In vitro anti-Leishmania activity of triclabendazole and its synergic effect with amphotericin B.

Introduction: Leishmaniasis is a neglected tropical disease, with approximately 1 million new cases and 30,000 deaths reported every year worldwide. Given the lack of adequate medication for treating leishmaniasis, drug repositioning is essential to save time and money when searching for new therapeutic approaches. This is particularly important given leishmaniasis's status as a neglected disease. Available treatments are still far from being fully effective for treating the different clinical forms of the disease. They are also administered parenterally, making it challenging to ensure complete treatment, and they are extremely toxic, in some cases, causing death. Triclabendazole (TCBZ) is a benzimidazole used to treat fasciolosis in adults and children. It presents a lower toxicity profile than amphotericin B (AmpB) and is administered orally, making it an attractive candidate for treating other parasitoses. The mechanism of action for TCBZ is not yet well understood, although microtubules or polyamines could potentially act as a pharmacological target. TCBZ has already shown antiproliferative activity against T. cruzi, T. brucei, and L. infantum. However, further investigations are still necessary to elucidate the mechanisms of action of TCBZ. Methods: Cytotoxicity assay was performed by MTT assay. Cell inhibition (CI) values were obtained according to the equation CI = (O.D treatment x 100/O.D. negative control). For Infection evaluation, fixated cells were stained with Hoechst and read at Operetta High Content Imaging System (Perkin Elmer). For growth curves, cell culture absorbance was measured daily at 600 nm. For the synergism effect, Fractional Inhibitory Concentrations (FICs) were calculated for the IC50 of the drugs alone or combined. Mitochondrial membrane potential (DYm), cell cycle, and cell death analysis were evaluated by flow cytometry. Reactive oxygen species (ROS) and lipid quantification were also determined by fluorimetry. Treated parasites morphology and ultrastructure were analyzed by electron microscopy. Results: The selectivity index (SI = CC50/IC50) of TCBZ was comparable with AmpB in promastigotes and amastigotes of Leishmania amazonensis. Evaluation of the cell cycle showed an increase of up to 13% of cells concentrated in S and G2, and morphological analysis with scanning electron microscopy showed a high frequency of dividing cells. The ultrastructural analysis demonstrated large cytoplasmic lipid accumulation, which could suggest alterations in lipid metabolism. Combined administration of TCBZ and AmpB demonstrated a synergistic effect in vitro against intracellular amastigote forms with cSFICs of 0.25. Conclusions: Considering that TCBZ has the advantage of being inexpensive and administrated orally, our results suggest that TCBZ, combined with AmpB, is a promising candidate for treating leishmaniasis with reduced toxicity.

Synergistic effect and ultrastructural changes in Trypanosoma cruzi caused by isoobtusilactone A in short exposure of time.

Butanolides have shown a variety of biological effects including anti-inflammatory, antibacterial, and antiprotozoal effects against certain strains of Trypanosoma cruzi. Considering the lack of an effective drug to treat T. cruzi infections and the prominent results obtained in literature with this class of lactones, we investigated the anti-T. cruzi activity of five butanolides isolated from two species of Lauraceae, Aiouea trinervis and Mezilaurus crassiramea. Initially, the activity of these compounds was evaluated on epimastigote forms of the parasite, after a treatment period of 4 h, followed by testing on amastigotes, trypomastigotes, and mammalian cells. Next, the synergistic effect of active butanolides against amastigotes was evaluated. Further, metacyclogenesis inhibition and infectivity assays were performed for the most active compound, followed by ultrastructural analysis of the treated amastigotes and trypomastigotes. Among the five butanolides studied, majoranolide and isoobtusilactone A were active against all forms of the parasite, with good selectivity indexes in Vero cells. Both butanolides were more active than the control drug against trypomastigote and epimastigote forms and also had a synergic effect on amastigotes. The most active compound, isoobtusilactone A, which showed activity against all tested strains inhibited metacyclogenesis and infection of new host cells. In addition, ultrastructural analysis revealed that this butanolide caused extensive damage to the mitochondria of both amastigotes and trypomastigotes, resulting in severe morphological changes in the infective forms of the parasite. Altogether, our results highlight the potential of butanolides against the etiologic agent of Chagas disease and the relevance of isoobtusilactone A as a strong anti-T. cruzi drug, affecting different events of the life cycle and all evolutionary forms of parasite after a short period of exposure.

Novel findings on the role of ficolins and colectins in the innate response against Leishmania braziliensis.

Leishmania (Viannia) braziliensis is the main agent of mucocutaneous Leishmaniasis, a neglected tropical disease that affects thousands of people in Brazil. It has been shown that complement plays a critical role at early stages of Leishmania infection and that is involved in the invasion of macrophages by the promastigotes. Ficolins and collectins are soluble pattern recognition and triggering molecules of the lectin complement pathway. We investigated here whether lectin pathway activators ficolin-1, ficolin-2, ficolin-3 and CL-11 bind to live L. braziliensis promastigotes in vitro. Promastigote forms in the stationary growth phase were incubated with normal human serum (NHS) or recombinant ficolins 1, 2 and 3, MBL and CL-11, and protein binding was evaluated by confocal microscopy and flow cytometry. Ficolins 1, 2 and 3, MBL and CL-11 were able to bind to the surface of live promastigotes after incubation with either NHS or recombinant proteins. A partial inhibition by N-acetyl-d-glucosamine characterizing the participation of acetylated groups in the deposition of ficolins and CL-11 to glycoconjugates on the surface of L. braziliensis was observed. These evidences highlight a role for the lectin pathway in the innate response to L. braziliensis.

Correlação entre a síndrome da dor femoropatelar com a flexibilidade dos músculos do quadril / Correlation between anterior knee pain with flexibility muscles hip

A Síndrome da dor femoro-patelar (SDFP) é uma das afecções mais comuns das articulações do joelho e tem como uma das suas principais funções a locomoção humana, enquanto o quadril é a maior junta de ajuste esférico do corpo humano. O objetivo deste estudo é conhecer a correlação da dor anterior do joelho com a flexibilidade e amplitude de movimento (ADM) do quadril em mulheres universitárias. Para tanto foi feito um estudo descritivo de corte transversal com uma população composta por 40 acadêmicas, divididas em 2 grupos de igual tamanho, sendo um composto por voluntarias com SDFP e o outro sem a síndrome. As voluntarias tinham idade entre 18 e 30 anos. Para avaliação da flexibilidade foi utilizado um flexímetro e da ADM o goniômetro universal. Após análise estatística dos resultados, verificou-se que a ADM de abdução de quadril apresentou-se diminuída, provavelmente devido à retração dos músculos adutores de quadril, ou mesmo pela perda da força muscular dos próprios abdutores. Com relação a flexibilidade, foi constatado a retração dos músculos flexores de quadril, havendo diferença significativa comparando o grupo com dor e o grupo sem dor anterior de joelho. Perante os resultados apresentados no presente estudo, pode-se concluir que existem alterações musculares referentes a flexibilidade e a ADM, nos grupos flexores e abdutores do quadril respectivamente, nos voluntários com presença de Síndrome da dor femoropatelar.

The syndrome Femoropatellar pain (PFPS) is one of the most common disorders of the knee and has as one of its main functions human locomotion, while the hip is the largest spherical fit joint of the human body. The aim of this study is to know the correlation of knee pain prior to the flexibility and range of motion (ROM) of the hip in college women. For this was made a descriptive cross-sectional study with a population composed of 40 academic, divided into two equal-sized groups, one composed of voluntary with PFPS and the other without the syndrome. The voluntary were aged between 18 and 30 years. To evaluate the flexibility we used a fleximeter and ADM universal goniometer. After statistical analysis, it was found that ADM hip abduction appeared was reduced, probably due to the retraction of the adductor muscles of the hip, or even the loss of muscle strength own abductors. With regard to flexibility, it was found the retraction of the flexor muscles of the hip, a significant difference comparing the group with pain and the group without previous knee pain. In view of the results presented in this study, it can be concluded that there are muscle changes for flexibility and ADM, the flexors and abductors of the hip groups respectively, in subjects with presence of patellofemoral pain syndrome.

Localização subcelular da actina 2 em Trypanosoma cruzi: Produção de soro policlonal e de parasitas transfectantes

O protozoário Trypanosoma cruzi possui um citoesqueleto diferenciado, baseado em um arcabouço de microtúbulos subpeliculares. Entretanto, o papel de outros componentes do citoesqueleto, como a actina, ainda permanece pouco explorado. Até o momento foram identificados no genoma de T. cruzi ao menos quatro genes codificadores para diferentes isoformas dessa proteína. No entanto, uma caracterização completa dessas isoformas ainda não foi realizada. Neste contexto, a presente dissertação teve por objetivo determinar a expressão e imunolocalização específica de uma das isoformas de actina, a actina 2. A partir de análise in silico encontramos que a actina 2 é uma isoforma exclusiva de T. cruzi, com 51% de identidade quando comparada à isoforma mais conservada em eucariontes (actina 1). Para obtenção do soro policlonal, uma sequência de 14 aminoácidos correspondente a uma região não conservada da actina 2 foi utilizada para imunizar camundongos. Ensaios de imunofluorescência demonstraram que a actina 2 é distribuída por todo o corpo celular do parasita, predominantemente na região perinuclear em formas epimastigotas. Em formas tripomastigotas uma minoria das células demonstrou essa marcação perinuclear, enquanto que em amastigotas a marcação mostrou-se dispersa pelo corpo do parasita. Ainda, a sequência da actina 2 foi inserida em um cassete baseado na tecnologia Gateway, contendo a etiqueta comercial FLAG (nas posições C e N-terminal) ou GFP. Os cassetes foram transfectados em formas epimastigotas e, após seleção, a presença do cassete foi confirmada por Western blot e PCR. A localização subcelular por imunofluorescência indireta da etiqueta FLAG (C-terminal) mostrou que a inserção da etiqueta não afetou o endereçamento da proteína, quando comparado aos resultados obtidos com o soro policlonal, diferentemente das outras construções obtidas. Ensaio de imunomarcação de formas epimastigotas por microscopia eletrônica de transmissão demonstrou uma marcação dispersa pelo corpo do parasita, confirmando os resultados obtidos por imunofluorescência. Nossos resultados indicam que o uso de parasitas geneticamente modificados e de anticorpos policlonais específicos permite uma avaliação mais precisa da imunolocalização de diferentes isoformas de uma mesma proteína de T. cruzi. Além disso, a imunolocalização da actina 2 difere daquela obtida com actina 1 por outros autores, que demonstraram uma marcação dispersa por todo o corpo do parasita, sugerindo assim que essas isoformas podem ter funções diferentes. Nossos dados abrem um leque de possibilidades no estudo da localização subcelular e função de cada uma das isoformas de actina de T. cruzi.