of T cruzi. In the peak of the parasitemia (12th day) the mice were sacrificed, and the sublingual glands were fixed in ALFAC. HE-stained histological sections were evaluated histometrically. The parasitemia was higher in females. Histopatologically, acini of the infected animals were smaller, with scanty production of secretion, and smaller striated ducts. The nuclei of the demilunes were smaller and showed amastigote nests in the cytoplasm. Karyometrically, nuclei of the acini, demilunes and striated ducts were smaller in the infected mice. Stereologically, it was observed that relative volumes of acini and ducts were smaller and, inversely, relative volumen were greater for the conjunctive tissue in the infected males. The surface densities of acini and ducts were bigger and the diameter and thickness of the wall were smaller in this group. On the other hand, relative volume of acini was smaller and those of the ducts and conjunctive tissue were bigger in the infected females. The diameter and thickness of the wall of acini were smaller, and those of the striated ducts were bigger in this group. The RAL strain of T cruzi caused general atrophy in the sublingual gland, with numerous nests of parasites in the glandular parenchyma.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD+ from NADH. The production of NADH stimulated by D-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K-m) values determined for D-glyceraldehyde-3-phosphate and NAD(+) were K-m = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.