Decreased cruzipain and gp85/trans-sialidase family protein expression contributes to loss of Trypanosoma cruzi trypomastigote virulence.

Two cell lines derived from a single Trypanosoma cruzi clone by long-term passaging generated a highly virulent (C8C3hvir) and a low virulent (C8C3lvir) cell line. The C8C3hvir cell line was highly infective and lethal to Balb/c mice, and the C8C3lvir cell line was three- to five-fold less infective to mouse cardiomyocytes than C8C3hvir. The highly virulent T. cruzi cell line abundantly expressed the major cysteine proteinase cruzipain (Czp), complement regulatory protein (CRP) and trans-sialidase (TS), all of which are known to act as virulence factors in this parasite. The in vitro invasion capacity and in vivo Balb/c mouse infectiveness of the highly virulent strain was strongly reduced by pre-treatment with antisense oligonucleotides targeting TS or CRP or with E64d. Based on these results, we conclude that decreased levels of TS, CRP and Czp expression could contribute to loss of T. cruzi trypomastigote virulence.

A cytoplasmic new catalytic subunit of calcineurin in Trypanosoma cruzi and its molecular and functional characterization.

Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca(2+)-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn(2+)/Ni(2+). The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication.

Aromatic glycosyl disulfide derivatives: evaluation of their inhibitory activities against Trypanosoma cruzi.

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(ß-D-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11 µM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.

A protein phosphatase 1 gamma (PP1γ) of the human protozoan parasite Trichomonas vaginalis is involved in proliferation and cell attachment to the host cell.

In this work, evidence for a critical role of Trichomonas vaginalis protein phosphatase 1 gamma (TvPP1γ) in proliferation and attachment of the parasite to the mammalian cell is provided. Firstly, proliferation and attachment of T. vaginalis parasites to HeLa cells was blocked by calyculin A (CA), a potent PP1 inhibitor. Secondly, it was demonstrated that the enzyme activity of native and recombinant TvPP1γ proteins was inhibited by CA. Thirdly, reverse genetic studies confirmed that antisense oligonucleotides targeted to PP1γ but not PP1α or ß inhibited proliferation and attachment of trichomonads CA-treated parasites underwent cytoskeletal modifications, including a lack of axostyle typical labelling, suggesting that cytoskeletal phosphorylation could be regulated by a CA-sensitive phosphatase where the role of PP1γ could not be ruled out. Analysis of subcellular distribution of TvPP1γ by cell fractionation and electron microscopy demonstrated the association between TvPP1γ and the cytoskeleton. The expression of adhesins, AP120 and AP65, at the cell surface was also inhibited by CA. The concomitant inhibition of expression of adhesins and changes in the cytoskeleton in CA-treated parasites suggest a specific role for PP1γ -dependent dephosphorylation in the early stages of the host-parasite interaction. Molecular modelling of TvPP1γ showed the conservation of residues critical for maintaining proper folding into the gross structure common to PP1 proteins. Taken together, these results suggest that TvPP1γ could be considered a potential novel drug target for treatment of trichomoniasis.

Molecular identification of the Diphyllobothrium species causing diphyllobothriasis in Chilean patients.

Diphyllobothriasis caused by the infection of adult Diphyllobothrium tapeworms sporadically occurs in Chile. The occurrence of the disease is closely linked to the consumption of raw or undercooked freshwater and marine fishes. Diagnosis of diphyllobothriasis has been based on laboratory examinations of the morphological characteristics of proglottids and eggs passed in the feces. Although determination of the parasite to the species level is possible through histologic examination of proglottid specimens, the parasites of patients who only discharge eggs cannot be diagnosed to the species level. Determining the species responsible for the infection of humans and other animals in affected areas is an important component of understanding the epidemiologic and enzootic characteristics of any infectious disease. We therefore compared the classification results obtained using a molecular approach with those obtained from morphological and histopathological examination of proglottids or eggs from five Chilean individuals with diphyllobothriasis. DNA analysis confirmed that the causative Diphyllobothrium species in Chile were first identified as Diphyllobothrium latum and Diphyllobothrium pacificum at least. Furthermore, mitochondrial cytochrome c oxidase subunit 1 gene analysis also supported the hypothesis that D. latum from Chile originated from Europe.

Enteroparasitosis en alumnos de educación básica de la ciudad de Antofagasta (2006 - 2007) / Enteroparasitism in students of basic education in two municipal schools of the city of Antofagasta (2006-2007)

In the northern part of Chile, due to its geoclimatic characteristics, enteroparasitosis in school children is mainly due to protozoos is such as Giardiosis and Blastocystosis, and Enterobiosis among helminthic infections. During the years 2006 and 2007, we made an enteroparasitosis survey of 92 school children in grades 1 to 8 in two municipal schools of Antofagasta. The global prevalence rate of parasites and commensals was 83.69%. We found frequencies of 68.48% for Blastocystis hominis, 8.70% for Giardia duodenalis, and 23.91% for Enterobius vermicularis. The frequencies for commensals were 18.48% for Entamoeba coli, 16.30% for Endolimax nana, 5.43% for Chilomastix mesnili and 3.26% for Lodamoeba bütschlii. We conclude that the frequency of infection by parasites and commensals among school children is related to the deficient sanitary conditions present in some sectors of the city of Antofagasta, associated with socioeconomic and cultural factors of the population.

En el norte de Chile por las características geoclimáticas, las enteroparasitosis en escolares se centra principalmente en protozoosis como la Giardiosis y Blastocistosis y la Enterobiosis entre las helmintiasis. Durante los años 2006 y 2007 se encuestó enteroparasitológicamente a 92 escolares que cursaban entre 1° y 8° básico en dos escuelas municipales de Antofagasta. La tasa de prevalencia global por parásitos y comensales fue de 83,69%. Con indicadores de 68,48% para Blastocystis hominis y 8,70% para Giardia duodenalis, y de 23,91% para Enterobius vermicularis. Entre los comensales se encontró Entamoeba coli (18,48%), Endolimax nana (16,30%), Chilomastix mesnili (5,43%) y Iodamoeba bütschlii (3,26%). Se concluye que la frecuencia de infecciones por parásitos y comensales entre los escolares estaría relacionada con las condiciones sanitarias deficitarias presentes en algunos sectores de la ciudad de Antofagasta, asociadas a factores socioeconómicos y culturales de la población.

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome.

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the alpha5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.

[Effectiveness of a repellent paint against the spider Loxosceles laeta]. / Evaluación experimental de la eficiencia de una pintura repelente para arañas del género Loxosceles.

BACKGROUND: Loxoscelism is a severe reaction to the bite of the spider Loxosceles laeta. In recent years, a paint with repellent properties has been promoted in the commerce. However, there are no reports of experiments evaluating its effectiveness. AIM: To evaluate experimentally the repellent properties of a paint against Loxosceles laeta. MATERIAL AND METHODS: Males, females and nymphs of L laeta were deposited in cockpits that allow the free displacement of the spider. Half of the cockpit was covered with repellent paint. Daily observations during one week, determined how frequently the spiders occupied the space covered with repellent paint. The experiments were run in triplicate. RESULTS: No statistical differences in the occupancy of spaces covered with repellent paint or not covered with it were observed for nymphs (87% and 67%, respectively), males (72% and 77%, respectively) or females (91% and 84%, respectively). CONCLUSIONS: The tested paint does not have a repellent action against the spider Loxosceles laeta.

Evaluación experimental de la eficiencia de una pintura repelente para arañas del género Loxosceles / Effectiveness of a repellent paint against the spider Loxosceles laeta

Background: Loxoscelism is a severe reaction to the bite of the spider Loxosceles laeta. In recent years, a paint with repellent properties has been promoted in the commerce. However, there are no reports of experiments evaluating its effectiveness. Aim: To evaluate experimentally the repellent properties of a paint against Loxosceles laeta. Material and methods: Males, females and nymphs of L laeta were deposited in cockpits that allow the free displacement of the spider. Half of the cockpit was covered with repellent paint. Daily observations during one week, determined how frequently the spiders occupied the space covered with repellent paint. The experiments were run in triplicate. Results: No statisticaldifferences in the occupancy of spaces covered with repellent paint or not covered with it were observed for nymphs (87% and 67%, respectively), males (72% and 77%, respectively) orfemales (91% and 84%, respectively). Conclusions: The tested paint does not have a repellent action against the spider Loxosceles laeta.

Calcineurin B of the human protozoan parasite Trypanosoma cruzi is involved in cell invasion.

During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca2+ concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca2+-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.

Experimental antihyperglycemic effect of diterpenoids of llareta Azorella compacta (Umbelliferae) Phil in rats.

Aqueous or ethanol infusions of Azorella compacta (llareta) in common with many other plants have been used as antidiabetic in the popular medicine in the altiplanic region of Chile. In order to determine if the diterpenic compounds chemically elucidated and isolated from this plant are responsible for this effect, streptozotocin diabetic rats (507 +/- 67 mg/mL glucose) were injected with two injections of diterpenic compounds mulinolic acid, azorellanol, and mulin-11,13-dien-20-oic acid at 180 mg/mL. Glycemia of animals treated with mulinolic acid and azorellanol was decreased to 243 +/- 2 and 247 +/- 14 mg/mL respectively, values very close to those reached by chlorpropamide injection used in controls. After 3 h treatment with mulin-11,13-dien-20-oic acid no effect was detected. The blood serum insulin in diabetic rats (146 +/- 58 pg/mL) was lower than in control rats. After injection of azorellanol, insulin was elevated to 247 +/- 23 pg/mL but with mulinolic acid, insulin was not changed. The antihyperglycemic effect of these compounds may explain the effectiveness of llareta in popular medicine. Because of the similarity to the hypoglycemic medication chlorpropamide, azorellanol could be acting on the beta cells of pancreatic islets, while mulinolic acid may act upon glucose utilization or production in the liver.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi.

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 M, these compounds displayed a strong lytic activity. It ranged from 88.4 0.6 to 99.0 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 M and 41-87 M, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 M. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 M. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi.

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 æM, these compounds displayed a strong lytic activity. It ranged from 88.4 ± 0.6 to 99.0 ± 1 percent for all strains and stages evaluate, with an IC50 /18 h values of 20-84 æM and 41-87 æM, respectively. The development of intracellular amastigotes was also inhibited by nearly 60 percent at 25 æM. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 æM. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy

Bioética como elemento integrador de la currícula en carreras del area de la salud / Bioethics as an integrative element in undergraduate health science curricula

Although education in ethics and bioethics is desirable for curricula in most of the health professions, there is a present no general consensus concerning the best mechanisms for their delivery. Bioethics, is an emergent discipline, iis important in providing interdisciplinary oersoectives not only valuable for professionals in practice, but even more importanly for students being trained in the health sciences. These students require experience offered by bioethics that provides practical support for their future professional performance. This article presents cirrucular innovation which integrates this new academic discipline into the mainstream of the health curriculum. It deals with universal principles of bioethics, particularly with regard to knowlege and aplication between students and professionals. This subject is controversial, and has been treated in diffrent ways in different institutions due to it's appeal to intellectual reflection and lack of objective criteria.

Aunque la enseñanza de la ética y bioética está incorporada en la curricula de la mayoría de las carreras de la salud, aún no hay un parecer unánime sobre cuál es el mejor modo de hacerlo. La bioética como disciplina emergente proporciona una instancia de reflexión interdisciplinaria no sólo válida entre los profesionales en ejercicio, sino que también con mucha mayor razón entre aquellos que se están formando en el pregrado y que necesitan las experiencias de enseñanza-aprendizaje que brinda la bioética como soporte práctico para su futuro desempeño laboral. En este artículo, a propósito de los principios universales de la bioética, especialmente sobre su conocimiento y aplicación entre los estudiantes y profesionales del área de la salud, se reflexiona sobre una innovación curricular que incorpore a esta nueva disciplina académica como eje integrador continuo de la curricula, tema largamente anhelado pero en pocas instituciones concretado, reconociéndole a la bioética su mayor acento en la reflexión intelectual.

Trichomonosis en adolescentes embarazadas de Antofagasta, Chile / Trichomonosis in pregnant adolescent from Antofagasta, Chile

Los problemas de salud de la adolescencia se caracterizan por una carga psicosocial elevada y un nivel de daño relativamente bajo término de morbilidad y mortalidad, sin embargo, la disminución de la edad promedio de la menarquia y el inicio precoz de actividad sexual coital son factores de riesgo para el embarazo y las enfermedades de transmisión sexual (ETS) entre los adolescentes. En este trabajo se investigó la infección por Trichomonas vaginalis y los factores epidemiológicos y obstétricos relacionados entre 300 adolescentes embarazadas de la ciudad de Antofagasta, cuyas edades variaron entre 12 y 18 años, de las cuales el 87,7 por ciento se concentró en el rango 15 y 17 años, en tanto que el 76,0 por ciento tuvo su menarquia entre los 12-14 años y el 27,3 por ciento inició su actividad sexual antes de los 15 años. Se determinó una tasa de infección por T. vaginalis de un 5,7 por ciento

Actividad de diterpenoides de azorella compacta, llareta, sobre amastigotes de trypanosoma cruzi / Activity of dipernoids isolated from azorella compacta, llareta, on trypanosoma cruzi amastigotes

The trypanocidad activity against amastigote forms of SPA-14, Tulahuen and G strains and CL Brener clone of Trypanosoma cruzi of diterpenoids isolated from Azorella compacta. Phil. (Llareta), a plant with ethnomedicinal prestige from prespanish age, was investigated. Amastigocidal activity was shown in azorellanol (2), diterpene isolated by first time, with an inhitory concentration 50 (IC) that varied between 60 M (CL Brener clone) and 84 M (SPA-14 strain), and in mulin -11,13 -dien-20-oico acid (5) with IC between 41 µM (G strain) and 87 mM (CL Brener clone). The cytotoxicity levels of both compounds against Hela and Vero cells and macrophages J144 are lower than nifurtimox and similar to gentian violet

Estudio epidemiológico de la pediculosis capitis y sarna en escolares básicos de Antofagasta, Chile, 1995 / Epidemiological study of pediculosis capitis and scabies in schoolchildren from Antofagasta, Chile, 1995

In order to contribute to a better knowledge of the pediculosis capitis and scabies during March-December 1995, 1122 primary schoolchildren under 14 years of age in the city-port of Antofagasta in northern Chile (20º South lat.), were examined. A total of 285 (25.4 percent) were found to be infested with Pediculus humanus capitis and only 20 (1.8 percent) with Sarcoptes scabiei. In general the rates of infestation to both ectoparasitic diseases were higher in groups of younger schoolchildren, also higher in women than in men and in those groups with high indexes of crowding and ignorance of the transmission mechanism of pediculosis capitis and scabies

Estado actual de la seroprevalencia de la infección chagásica humana y canina en la comuna de San Pedro de Atacama, II Región de Antofagasta en Chile 1995 / Present status of seroprevalence of canine and human chagasic infection in San Pedro de Atacama, II Región, Antofagasta, Chile 1995

In order to assess the impact of a control program against triatoma infestans launched in 1988, based on insecticide spraying of dwellings, a serological survey for chagasic infection was carried out during 1995 in three localities from San Pedro de Atacama county (22º55' south lat., 68º12' west long.), II Region of Antofagasta in northern Chile. Blood samples from 531 children and adolescents and 65 dogs were subjected to ELISA test and indirect inmunofluorescent test for Chagas's disease respectively. Tests resulted positive in 12 (2,3 percent) persons, all above 5 years old, in contrast with the 16,8 percent serological positivity observed in 1985. Three (4,6 percent) dogs (two 0-12 months old) resulted positive. These results indicate that dwellings sprayings with long-term activity insecticides against T. infestans is a good tool to prevent new human infections with T. cruzi. However, active vector transmissiom among domestic animals (canines) could be recently acquired.