Antileishmanial activity and cytotoxicity of ent-beyerene diterpenoids.

We describe the in vitro activity of two natural isomeric ent-beyerene diterpenes, several derivatives and synthetic intermediates. Beyerenols 1 and 2 showed EC50 of 4.6 ±â€¯9.4 and 5.3 ±â€¯9.4 µg/mL against amastigotes of L. (V) brazilensis, with SI of 5.1 and 7.7, respectively. Beyerenol 1 was synthesized from stevioside. In vivo experiments with bereyenols showed cure in 50% of hamsters infected with L. (V) brazilensis topically applied as Cream I (beyerenol 1, 0.81%, w/w) and Cream III (beyerenol 2, 1.96%, w/w). These results suggest that beyerenols are potential candidates for cutaneous leishmaniasis chemotherapy by topical application. In vitro assays of amastigotes of L. (V) brazilensis showed EC50 of 1.1 ±â€¯0.1 and 1.3 ±â€¯0.04 µg/mL, with SI of 3.1 and 3.5 for hydrazone intermediates 10 and 11, respectively.

Agreement between cranial and facial classification through clinical observation and anthropometric measurement among Envigado school children.

BACKGROUND: To evaluate the agreement between cranial and facial classification obtained by clinical observation and anthropometric measurements among school children from the municipality of Envigado, Colombia. METHODS: This cross-sectional study was carried out among 8-15-year-old children. Initially, an indirect clinical observation was made to determine the skull pattern (dolichocephalic, mesocephalic or brachycephalic), based on visual equivalence of right eurion- left eurion and glabella-opisthocranion anthropometric points, as well as the facial type (leptoprosopic, mesoprosopic and euryprosopic), according to the left and right zygomatic, nasion and gnation points. Following, a direct measurement was conducted with an anthropometer using the same landmarks for cranial width and length, as well as for facial width and height. Subsequently, both the facial index [euryprosopic (≤80.9%), mesoprosopic (between 81% - 93%) and leptoprosopic (≥93.1%)] and the cranial index [dolichocephalic (index ≤ 75.9%), mesocephalic (between 76% - 81%), and brachycephalic (≥81.1%)] were determined. Concordance between the indices obtained was calculated by direct and indirect measurement using the Kappa statistic. RESULTS: A total of 313 students were enrolled; 172 (55%) were female and 141 (45%) male. The agreement between the direct and indirect facial index measurements was 0.189 (95% CI 0.117-0261), and the cranial index was 0.388 (95% CI 0.304-0.473), indicating poor concordance. CONCLUSIONS: No agreement was observed between direct measurements conducted with an anthropometer and indirect measurements via visual evaluation. Therefore, the indirect visual classification method is not appropriate to calculate the cranial and facial indices.

Cutaneous leishmaniasis in the dorsal skin of hamsters: a useful model for the screening of antileishmanial drugs.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l0(7) promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity.