Adriana Restrepo

In Vitro and In Vivo Cytotoxicities and Antileishmanial Activities of Thymol and Hemisynthetic Derivatives

ABSTRACT The in vitro and in vivo antileishmanial and cytotoxic activities of thymol and structural derivatives in comparison to those of Glucantime were studied. The results showed here suggest that thymol and hemisynthetic derivatives have promising antileishmanial potential and could be considered as new lead structures in the search for novel antileishmanial drugs.

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.

Distribution and Status of Podocnemis lewyana in the Magdalena River Drainage of Colombia

ABSTRACT We obtained evidence of the continued presence of Podocnemis lewyana in 18 different sites within the Magdalena River drainage of northern Colombia. However, abundances at most sites were low, even in areas where the species had previously been reported as common. Although hunting of adults is no longer commercially viable, local people consume individuals that are captured incidentally while fishing. Hunting of eggs during the incubation period each year continues to be intensive. Turtle abundances were negatively related to human densities throughout this area. In the Chicagua River, where turtle abundances were highest, we conducted standardized shoreline censuses of basking adults and documented abundances of approximately 6 individuals/km2. Turtles often basked on mud banks, beaches, or emergent logs in aggregations made up of all size classes. Pilot trapping efforts also succeeded in capturing all size classes of turtles, which suggests that more intensive monitoring programs would be feasible. Given the evidence of declines in most areas of the range of this species, the current lack of regulation of its exploitation, and the projections for continued human population growth in this area, we recommend its International Union for Conservation of Nature (IUCN) status be changed from Endangered to Critically Endangered (CR A2acd).

Reproductive Ecology of the Magdalena River Turtle (Podocnemis lewyana) in the Mompos Depression, Colombia

Abstract We studied various aspects of the reproductive biology of the Magdalena River turtle (Podocnemis lewyana) during 2 nesting seasons in 2 locations in the Mompos Depression in northern Colombia. We monitored 53 nests; 22 that completed incubation in situ, 24 that were transferred to protected areas (transferred nests), and 7 that were constructed using eggs removed from females that had been consumed by local people (“oviductal” nests). For each nest, we registered the oviposition date, distance to the nearest body of water, soil type, vegetative cover, exposure to sunlight, depth to the first egg, and maximum depth of the nest chamber. We also quantified egg dimensions and clutch sizes, mean incubation temperatures and duration of incubation periods, hatching success rates, and sex ratios. Most nests were located within 15 m of the shoreline, in open or grassy areas with permanent exposure to sunlight, and in sandy soils. The 3 nest types (natural, transferred, and oviductal) did not differ in hatching success rates. In both seasons, the majority of the nests produced hatchlings of both sexes, but the sex ratios differed between the nesting beaches and seasons. Incubation temperatures influenced incubation periods and hatching success rates, but not hatchling sizes or weights. The management implications of the findings are discussed.

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.

Nest Site Selection by Colombian Slider Turtles, Trachemys callirostris callirostris (Testudines: Emydidae), in the Mompos Depression, Colombia

ABSTRACT Nest-site selection by female Trachemys callirostris callirostris was studied on an island in the Pijiño wetland of the Mompos Depression of Colombia. We quantified distances of nests to the shoreline and compared vegetative cover and soil types at nest locations and at randomly selected points on the island. Nests sites were concentrated in the first 5 m from the shoreline and were always located under vegetative cover. Such cover might reduce thermal stress to nesting females, lower the probability of nest detection by predators, and buffer the incubating eggs from environmental extremes. Females preferentially nested under water hyacinth, but it was not clear whether they selected directly for this vegetative cover type or simply preferred dirt substrates where it dominated, because dirt soils contained higher humidity.

In vitro and in vivo antileishmanial activity of Artemisia annua L. leaf powder and its potential usefulness in the treatment of uncomplicated cutaneous leishmaniasis in humans

INTRODUCTION: Cutaneous leishmaniasis (CL) is a tropical disease that affects millions of individuals worldwide. The current drugs for CL may be effective but have serious side effects; hence, alternatives are urgently needed. Although plant-derived materials are used for the treatment of various diseases in 80% of the global population, the validation of these products is essential. Gelatin capsules containing dried Artemisia annua leaf powder were recently developed as a new herbal formulation (totum) for the oral treatment of malaria and other parasitic diseases. Here, we aimed to determine the usefulness of A. annua gel capsules in CL. METHODS: The antileishmanial activity and cytotoxicity of A. annua L. capsules was determined via in vitro and in vivo studies. Moreover, a preliminary evaluation of its therapeutic potential as antileishmanial treatment in humans was conducted in 2 patients with uncomplicated CL. RESULTS: Artemisia annua capsules showed moderate in vitro activity in amastigotes of Leishmania (Viannia) panamensis; no cytotoxicity in U-937 macrophages or genotoxicity in human lymphocytes was observed. Five of 6 (83.3%) hamsters treated with A. annua capsules (500mg/kg/day) for 30 days were cured, and the 2 examined patients were cured 45 days after initiation of treatment with 30g of A. annua capsules, without any adverse reactions. Both patients remained disease-free 26 and 24 months after treatment completion. CONCLUSION: Capsules of A. annua L. represent an effective treatment for uncomplicated CL, although further randomized controlled trials are needed to validate its efficacy and safety.

Short-Term Population Dynamics of Three Frog Species in the Northern Andes, Colombia

Abstract. To assess the conservation status of endemic species we examined the population trends in three frog species with different life histories. We performed a mark-recapture field experiment in a protected area on the eastern flank of the Cordillera Central in the northern Andes of Colombia. We marked and followed 123 individuals of Pristimantis jaguensis, 178 Colostethus aff. fraterdanieli, and 83 Dendrobates truncatus. The survival probability and the population growth rate of P. jaguensis varied as a function of capture occasion, while the recapture probability was constant. In the two dendrobatids, C. aff. fraterdanieli and D. truncatus, survival and recapture probabilities were constant over time and the population growth rate indicated the populations were stables during monitoring. Long-term monitoring programs are recommended to establish with certainty the conservation status of each species. However, in the perspective of the current biodiversity crisis in which rapid management decisions must be made, it is necessary to draw on the available data even if its limited. Although our demographic dataset is limited in time, we demonstrated how this information constitutes a starting point to understand the conservation status of these frog populations and to establish standard methods to continue long-term monitoring programs.

Out of Amazonia: the unexpected trans-Andean distribution of Cochranella resplendens (Lynch and Duellman, 1978) (Anura: Centrolenidae)

The glassfrog genus Cochranella, with nine recognized species, is distributed in the lowlands and mid elevation of the Neotropical forests, from Nicaragua to Bolivia (Guayasamin et al. 2009; Twomey et al. 2014). Four species are trans-Andean-C. granulosa (Taylor 1949) occurs in the lowlands and mountains, at mid elevation, of Central America, C. litoralis (Ruiz-Carranza & Lynch 1996) and C. mache Guayasamin & Bonaccorso 2004 occur in the Pacific lowlands and the western cloud forests of Colombia and Ecuador, and C. euknemos (Savage & Starrett 1967) occurs both in Central America and South America (northwestern Colombia).-The other five species have cis-Andean distributions in the Amazonian slopes and lowlands, from Colombia to Bolivia: C. nola Harvey 1996, C. guayasamini Twomey, Delia & Castroviejo-Fisher 2014, C. resplendens (Lynch & Duellman 1973), C. erminea Torres-Gastello, Suárez-Segovia & Cisneros-Heredia 2007, and C. phryxa Aguayo-Vedia & Harvey 2006. In Colombia, C. resplendens is known from the foothills of the Amazon versant in Caquetá (Malambo et al. 2013) and Putumayo (Lynch & Duellman 1973; Ruiz-Carranza et al. 1996). The species is also known from Ecuador (Lynch & Duellman 1973) and Peru (Twomey et al. 2014). Here, we report two new records of Cochranella resplendens, extending the species distribution beyond the Amazonian lowlands into the northern Cordillera Central in Colombia.

Amphibians and reptiles from two localities in the northern Andes of Colombia

The northern Andes are a geographically, climatically, and ecologically complex area that harbors a high number of species and presents a high level of endemism. However, the landscape of this region has been highly transformed by human activities, including agriculture, cattle grazing, urbanization, and recently, the construction of large infrastructure projects. This study presents a list of amphibians and reptiles compiled from field surveys carried out between 2006 and 2016 in the protected areas surrounding 2 hydroelectric projects located on the eastern flank of the northern Cordillera Central in Colombia. The checklist is comprised of 101 species (43 amphibians and 58 reptiles), including 17 endemic to Colombia and 4 threatened species. The high species density, high representativeness of the regional biodiversity, and high level of amphibian endemism make these 2 protected areas important for conservation of the regional herpetofauna.