RESUMEN

Leishmaniasis is a neglected disease caused by flagellated parasites of the Leishmania genus affecting more than 10 million people worldwide. Current treatments for leishmaniasis involve the administration of poorly tolerated drugs with toxic side effects in patients. There is an imperative necessity for novel compounds to treat this disease. One of the most used strategies in the search for different antiparasitic compounds is the screening of purified plant molecules. The diterpenes 12-hydroxy-11,14-diketo-6,8,12-abietatrien-19,20-olide (HABTO) and 5-epi-icetexone (ICTX) isolated from Salvia cuspidata were shown to be effective against Leishmania amazonensis in vitro and in vivo. They displayed an antiproliferative effect against L. amazonensis promastigotes. They also induce an increase in ROS levels and affect the mitochondrial activity of parasites. HABTO and ICTX in an in vivo model of cutaneous leishmaniasis decrease footpad swelling, parasite load, and splenic index. Moreover, they induce significant reduction in the O.D. of total anti-Leishmania IgG and IgG1 subtype antibody responses against L. amazonensis compared to the PBS group but maintain high levels of IgG2a. This suggests that in HABTO- or ICTX-treated mice, there is a slowdown in the progression of the disease. These terpenes could be considered as possible novel antileishmanial agents against L. amazonensis and thus treat cutaneous leishmaniasis.

Asunto(s)

RESUMEN

Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.

Asunto(s)

RESUMEN

Background and aim: Prosopis strombulifera (Lam.) Benth is a rhizomatous shrub native from different zones of Argentine Republic. P. strombulifera aqueous extract (PsAE) has different effects and several biological activities have been reported. The goal of this study was to analyze the activity of PsAE on a murine model of cutaneous leishmaniasis caused by Leishmania amazonensis. Experimental procedure: PsAE was orally administered at 150 mg/animal/day on BALB/c mice infected in the right footpad (RFP) with 1 × 105 promastigotes of L. amazonensis. As a chemotherapeutic control of treatment, animals receive a commercial form of meglumine antimoniate (MA) (Glucantime®, Aventis, Paris, France). Results and conclusion: We observe that the size of RFP lesions of infected mice without treatment showed a grade of inflammation, ulceration and necrosis at the site of infection much greater than that observed with PsAE or MA treatment. Moreover, PsAE was capable of decreasing parasite burden and splenic index. Furthermore, PsAE treated mice showed a significant decrease in O.D. of total anti-Leishmania IgG antibody responses against L. amazonensis. This decrease was similar to those observed when the reference drug, MA, was used. This would indicate that PsAE treatment inhibits or delays disease progression in mice. In conclusion, our findings suggest that PsAE could be a potential candidate to be used, as a new therapeutic strategy, to treat cutaneous leishmaniasis caused by L. amazonensis.

RESUMEN

Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.

RESUMEN

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Asunto(s)

RESUMEN

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Asunto(s)

RESUMEN

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Asunto(s)

RESUMEN

BACKGROUND: Embelin (EMB), obtained from Oxalis erythrorhiza Gillies ex Hooker et Arnott (Oxalidaceae), was reported against Trypanosoma cruzi and Leishmania spp. Additionally, antiprotozoan activity against Plasmodium falciparum was reported for its methylated derivative (ME). PURPOSE: To evaluate the potential anti-Trypanosoma cruzi activity of EMB, ME and 2,5-di-O-methylembelin (DME) and analyze the possible mechanism of action. STUDY DESIGN/METHODS: EMB was isolated by a chromatographic method from the air-dried ground whole plant. To evaluate the effects of methylation, ME and DME were synthesized and tested against T. cruzi epimastigotes and trypomastigotes. The most active compound ME was evaluated against amastigotes. Ultrastructural alterations, ROS generation and the effect on mitochondrial activity of ME were measured. RESULTS: Compounds inhibited the proliferation of epimastigotes. ME was also active against intracellular amastigotes. Mitochondrial alterations were observed by TEM. Additionally, ME modified the mitochondrial activity, and induced an increase in ROS levels. These evidences postulate the mitochondrion as a possible target of ME. CONCLUSION: ME inhibited amastigotes proliferation, thus being a potential lead compound for the treatment of Chagas' disease.

Asunto(s)

RESUMEN

Secochiliolide acid (1) isolated from the Patagonian shrub Nardophyllum bryoides, was used as a scaffold for the preparation of a series of nine derivatives. Compound 1 and its derivatives were tested against Trypanosoma cruzi epimastigotes grown in liquid media. It was first observed that secochiliolide acid (1) inhibited the proliferation of the parasites, with an IC50 of 2 µg/mL. Six of the synthesized derivatives were also active with IC50's between 2 and 7 µg/mL which are comparable to that of the commercial drug benznidazole (2.5 µg/mL). These results indicate that the carboxyl group is not essential for the bioactivity of 1, while the presence of the tetrasubstituted exocyclic double bond seems to be important. Moreover, the presence of the furan and spirolactone rings is not essential for the bioactivity per se, but is important in combination with other structural fragments present in the molecule.

Asunto(s)

RESUMEN

Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host's cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc), dehydroleucodine (DhL), psilostachyin (Psi), and, at lesser extent, psilostachyin C (Psi C) is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.

RESUMEN

Chagas disease is a major health problem in Latin America and is caused by the parasitic protozoan Trypanosoma cruzi. Although many drugs have been used to alleviate the disease, these have been ineffective in the chronic phase and have also presented numerous side effects on patients. In this study we tested the effect of three sesquiterpene lactones (dehydroleucodine, helenalin and mexicanin) and a diterpene (5-epi-icetexone) on parasites (Y-strain) grown in host cells. At 48 h of treatment, the number of amastigotes inside the cells was lower than in the controls. This effect was observable at concentrations of 1.5-3.8 µM, which are of low cytotoxicity to host cells. In addition, the compounds caused a decrease in the percentage of infected cells. The treatments also reduced the presence of trypomastigotes in the extracellular medium. In all cases, helenalin was the most potent. The number of parasites per cell at 24h indicates the occurrence of multiple infection, which would also be affected by the compounds. However, we should not discard an effect on the proliferation and survival of parasites within the host cells. On the other hand, an additional effect on the differentiation of parasites and/or the survival of extracellular trypomastigotes might be possible. We conclude that these compounds are very effective against T. cruzi possibly by multiple mechanisms.

Asunto(s)

RESUMEN

Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide. We observed that the diterpene does not affect the growth of the parasites when added within 10 h after removal of hydroxyurea, but the compound was effective on growth when added to the cultures after 12 h. Thymidine incorporation was somewhat inhibited when the diterpene was added at 12 h after removal of hydroxyurea, possibly on the transition S/G2. Using transmission electron microscopy we observed that the diterpene induced a delay in the progression of cell division. We conclude that the compound, at cytostatic dose, affects the cell cycle of T. cruzi, possibly in the transition S/G2 phase and cell division. Further studies will focus to identify the molecular targets for the action of 5-epi-icetexone.

Asunto(s)