BackgroundChagas Disease caused by Trypanosoma cruzi infection, is one of the most important neglected tropical diseases (NTD), without an effective therapy for the successful parasite eradication or for the blocking of the disease’s progression, in its advanced stages. Due to their low toxicity, wide pharmacologic spectrum, and potential synergies, medicinal plants as Lippia alba, offer a promising reserve of bioactive molecules. The principal goal of this work is to characterize the inhibitory properties and cellular effects of the Citral and Carvone L. alba chemotype essential oils (EOs) and their main bioactive terpenes (and the synergies among them) on T. cruzi forms.MethodsTwelve L. alba EOs, produced under diverse environmental conditions, were extracted by microwave assisted hydrodistillation, and chemically characterized using gas chromatography coupled mass spectrometry. Trypanocidal activity and cytotoxicity were determined for each oil, and their major compounds, on epimastigotes (Epi), trypomastigotes (Tryp), amastigotes (Amas), and Vero cells. Pharmacologic interactions were defined by a matrix of combinations among the most trypanocidal terpenes (limonene, carvone; citral and caryophyllene oxide). The treated cell phenotype was assessed by fluorescent and optic microscopy, flow cytometry, and DNA electrophoresis assays.ResultsThe L. alba EOs displayed significant differences in their chemical composition and trypanocidal performance (p = 0.0001). Citral chemotype oils were more trypanocidal than Carvone EOs, with Inhibitory Concentration 50 (IC50) of 14 ± 1.5 μg/mL, 22 ± 1.4 μg/mL and 74 ± 4.4 μg/mL, on Epi, Tryp and Amas, respectively. Limonene exhibited synergistic interaction with citral, caryophyllene oxide and Benznidazole (decreasing by 17 times its IC50) and was the most effective and selective treatment. The cellular analysis suggested that these oils or their bioactive terpenes (citral, caryophyllene oxide and limonene) could be inducing T. cruzi cell death by an apoptotic-like mechanism.ConclusionsEOs extracted from L. alba Citral chemotype demonstrated significant trypanocidal activity on the three forms of T. cruzi studied, and their composition and trypanocidal performance were influenced by production parameters. Citral, caryophyllene oxide, and limonene showed a possible induction of an apoptotic-like phenotype. The best selective anti-T. cruzi activity was achieved by limonene, the effects of which were also synergic with citral, caryophyllene oxide and benznidazole.

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Background Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in Chronic Chagas Disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes, as citral (neral plus geranial), limonene, and caryophyllene oxide, presents in essential oils (EO) extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages. Methods T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oils fractions derived from Carvone and Citral-L. alba chemotypes, respectively. Results ACT1 (IC50 = 45 ± 1.7 μg/mL) and ACT2 (IC50 = 80 ± 1.9 μg/mL) exhibit similar trypanocidal effects to Benznidazole (BZN) (IC50 = 48 ± 2.5 μg/mL), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN (∑FIC = 0.52 ± 0.13 μg/mL) or ACT2 (∑FIC = 0.46 ± 1.7 μg/mL). ACT1 also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The ACT1 + ACT2 and ACT1 + BZN experimental treatments reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) and increased the anti-inflammatory cytokines (IL-4 and IL-10). Conclusion Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising L. alba essential oil fraction for further studies in drug development towards the Chagas disease control.

Background: Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in chronic Chagas disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes (citral, limonene, and caryophyllene oxide) presents in essential oils extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages.Methods: T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oil fractions derived from Carvone and Citral-L. alba chemotypes, respectively.Results: ACT1 and ACT2 exhibit similar trypanocidal effects to Benznidazole (BZN), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN or ACT2. This compound also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The experimental treatments (ACT1+ACT2 and ACT1+BZN) reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α), while increased the anti-inflammatories (IL-4 and IL-10).Conclusion: Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising compound for further studies in drug development towards the Chagas disease control.

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This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex- PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

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Introducción. Las candidiasis son un grupo de infecciones oportunistas causadas por levaduras del género Candida. C. albicans es la especie de mayor prevalencia en infecciones superficiales y profundas, sin embargo en la actualidad la frecuencia de especies no albicans, ha incrementado considerablemente su relevancia clínica en la última década, haciendo obligatoria la utilización de técnicas diagnósticas que permitan la identificación de especies para el manejo terapéutico adecuado de los pacientes.Objetivo. Diseñar y optimizar una técnica de PCR múltiplex considerando parámetros termodinámicos, para la identificación simultánea de cinco especies de Candida relevantes en la etiología de candidiasis humana.Materiales y métodos. Para el diseño de los cebadores se consideraron restricciones físicas y termodinámicas que afectan la PCR múltiplex, usando Gene Runner y Mult-PSOS. Como secuencias base se utilizaron: región transcrita interna 2 (ITS2) (AJ249486.1) para C. albicans y topoisomerasa II (TOPII) para C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1) y C. guillermondii (AB049145.1). Como moldes fueron utilizados extractos de ADN total obtenidos de cepas ATCC y aislamientos clínicos de las especies de Candida.Resultados. Se diseñaron 10 cebadores para la amplificación simultánea de las especies de Candida. El patrón de bandas obtenido fue: C. albicans (206pb), C. guillermondii (244pb), C. tropicalis (474pb), C. parasilopsis (558pb) y C. krusei (419pb).Conclusión. El ensayo de PCR múltiplex diseñado permitió la amplificación simultánea y eficiente de todos los amplicones correspondientes a las especies de Candida estudiadas, las cuales presentaron una adecuada resolución en gel de agarosa al 1,3%.