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In JoVE (1)
Other Publications (10)
Articles by Sara M. Robledo in JoVE
JoVE Immunology and Infection
Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs
1Program for the Study and Control of Tropical Diseases -PECET-School of Medicine, University of Antioquia, 2School of Agrarian Sciences, University of Antioquia
Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.
Other articles by Sara M. Robledo on PubMed
[Leishmania: Role of P Glycoprotein in Drug Resistance and Reversion Strategies]
Protozoan parasites are important causative agents of morbidity and mortality throughout the world--a problem further complicated by the emergence of drug resistance in these parasites. Mechanisms of drug resistance include the following: decreased uptake of the drug into the cell, loss of drug activation, alterations in the drug target, and over-expression of a well-known multiple drug transporter proteins. In this review, two critical components of resistance are stressed: (1) the role of ATP binding cassette proteins, such as P-glycoproteins, in mediating drug resistance in Leishmania and other protozoans, followed by development of cross-resistance to many structurally and functionally unrelated drugs, and (2) some concepts concerning the reversal mechanism of multidrug resistance by drugs and natural products. Several modulators or chemosensitizers alter the capacity of P-glycoproteins to maintain subtoxic intracellular drug concentrations. Calcium channel blockers such as verapamil act in this mode; however, high concentrations are required for an efficient and effective inhibition and, in addition, produce undesirable side effects. The discovery of new, natural product modulators of P-glycoproteins is stressed. This category of modulators offer potentially improved efficacy and lowered toxicity for the mammalian host.
Leishmania (Viannia) Panamensis: an in Vitro Assay Using the Expression of GFP for Screening of Antileishmanial Drug
Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.
Association of Acenaphthoporphyrins with Liposomes for the Photodynamic Treatment of Leishmaniasis
Acenaphthoporphyrins are potential photosensitizers for photodynamic therapy, but their hydrophobicity limits their potential. Liposomes have been widely investigated as delivery vehicles that can transport hydrophobic drugs in biological systems. Here we study the association of acenaphthoporphyrins with liposomes made up of dimyristoyl phosphatidylcholine (DMPC), and to liposomes made up of a mixture of DMPC, cholesterol (Chol) and distearoyl phosphatidylglycerol (DSPG) in a 2:1:0.8 molar ratio to evaluate how liposome composition affects association constants. In liposomes consisting only of DMPC, the smaller monoacenaphthoporphyrin had the largest association constant of 5.5 x 10(4) m(-1) while the larger adj-diacenaphthoporphyrin and opp-diacenaphthoporphyrin (ODP) had smaller association constants at 1.8 x 10(4) and 1.5 x 10(4) m(-1), respectively. The addition of liposomal Chol and DSPG has little effect on the magnitudes of the association constants. Polarization studies show that the acenaphthoporphyrins are driven far into the lipid bilayer to minimize polar-nonpolar interactions. Confocal microscopy confirms that the DMPC liposomes transport the porphyrins into promastigotes of Leishmania tarentolae. The compounds associated with DMPC:Chol:DSPG liposomes are effective in vitro against axenic and intracellular amastigotes of the pathogenic Leishmania panamensis. The effectiveness of the compounds is enhanced upon exposure of cultures to visible light.
Leishmania Tarentolae: Utility As an in Vitro Model for Screening of Antileishmanial Agents
Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.
Transfusion-transmitted Visceral Leishmaniasis Caused by Leishmania (Leishmania) Mexicana in an Immunocompromised Patient: a Case Report
Transfusion-transmitted leishmaniasis is an increasing problem in areas where visceral and cutaneous leishmaniases are endemic.
In Vitro and in Vivo Studies of the Utility of Dimethyl and Diethyl Carbaporphyrin Ketals in Treatment of Cutaneous Leishmaniasis
Carbaporphyrin ketals are porphyrinoid compounds in which a pyrrole ring of a typical porphyrin macrocycle has been replaced by a ketal-substituted indene ring. It was recently demonstrated that these compounds are effective in vitro against Leishmania tarentolae. Their in vitro effectiveness is increased when they are exposed to visible light; they act as photosensitizers capable of mediating the production of reactive oxygen species (ROS). Following on this evidence, the effectiveness and cytotoxicity of the dimethyl and diethyl carbaporphyrin ketals (CKOMe and CKOEt, respectively) were determined in vitro using pathogenic Leishmania species with and without exposure to visible light (2 and 4 h). The effectiveness against various pathogenic Leishmania species was determined to be in a micromolar range. Additionally, the effect of encapsulating the carbaporphyrin ketals in liposome formulations was tested. Liposomal delivery diminished their toxicity, while the effectiveness was enhanced upon exposure to visible light (photodynamic effect). The cytotoxicity levels for human U937 cells and hamster peritoneal macrophages were in the ranges of 0.3 to 9 μM and 7 to 330 μM, respectively. When tested in vivo, using a hamster (Mesocricetus auratus) model of cutaneous leishmaniasis, CKOMe was active even in the dark, suggesting that the compound, once metabolized in the animal tissue, produces an active ingredient that does not seem to be photosensitive. Reduction in lesion size, histopathologic analyses, and smears confirmed the in vivo effectiveness of the compound, since the parasitic load was diminished without noticeable toxic effects.
New Application for Expanded Porphyrins: Sapphyrin and Heterosapphyrins As Inhibitors of Leishmania Parasites
Sapphyrins and a series of related porphyrinoid macrocycles have been investigated as potential agents for the treatment of leishmaniasis. The effectiveness of the compounds was evaluated in vitro upon incubation with Leishmania tarentolae or L. panamensis amastigotes and promastigotes. Their effectiveness was also assessed against intracellular L. panamensis. The cytotoxicity of the compounds was evaluated in vitro using the U937 human promonocyte cell line. Effectiveness and cytotoxicity were assessed in the presence and absence of visible light to assess the photodynamic activity of the compounds. Sapphyrin and two related heterosapphyrins were shown to be particularly effective as inhibitors of Leishmania. A photodynamic effect was observed, which may be attributed to the formation of reactive oxygen species. Yields of singlet oxygen ((1)O(2)) produced were determined in ethanol solutions by direct measurement of (1)O(2) phosphorescence. Confocal microscopy demonstrated that sapphyrin and related macrocycles were taken up by the Leishmania cells and that their presence induces the formation of mitochondrial superoxide. Sapphyrins have been widely investigated as anticancer agents and we here show activity against the Leishmania parasites.
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.
In Vitro and In Vivo Efficacy of Ether Lipid Edelfosine Against Leishmania Spp. and SbV-Resistant Parasites
The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.
