Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Posible transmisión oral de la enfermedad de Chagas en trabajadores del sector de los hidrocarburos en Casanare, Colombia, 2014 / Possible oral transmission of Chagas disease among hydrocarbons sector workers in Casanare, Colombia, 2014

RESUMEN Introducción. Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, puede transmitirse por vía oral tras la ingestión de alimentos o bebidas contaminadas. En la semana epidemiológica 14 del 2014, se notificaron dos casos de enfermedad aguda de Chagas en Paz de Ariporo, Casanare, entre trabajadores del sector de los hidrocarburos, episodio que motivó la investigación epidemiológica en el área. Objetivo. Caracterizar la población afectada, establecer medidas de control y confirmar el brote. Materiales y métodos. Se hizo una investigación de brote con los siguientes componentes: a) búsqueda de personas sintomáticas (cuadro clínico sugerente de enfermedad de Chagas según la definición de caso), para remitirlas a atención médica; b) aplicación de una encuesta entomológica en 192 de 197 viviendas; c) inspección sanitaria y análisis microbiológico de muestras de alimentos, y d) estudio de reservorios. La organización y el análisis de los datos se hicieron mediante estadística descriptiva con el programa Epi-Info 7.1.5. Asimismo, se establecieron los índices de infestación en el domicilio y el peridomicilio. Resultados. Se registraron 552 personas expuestas y se confirmaron por laboratorio 40 casos (7,2 %); siete casos se dieron en mujeres (17,5 %) y 33 en hombres (82,5%), es decir, en una relación de 1:5. La edad promedio fue de 39,1 (± 10,8) años, la tasa de ataque, de 7,2 %, y la letalidad, de 5 % (2/40). Los signos y síntomas incluyeron fiebre en el 100 % de los casos, cefalea en el 80 %, mialgias y artralgias en el 65 %, edema facial en el 55 %, y dolor abdominal en el 37,5 %. El tiempo promedio de incubación fue de 17 (3-21) días. El índice de infestación de Rhodnius prolixus fue de 3,3 % en el domicilio y de 2,2 % en el peridomicilio. En los cinco restaurantes inspeccionados, se encontraron condiciones sanitarias deficientes y alimentos con niveles de contaminación microbiológica inaceptables. Por último, un perro y dos zarigüeyas fueron positivos para los anticuerpos IgG en la prueba ELISA. Conclusiones. Mediante el análisis de las características epidemiológicas, ambientales y sanitarias, se confirmó un brote agudo de enfermedad de Chagas por exposición ocupacional y de posible transmisión oral, que podría ser el de mayor proporción reportado hasta la fecha en Colombia.

ABSTRACT Introduction: Trypanosoma cruzi, the etiological agent for Chagas disease, can be transmitted by oral intake of contaminated food or drinks. During epidemiological week 14 of 2014, two cases of acute Chagas disease were notified among hydrocarbons sector workers in Paz de Ariporo, Casanare. Objective: To characterize the affected population, to establish control and prevention measures and to confirm the outbreak. Materials and methods: We conducted an outbreak investigation that included the following components: a) Search for symptomatic people compatible with Chagas disease according to the case definition for their referral to medical services; b) entomological survey (192/197 houses); c) sanitary inspection and microbiological analysis of food samples; and d) study of reservoirs. Data management and analysis were done with Epi-Info 7.1.5 using descriptive statistics. We also calculated intradomicile and peridomicile triatomine infestation indexes. Results: We detected 552 exposed people; 40 had the disease (7.2%), of whom seven were women (17,5%) and 33, men (82.5%), i.e., a male-female ratio of 5:1. The mean age was 39.1 ± 10.8 years; the attack rate was 7.2% and lethality, 5% (2/40). Symptoms included fever (100% of cases), headache (80%), myalgia and arthralgia (65%), facial edema (55%), and abdominal pain (37.5%). The mean incubation time was 17 days (range: 3-21). Rhodnius prolixus domiciliary infestation index was 3.3 % and 2.2% in the peridomicile. In the five restaurants inspected sanitary conditions were deficient and food samples were microbiologically non-conforming. We found a dog and two opossums positive for IgG antibodies by ELISA. Conclusions: Environmental, sanitary and epidemiological conditions at the place confirmed an outbreak of Chagas diseases related to occupational exposure, possibly by oral transmission, which may be the largest to date in Colombia.

Diversidad de Triatominae (Hemiptera: Reduviidae) en Santander, Colombia: implicaciones epidemiológicas / Diversity of Triatominae (Hemiptera: Reduviidae) in Santander, Colombia: Epidemiological implications

Resumen Introducción: Los triatominos domiciliados y silvestres constituyen un problema de impacto epidemiológico en el departamento de Santander, pues se han asociado recientemente con brotes agudos de la enfermedad de Chagas, por lo cual el análisis de su diversidad y variación temporal contribuye al conocimiento de su biología y ecología en una de las áreas más endémicas del país. Objetivo: Analizar la diversidad de triatominos en dos regiones de Santander. Materiales y métodos: Se analizó la información de la base de datos del Laboratorio de Entomología del Centro de Investigaciones en Enfermedades Tropicales de la Universidad Industrial de Santander (CINTROP-UIS), la cual contiene registros de triatominos en Santander. La información se separó en dos regiones, el Magdalena Medio y la zona andina, para cada una de las cuales se diseñaron curvas de acumulación de especies y de rango de abundancia, se calcularon los índices de diversidad y de igualdad, se analizó la colonización y se evaluó la variación temporal o persistencia de la comunidad. Resultados: El 95 % de los triatominos provenía de la zona andina y, el 4,57 %, del Magdalena Medio, con nueve y diez especies, respectivamente. Se encontró mayor diversidad y riqueza en el Magdalena Medio en comparación con la zona andina. Las especies dominantes en la zona andina fueron Rhodnius prolixus y Triatoma dimidiata, mientras que en Magdalena Medio fueron Rhodnius pallescens y Panstrongylus geniculatus. La variación temporal mostró persistencia de las comunidades en el tiempo. Conclusiones:. Los resultados evidenciaron diferencias en la diversidad de las dos regiones, además del potencial de las especies silvestres para ocupar ecótopos artificiales. La intrusión de triatominos y la reciente incriminación de especies silvestres en la transmisión de Trypanosoma cruzi, indican la necesidad de un mayor conocimiento de la ecología de estos vectores para orientar las estrategias de control.

Abstract Introduction: Domestic and wild triatomines in the department of Santander have an epidemiological impact, as recently they have been linked to outbreaks of acute Chagas disease. The analysis of their diversity and temporal variation contributes to the understanding of their biology and ecology in one of the most endemic areas of the country. Objectives: To analyze triatominae diversity in two regions of Santander. Materials and methods: We analyzed the triatomine records for Santander contained in the CINTROPUIS entomology lab database. We grouped the information for two regions: the Middle Magdalena area and the Andean region, and for each one we designed species accumulation and range-abundance curves, we calculated diversity and equality indices, and we analyzed colonization and temporal variation or persistence of the community. Results: Ninety five percent of triatomines came from the Andean area and 4.57% from Magdalena Medio, with nine and ten species each. The dominant species in the Andean area were Rhodnius prolixus and Triatoma dimidiata while in Magdalena Medio they were Rhodnius pallescens and Panstrongylus geniculatus. We found a greater diversity and richness in Middle Magdalena compared to the Andean area. The temporal variation showed persistence of communities over time. Conclusions: Results revealed differences in the diversity of the two regions and the potential of wild species to occupy artificial ecotopes. Triatomines intrusion and the recent involvement of wild species in the transmission of Trypanosoma cruzi emphasize the need to further investigate the ecology of these vectors in order to guide population control strategies.

Riesgo de transmisión de la enfermedad de Chagas por intrusión de triatominos y mamíferos silvestres en Bucaramanga, Santander, Colombia / Risk of transmission of Chagas disease by intrusion of triatomines and wild mammals in Bucaramanga, Santander, Colombia

Introducción: La notificación de triatominos en las viviendas de algunos barrios de Bucaramanga motivó la realización de este estudio. Objetivo: Evaluar la intrusión de triatominos y mamíferos, así como algunos factores de riesgo para la enfermedad de Chagas en viviendas urbanas. Materiales y métodos: En un barrio de Bucaramanga, Santander, se recolectaron triatominos mensualmente durante un año con participación comunitaria mediante búsqueda manual en el alumbrado público, y el uso de trampas de luz, cebo animal y atrayentes químicos en el bosque cercano. Los reservorios se recolectaron con trampas cebadas. Los insectos y mamíferos se determinaron y examinaron para establecer su infección natural. Los factores de riesgo de las viviendas se midieron mediante una encuesta sobre factores sociales y ambientales. Resultados: Se recolectaron 11 adultos de Panstrongylus geniculatus y 63 de Rhodnius pallescens en el bosque, en zonas de recreación en el peridomicilio y en el domicilio, incluidas dos hembras y 21 ninfas de R. pallescens en dormitorios. Se capturaron dos ejemplares de Didelphis marsupialis en el bosques adyacente. De los 11 individuos de P. geniculatus capturados, se examinaron nueve, de los cuales cinco fueron positivos para Trypanosoma cruzi (56 %); de los 63 individuos de R. pallescens capturados, se examinaron ocho, cuatro de los cuales fueron positivos para T. cruzi (50 %). De dos especímenes de D. marsupiales capturados, uno fue examinado y se encontró que era positivo para T. cruzi. No se pudo establecer un factor de riesgo significativo, sin embargo, las viviendas con reporte de triatominos se encontraban más cerca del bosque adyacente. Conclusiones: El hallazgo de especies de triatominos intrusivas y de mamíferos con T. cruzi en el domicilio y el peridomicilio, así como en los bosques periurbanos, demuestra el riesgo de infección en las poblaciones que habitan en viviendas urbanas adyacentes a los ecótopos donde se mantiene el ciclo silvestre.

Abstract Introduction: Notice of triatomines in dwellings of some neighborhoods in Bucaramanga motivated the realization of this study. Objetive: To evaluate the intrusion of triatomines and mammals, as well as some risk factors in urban dwellings. Materials and methods: Triatomines were collected in a neighborhood in Bucaramanga, Santander, on a monthly basis during one year with participation of the community. Collection included manual search in lamp posts, use of light traps, animal bait, and chemical attractants in nearby forests. Reservoirs were collected with bait traps. Insects and mammals were identified and examined in order to determine their natural infection. Risk factors in homes were assessed by means of a social-environmental survey. Results: Eleven adult specimens of Pastrongylus geniculatus, as well as 63 of Rhodnius pallescens were collected in the forest, recreational peridomiciliary areas, and houses. Even two females and 21 nymphs of R. pallescens were found in bedrooms. Two specimens of Didelphis marsupialis were captured in neighboring forests. Out of the eleven P. geniculatus captured, nine were examined. Of these, five were positive for Trypanosoma cruzi. It was not possible to establish a significant risk factor; however, the dwellings with report of triatomines were located nearer to the adjacent forest. Conclusions: The finding of intrusive triatominae species and mammals with T. cruzi in intradomiciliary and peridomiciliary areas and periurban forests prove the potential risk to acquire infection from these populations that dwell in urban housing adjacent to these ecotopes where the sylvan cycle is kept.

Search for a platelet-activating factor receptor in the Trypanosoma cruzi proteome: a potential target for Chagas disease chemotherapy

Chagas disease (CD) causes the highest burden of parasitic diseases in the Western Hemisphere and is therefore a priority for drug research and development. Platelet-activating factor (PAF) causes the CD parasite Trypanosoma cruzi to differentiate, which suggests that the parasite may express PAF receptors. Here, we explored the T. cruzi proteome for PAF receptor-like proteins. From a total of 23,000 protein sequences, we identified 29 hypothetical proteins that are predicted to have seven transmembrane domains (TMDs), which is the main characteristic of the G protein-coupled receptors (GPCRs), including the PAF receptor. The TMDs of these sequences were independently aligned with domains from 25 animal PAF receptors and the sequences were analysed for conserved residues. The conservation score mean values for the TMDs of the hypothetical proteins ranged from 31.7-44.1 percent, which suggests that if the putative T. cruzi PAF receptor is among the sequences identified, the TMDs are not highly conserved. These results suggest that T. cruzi contains several GPCR-like proteins and that one of these GPCRs may be a PAF receptor. Future studies may further validate the PAF receptor as a target for CD chemotherapy.

El rol de tres pruebas de ELISA con antígenos de promastigotes de Leishmania braziliensis, L. amazonensis y L. guyanensis en el diagnóstico de leishmaniasis tegumentaria / Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis

Es importante conocer si la variabilidad de especies de Leishmania circulantes en una región afecta la performance de las pruebas de ELISA estandarizadas para el diagnostico de la leishmaniasis. El objetivo de este trabajo fue analizar la reactividad de la prueba de ELISA utilizando homogenados de promastigotes de Leishmania (V.) braziliensis (ELISAb), L (L) amazonensis (ELISAa) y L (V.) guyanensis (ELISAg) frente a distintos grupos de sueros. Se estudiaron muestras de personas con leishmaniasis cutánea (n = 37), leishmaniasis mucocutánea (n = 8), no infectados (n = 52), infectadas por Trypanosoma cruzi (n = 11) e infecciones mixtas (n = 14). Se calcularon las sensibilidades, especificidades, cut off, valores predictivos, y se compararon las tres pruebas usando ANOVA, índice de concordancia kappa, comparación de curvas ROC e intervalos de confianza construidos por el método de bootstrap. Se encontraron diferencias significativas al comparar los niveles de DO de los sueros de pacientes con leishmaniasis cutánea respecto a los controles negativos, pero no se encontraron diferencias entre pruebas. Las sensibilidades calculadas fueron de 84.6% para ELISAb y ELISAa y de 88.5 para ELISAg, mientras que el valor de especificidad para las tres pruebas fue de 96.2. El índice de concordancia kappa y la comparación de curvas ROC mostraron performances similares para las tres pruebas (p = 0.225). La elevada reactividad obtenida para estas ELISAs frente a sueros de pacientes con leishmaniasis mucocutánea indica un importante potencial de esta técnica como complemento en el diagnóstico de la enfermedad.

It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V.) braziliensis (ELISAb), Leishmania (L) amazonensis (ELISAa) and Leishmania (V.) guyanensis (ELISAg) against different sera groups. Samples from individuals with cutaneous leishmaniasis (n = 37), mucocutaneous leishmaniasis (n = 8), healthy controls (n = 52), persons infected with Trypanosoma cruzi (n = 11) and mixed infections (n = 14) were included in the study. We calculated sensitivities, specificities, cut offs, and predictive values for the three tests and compared them using ANOVA, kappa index, ROC curves comparison, and confidence intervals calculated by the bootstrap method. Significant differences were found when comparing the OD levels of sera from patients with cutaneous leishmaniasis against healthy controls, but there were no differences when comparing the different ELISAs. The sensitivities calculated for ELISAb and ELISAa were 84.6 and of 88.5% for ELISAg, while the value of specificity for the three tests was 96.2. The kappa index (0.87) and comparison of ROC curves showed similar performance for the three ELISAs (p = 0.225). The high reactivity obtained for these ELISAs in sera of patients with mucocutaneous leishmaniasis indicates this test as an important complement in the diagnosis of the disease.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.

á-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an á-N-acetylglucosamine (á-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Atividade terapêutica do 5-nitrofuraldeído-semicarbazona (nitrofurazona) em esquemas de duração prologada na infeccção experimental do camundongo pelo Trypanosoma cruzi / Therapeutic activity of 5-nitrofuraldeído-semicarbazone (nitrofurazone) in schemes prolong the duration of infectious disease in experimental mice by Trypanosoma cruzi

Revisões históricas aos avanços científicos para o controle da doença, o Simpósio Internacional Comemorativo do Centenário da Descoberta da Doença de Chagas (1909-2009).

Clonamiento y expresión heterologa en saccharomyces cerevisiae de la acuaporina 2 de trypoanosoma cruzy (TcAQP2) / Cloning and heterologous expression in saccharomyces cerevisiae of the aquaporin 2 of trypanosoma cruzi (TcAQ2)

Trypanosoma cruzi es el agente causal de la Enfermedad de Chagas, una entidad endémica no controlada y en crecimiento en Latinoamérica. El parásito, durante su ciclo de vida, se ve sometido a cambios bruscos en la osmolaridad. Tales situaciones de estrés osmótico ameritan sistemas fisiológicos que le permitan a estas células adaptarse y sovrevivir a la nueva situación. En distintos organismos, no de los elementos primordiales que intervienen en osmorregulación son las acuaporinas, proteínas de membrana pertenecientes a la familia MIP ("major intrinsec protein"), que permiten el paso selectivo de agua (acuaparinas clásicas) y otras moléculas no cargadas (acuagliceroporinas) a través de membranas biológicas, en función de gradientes osmóticos. En el genoma de T. cruzi existen cinco genes que presentan similitud con proteínas de la familia MIP. Una de estás ya ha sido caracterizada, TcAQP1 (Trypanosoma cruzi, acuaporina 1), un canal permeable solo al agua. En este artículo de investigación se inició el estudio de un segundo gen con similitud a acuaporina, al cual hemos denominado TcAQP2. El gen TcAQP2 codifica para una proteína de 276 aminoácios. Por medio de herramientas de bioinformática se realizó la predicción de las estructura de dicha proteína; posee características que permiten ubicarla dentro de la familia MIP. Se realizó el clonamiento y secuenciación de la TcAQP2, detectándose diez mutaciones silentes. Este hecho sugiere que se trata de mutaciones propias de la cepa de T. cruzi CL Brener utilizada en el desarrollo experimental de este trabajo. Posteriormente se realizó el subclonamiento de TcAQP2 en el plásmido de expresión en el modelo de levadura pYES.2 y se transformó en las células de saccharomyces cerevisiae. La expresión funcional de la TcAQP2 en dichas células, muestra evidencias de que está proteína interviene en osmorregulación cuando las células son expuestas a choques hiper-osmóticos e hipo-osmóticos...

Trypanosoma cruzi, the etiological agent of changes disease, es prevalent throughout Latin America. During its life cycle, the parasite undergoes extreme fluctuations in osmolarity, consequentrly physiological systems are required to assure its survival. In many organisms one of the more important systems involved in osmoregulation are aquaporins. These proteins are members of the major intrinsic protein family (MIP). Aquaporins can be divided in two groups according to its permeablity: the first one is only permeable to water (orthodox aquiaporins and the second one is permeable to water, glycerol, and other small, uncharged molecules (aquaglyceroporins). In the T cruzi genome there are 5 genes that encode proteins similar to aquaporins. One of these genes (Trypanosoma cruzi aquaporin 1, TcAQp1) has been already characerized as a channel that is only permeable to water. It was also demonstrated that TcAQP1 is involved in parasite osmoregulation. In this work, we started to investigate other gene with aquaporin similarity, which we named Trypanosoma cruzi aquaporin 2 (TcAQP2). The gene TcAQP2 encodes a protein of 276 aminoacids. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA), which is the classical conserve motif in the MIP family proteins. Cloning and sequencing of the TcAQP2 gene allowed the detection of ten silent mutations in all clones analyzed, as compare to the sequence reported in data bank of the parasite, suggesting that they belong to the CL Brener strain used in this study. In order to analyze the TcAQP2 function, subcloning in the plasmid for heterologous expression in yeast (pYES.2) of this gene was performed. After transformation and functional expression alf TcAQP2 in Saccharomyces cerevisiae, cells were exposed to osmotic stress. The results indicate that this protein is involved in osmoregulation, and suggest that TcAQP2 participate as a channer for water and/or glycerol in...

Busca de substâncias antitumorais e tripanosomicidas: estudo químico de Lentinus strigosus (Basidiomycota) e Habenaria petalodes(Orquidaceae)

As espécies estudadas neste trabalho foram selecionadas a partir de um estudo de triagem intitulado “Bioprospecção da Biodiversidade Mineira e Desenvolvimento de Novas Drogas em Minas Gerais”, em que extratos de espécies vegetais e fungos basidiomicetos, coletados em ecossistemas locais, foram submetidos a diversos ensaios biológicos. O extrato acetato em etila do meio líquido do fungo Lentinus strigosus inibiu a enzima tripanotiona redutase (TR) de Trypanosoma cruzi e seu fracionamento biomonitorado levou ao isolamento de quatro substâncias; neopanepoxidol, 6,7-epóxi-4(15)-hirsuteno-1,5-diol, hipnofilina e panepoxidona, sendo as duas últimas responsáveis pelas atividades biológicas do extrato. Esses dois metabólitos terpênicos apresentaram atividade citotóxica para três linhagens tumorais humanas (UACC-62 -melanoma; TK-10 - renal e MCF-7 - mama; CI de 3,2 μM a 11,0 μM), inibiram a proliferação de linfócitos induzidos por. fitohemaglutinina (CI de 5,9 μM e 10,0 μM), a enzima TR (CI de 48,5 μM a 1,0 μM) e o crescimento de formas amastigotas de T. cruzi (CI de 6,0 μM e 2,5 μM). Panepoxidona e hipnofilina já foram descritas anteriormente, porém, é a primeira vez que se relata o isolamento desses metabólitos em L. strigosus.

O extrato bruto etanólico de Habenaria petalodes (Orquidaceae) apresentou atividade citotóxica para três linhagens tumorais humanas a 20 μg/ mL (UACC-62 - melanoma; TK-10 -renal e MCF-7 - mama). O extrato etanólico bruto de toda a planta foi extraído com diclorometano (fração apolar) e metanol/água (fração polar) por extração líquidolíquido. A fração diclorometânica apresentou actividade citotóxica para as três linhagens de células tumorais a 20 μg/ mL e foi selecionada para posterior fracionamento biomonitorado devido a sua baixa massa e complexidade química. Devido à ausência de relatos sobre os constituintes químicos, as frações polares de Habenaria petalodes (Orquidaceae) foram priorizadas para estudo fitoquímico. A fração polar foi extraída em cartucho Sep-Pak C e forneceu as subfrações, metanólica e aquosa que foram fracionadas por técnicas cromatográficas, obtendose três derivados do ácido succínico; loroglossina, militarina e dactilorina acetilada no anel 2 e, três flavonóides: isoquercitrina, isoramnetina 3-O-β-glicopiranosídeo e 50 50 50 50. 18 isoramnetina 3,7-di-O-β-glicopiranosídeo, isolados pela primeira vez nesta espécie vegetal

Busca de substâncias antitumorais e tripanosomicidas: estudo químico de Lentinus strigosus (Basidiomycota) e Habenaria petalodes(Orquidaceae) / Search and trypanocidal antitumor substances: chemical study of Lentinus strigosus (Basidiomycota) and Habenaria petalodes (Orquidaceae)

As espécies estudadas neste trabalho foram selecionadas a partir de um estudo de triagem intitulado “Bioprospecção da Biodiversidade Mineira e Desenvolvimento de Novas Drogas em Minas Gerais”, em que extratos de espécies vegetais e fungos basidiomicetos, coletados em ecossistemas locais, foram submetidos a diversos ensaios biológicos. O extrato acetato em etila do meio líquido do fungo Lentinus strigosus inibiu a enzima tripanotiona redutase (TR) de Trypanosoma cruzi e seu fracionamento biomonitorado levou ao isolamento de quatro substâncias; neopanepoxidol, 6,7-epóxi-4(15)-hirsuteno-1,5-diol, hipnofilina e panepoxidona, sendo as duas últimas responsáveis pelas atividades biológicas do extrato. Esses dois metabólitos terpênicos apresentaram atividade citotóxica para três linhagens tumorais humanas (UACC-62 -melanoma; TK-10 - renal e MCF-7 - mama; CI de 3,2 μM a 11,0 μM), inibiram a proliferação de linfócitos induzidos por. fitohemaglutinina (CI de 5,9 μM e 10,0 μM), a enzima TR (CI de 48,5 μM a 1,0 μM) e o crescimento de formas amastigotas de T. cruzi (CI de 6,0 μM e 2,5 μM). Panepoxidona e hipnofilina já foram descritas anteriormente, porém, é a primeira vez que se relata o isolamento desses metabólitos em L. strigosus.

O extrato bruto etanólico de Habenaria petalodes (Orquidaceae) apresentou atividade citotóxica para três linhagens tumorais humanas a 20 μg/ mL (UACC-62 - melanoma; TK-10 -renal e MCF-7 - mama). O extrato etanólico bruto de toda a planta foi extraído com diclorometano (fração apolar) e metanol/água (fração polar) por extração líquidolíquido. A fração diclorometânica apresentou actividade citotóxica para as três linhagens de células tumorais a 20 μg/ mL e foi selecionada para posterior fracionamento biomonitorado devido a sua baixa massa e complexidade química. Devido à ausência de relatos sobre os constituintes químicos, as frações polares de Habenaria petalodes (Orquidaceae) foram priorizadas para estudo fitoquímico. A fração polar foi extraída em cartucho Sep-Pak C e forneceu as subfrações, metanólica e aquosa que foram fracionadas por técnicas cromatográficas, obtendose três derivados do ácido succínico; loroglossina, militarina e dactilorina acetilada no anel 2 e, três flavonóides: isoquercitrina, isoramnetina 3-O-β-glicopiranosídeo e 50 50 50 50. 18 isoramnetina 3,7-di-O-β-glicopiranosídeo, isolados pela primeira vez nesta espécie vegetal

A alpha-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

alpha-glycerophosphate dehydrogenase (alpha-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance

Lipids shed into the culture medium by trypomastigotes of Trypanosoma cruzi

Trypomastigote forms of Trypanosoma cruzi were metabolically labeled with [14C]-ethanolamine and [3H]-palmitic acid. Lipids shed to the culture medium were analyzed and compared with the parasite components. Phosphatidylcholine and lysophosphatidylcholine accounted for 53 per cent of the total incorporated precursor. Interestingly, phosphatidylethanolamine and its lyso derivative lysophosphatidylethanolamine, although present in significant amounts in the parasites, could not be detected in the shed material. Shed lipids were highly enriched in the desaturated fatty acids C16:1 and C18:1 when compared to the total fatty acid pool isolated from the parasites.

IL-12 aumenta a eficácia do Benzonidazol no tratamento da Doença de Chagas experimental

Neste estudo nós avaliamos o papel das citocinas IFN[gama] e IL-12 na mediação e/ou aumento da atividade tripanosomicida in vivo do derivado nitroheterocíclico, Benzonidazol (Bz), durante a fase aguda da infecção da doença de Chagas experimental. Nossos estudos mostratam que o tratamento com anticorpos monoclonais (mAbs) neutralizantes anti-IL-12 e anti-IFN[gama], não tiveram um efeito aparente quando a dose ótima (100mg/Kg) de Bz foi utilizada. Enquanto isso, o tratamento com Acm neutralizantes anti-IL-12 ou anti-IFN[gama] aumentou a parasitemia e acelerou a mortalidade dos camundongos tratados com dose sub-ótima (25mg/Kg) de Bz. Simultaneamente, nossos experimentos, in vitro e in vivo, mostraram que cepas resistentes induzem pouca produção de IL-12 por macrófagos inflamatórios, quando comparadas com as cepas susceptíveis parcialmente resistente ao tratamento com Bz. Estes resultados sugerem que a ativação do compartimento imune celular, no estágio inicial da infecção com T.cruzi, pode favorecer a atividade do Bz in vivo. Para testarmos esta hipótese, tratamos comundongos infectados com a cepa Colombiana (resistente a droga) de T.cruzi com 100mg/Kg de Bz combinado com diferentes concentrações de IL-12r. Nossos resultados, baseados em métodos parasitológicos, sorológicos e pela Reação em Cadeia da Polimerase (PCR), mostraram que obteve-se uma alta porcentagem de cura em camundongos recebendo a terapia Bz combinado com IL-12r, quando comparados com os camundongos tratados com a mesma dose de Bz somente.