RESUMO
The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated Leishmania braziliensis (Lb) and Leishmania infantum (Li) that constitutively express the tandem tomato red fluorescent protein (tdTomato) as a model for large-scale screens of anti-Leishmania compounds. Confocal microscopy of Lb and Li::tdTomato revealed red fluorescence distributed throughout the entire parasite, including the flagellum, and flow cytometry confirmed that the parasites emitted intense fluorescence. We evaluated the infectivity of cloned promastigotes and amastigotes constitutively expressing tdTomato, their growth profiles in THP-1 macrophages, and susceptibility to trivalent antimony, amphotericin, and miltefosine in vitro. The phenotypes of mutant and wild-type parasites were similar, indicating that the constitutive expression of tdTomato did not interfere with the evaluated parameters. We applied our validated model to a repositioning strategy and assessed the susceptibility of the parasites to eight commercially available drugs. We also screened 32 natural plant and fungal extracts and 10 pure substances to reveal new active compounds. The infectivity and Glucantime treatment efficacy of BALB/c mice and golden hamsters infected with Lb and Li::tdTomato mutant lines, respectively, were very similar compared to animals infected with wild-type parasites. Standardizing our methodology would offer more rapid, less expensive, and easier assays to screen of compounds against L. braziliensis and L. infantum in vitro and in vivo. Our method could also enhance the discovery of active compounds for treating leishmaniasis.
Assuntos
Antiprotozoários , Leishmania braziliensis , Leishmania infantum , Leishmaniose , Cricetinae , Animais , Camundongos , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Fluorescência , Leishmaniose/tratamento farmacológico , Leishmania infantum/genética , Leishmania braziliensis/genética , Mesocricetus , Camundongos Endogâmicos BALB CRESUMO
Natural products derived from plants can be used as photosensitizers for antimicrobial photodynamic therapy (aPDT) combining key therapeutic strategies for tissue repair while controlling microorganisms' growth. We investigated a standardized extract of pequi peels (Caryocar brasiliense Cambess) as a brownish natural photosensitizer for aPDT using blue light. Three concentrations of the pequi extract (PE; 10, 30, or 90 µg/mL) were tested solely or associated with blue laser (445 nm, 100 mW, 138 J/cm2 , 6 J, 60 s). In vitro, we quantified reactive oxygen species (ROS), assessed skin keratinocytes (HaCat) viability and migration, and aPDT antimicrobial activity on Streptococcus or Staphylococcus strains. In vivo, we assessed wound closure for the most active concentration disclosed by the in vitro assay (30 µg/mL). Upon aPDT treatments, ROS were significantly increased in cell monolayers regardless of PE concentration. PE at low doses stimulates epithelial cells. Although PE stimulated cellular migration, aPDT was moderately cytotoxic to skin keratinocytes, particularly at the highest concentration. The antimicrobial activity was observed for PE at the lowest concentration (10 µg/mL) and mostly at PE 10 µg/mL and 30 µg/mL when used as aPDT photosensitizers. aPDT with PE 30 µg/mL presents antimicrobial activity without compromising the initial phases of skin repair.
RESUMO
Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. Only two drugs are available, with the drawback of low rate of cure in the chronic phase of the disease and undesirable side effects. These facts highlight the need to find new compounds for Chagas disease chemotherapy. We describe the isolation and identification of an inseparable mixture of two new trixikingolides from Trixis vauthieri, a plant from family Asteraceae, which present outstanding in vitro trypanocidal activity, with IC50 value of 0.053 µM against the intracellular trypomastigotes and amastigotes forms of T. cruzi infecting L929 cells. The IC50 of the mixture against the host cells is 68 times higher and about 70 times more potent than benznidazole, the reference drug used as control at the experiments. The next step, which depends on obtaining larger quantities of the mixture, is to test it on mice infected with T. cruzi.