Development of a target-free high-throughput screening platform for the discovery of antileishmanial compounds.

Leishmania parasites are the causative agents of a wide spectrum of human diseases. The clinical manifestations of leishmaniasis range from self-healing skin lesions to fatality. The World Health Organization has classed leishmaniasis as a category 1 neglected tropical disease. Leishmaniasis represents a major international health challenge, affecting 12 million people per year and with nearly 310 million people at risk. The first-line chemotherapies used to treat leishmaniasis are intravenous pentavalent antimonials; however, these drugs are highly toxic. As the use of oral treatment options such as paromomycin and miltefosine has increased, the incidence of disease relapse has increased and drug resistance to antimonials has developed, emphasizing the importance of identifying new chemotherapies. A novel, target-free fluorometric high-throughput screen with an average Z-score of 0.73 +/- 0.13 has been developed to identify small molecules with antileishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVER-set™ library cassette #5 yielded 210 compounds that killed 80% of parasites, resulting in a hit rate of 2.1%. One hundred and nine molecular scaffolds were represented within the hit compounds, and one scaffold that exhibited potent antileishmanial activity was 2,4-diaminoquinazoline. Host cell toxicity was determined prior to in-vitro infection of human THP-1 macrophages with Leishmania donovani mCherry expressing promastigotes; successful drug treatment was considered when the half maximal inhibitory concentration was <10 µM. BALB/c mice were infected with Leishmania major mCherry promastigotes and treated with small molecules that were successful during in-vitro infections. Several small molecules tested were as efficacious at resolving cutaneous leishmaniasis lesions in mice as known antimonial treatments.

In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.

Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instruments.

Estudio cualitativo del uso de plantas medicinales en forma complementaria o alternativa con el consumo de fármacos en la población rural de la ciudad de Bulnes, Región del Bío-Bío, Chile / Qualitative study of use medicinal plants in a complementary or alternative way with the use of among of rural population of the Bulnes City, Bío-Bío Region, Chile

In a rural population of Bulnes, Bio-Bio Region of Chile, it was assessed the daily consumption of medicinal plants and drugs. Through 168 interviews the consumption of 5 medicinal plants was evaluated, considering their application, used plant parts, type of consumption, preparation, age range of people consuming mainly medicinal plants, use of other pharmacological treatments, main diseases present in the population under study and the type of drugs consumed. The obtained results demonstrate that higher consumed medicinal plants were peppermint (Mentha piperita) chamomile (Matricaria recutita), rute (Ruta graveolens), oregano (Origanum vulgare) and pennyroyal (Mentha pulegium). The consumption of medicinal plants was mainly complementary. The therapeutic use of medicinal plants in the rural population of Bulnes, is mainly digestive and it is not necessarily related to those diseases with more prevalence or most commonly observed in the population. It is important to consider future studies to determine if medicinal plants are more likely used to reduce the digestive discomfort which arises as a lateral damage of pharmaceutical drugs.

En una población rural de la Comuna de Bulnes, VIII región de Chile, fue valorado el consumo de plantas medicinales y fármacos. A través de 168 encuestas fue valorado el consumo de 5 plantas medicinales, su aplicación, las partes mas usadas de las plantas, tipo de consumo, preparación, porcentaje del rango etáreo que consume mayoritariamente plantas medicinales, porcentaje de la población sin y con consumo de fármacos, el porcentaje de las enfermedades presentes en la población en estudio y de los fármacos consumidos. Se obtuvo que las plantas medicinales más consumidas fueron la menta (Mentha piperita), manzanilla (Matricaria recutita), ruda (Ruta graveolens), orégano (Origanum vulgare) y poleo (Mentha pulegium). El consumo de las plantas medicinales fue principalmente de tipo complementario. El uso terapéutico de las plantas medicinales en la población rural de Bulnes, es principalmente de tipo digestivo no relacionándose con las enfermedades de mayor presencia en la población. Dicho uso es probablemente para disminuir las molestias digestivas causadas por el consumo de fármacos. Es necesario realizar mas estudios tendientes a esclarecer este punto de importancia relevante.

Circuits and projections of cat stellate ganglion.

BACKGROUND: Although connections of stellate ganglion (SG) have been widely explored, some features of pathways and projections remain unknown, such as the source and fate of preganglionic axons present in output branches, including both synaptically interrupted and traversing pathways as well as axon composition (efferent and afferent) of these output nerves. METHODS: Circuits and central projections of cat SG were investigated using horseradish peroxidase (HRP) tracer and electrophysiologic techniques including stimulation of ganglionic branches during recording of genesis of compound action potentials in other nerves or centrally evoked responses. RESULTS: All branches of SG including vertebral nerve are mixed, i.e., they contain axons that synapse in the periphery or traverse ganglia. A novel synaptically interrupted pathway bi-directionally coursing along subclavian branches and inferior cardiac nerve was identified. Preganglionic axons traversing stellate ganglion course in communicating branch to vagus nerve and to inferior cardiac nerve, a small number of these preganglionic axons traversing stellate ganglion reach cervical sympathetic trunk via subclavian branches. For the first time, a small number of preganglionic traversing pathways were also detected in vertebral nerve. Afferent axons with somata located in C8-T7 dorsal root ganglia, identified in all branches of SG, projected centrally to neurons in thalamus and somatosensory zones of cerebral cortex and coincided with afferent projections of brachial plexus. CONCLUSIONS: Present data contribute to the morphologic description of autonomic regulation of thoracic organs, including centrally independent peripheral autonomic axon reflexes.

Occurrence, co-occurrence and topographic distribution of choline acetyl transferase, Met-enkephalin and neurotensin in the stellate ganglion of the cat.

The presence of the classical ganglionic transmitter acetylcholine (ACh), its occurrence and possible co-occurrence with the neuromodulator peptides methionine enkephalin (Met-ENK) and neurotensin (NT), as well as the possible coexistence of these peptides in the preganglionic axon terminals of the cat stellate ganglia were investigated with light and confocal microscopy using immunofluorescence. Choline acetyltransferase (ChAT), Met-ENK, and NT immunoreactivity was detected with light microscopy in axon terminals near tyrosine hydroxylase (TH) immunoreactive (IR) cells. Cell bodies immunopositive for ChAT or Met-ENK were also detected and were TH-negative or TH-positive. Denervation by sectioning preganglionic axons produced two effects: the almost complete elimination of IR fibers and an increase in the number of ChATIR and Met-ENKIR cell bodies, together with the appearance of NTIR cell bodies. Preganglionic ChATIR fibers and boutons form a dense network throughout the entire ganglion, with a homogeneous regional distribution. ChAT, Met-ENK, and NT are essentially stored in different nerve endings, although a low level of co-occurrence was detected. NTIR and Met-ENKIR networks of boutons were observed to have independent and somewhat complementary regional distributions. Further analysis with simultaneous triple labeling for NT, Met-ENK, and TH, and confocal microscopy showed fibers and boutons containing Met-ENK or NT reached distinct neurons separately, or both converge onto the same cells. This finding suggests that modulation (the facilitation-inhibition balance) of ganglionic transmission is achieved mainly by the selective and complementary innervation of boutons containing NT (facilitation) and Met-ENK (inhibition) and only rarely by terminals which coexpress both peptides.