Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action.

BACKGROUND: Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE: The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS: Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS: The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS: Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action

BACKGROUND Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

First epidemiological description of tegumentar and visceral Leishmaniasis in Patrocínio municipality, Minas Gerais (2000-2017) / Primeira descrição epidemiológica de leishmaniose tegumentar e visceral no município de Patrocínio, Minas Gerais (2000-2017)

Leishmaniasis is a disease that can affect visceral organs (visceral leishmaniasis; VL) or mucous membranes and skin, causing lesions of different forms and levels of severities (tegumentary leishmaniasis; TL). Like several others, leishmaniasis is a neglected disease, as the pharmaceutical industry seems to show little to no interest in developing new drugs targeting the disease. This study aims to trace the epidemiological profile of leishmaniasis in the Municipality of Patrocínio, State of Minas Gerais, over a period of time. Secondary data of reported cases from 2000 to 2017 were analyzed as provided by the Patrocinio Health Department. As no literature was found on the status of such a disease in Patrocinio, it is important to trace the epidemiological profile of leishmaniasis in the area. The findings pointed out that the disease affected predominantly male in the economically active population, mainly from the urban area, and that it had no relationship with professional activity. Twenty-two cases of leishmaniasis (15 of TL and 7 of VL) were reported, all of which were treated and cured. Five cases of TL and 1 case of VL were autochthonous, and confirmed cases of canine infection took place in 2011, 2016 and 2017.KEYWORDS: Alto Paranaíba. Autochthonous. Human leishmaniasis. Triângulo Mineiro. INTRODUCTION Leishmaniasis is a parasitic disease caused by protozoa of the Leishmania genus, which are transmitted from one host to another by phlebotomine sandflies (NAGLE et al., 2014). It may affect both visceral organs and skin surfaces (HANDLER et al., 2015) and lead to four major clinical syndromes: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis or kala-azar (VL) and post-kala-azar dermal leishmaniasis (PKDL) (ANVERSA et al., 2018). Leishmaniasis is treated as a public health problem due to its high incidence and the disorders it can cause to the affected individuals. TL is characterized by ulcers on the face and/or extremities and nasal/oral/pharyngeal mucous (HANDLER et al, 2015), which can cause deformities such as disfiguring and permanent scars with partial or total mutilation of the nasopharyngeal mucosa, with further social, economic and psychological implications (ANVERSA et al., 2018, SUNYOTO; BOELAERT; MEHEUS, 2019). The mortality rate for VL, a more severe form, is 10%, making it the second most deadly tropical parasitic infection in the world after malaria (HANDLER et al., 2015). Updated regional information on the disease is important for epidemiological surveillance. However, the actual number of leishmaniasis cases is unknown due to underreporting, lack of epidemiological surveillance system or adequate diagnostic methods. Most data on incidence rates are based on estimates (PACE, 2014). Around 1.5 to 2 million new cases of leishmaniasis are estimated to occur annually worldwide: 500,000 are VL; 1 to 1.5 million cases are related to cutaneous or mucocutaneous leishmaniasis. Both forms commonly occur in Brazil (ANVERSA et al., 2018; WHO, 2020). Silva (2017) reported that the highest incidence rates of VL in the State of Minas Gerais, Brazil, from 2002 to 2013 were concentrated in the mesoregions in the North (Noroeste de Minas, Norte de Minas, and Jequitinhonha), in the East (Vale do Rio Doce) and in the center of the state (Central Mineira and Metropolitana de Belo Horizonte). The other mesoregions (Campo das Verentes, Oeste de Minas, Sul/Sudoeste de Minas, Triângulo Received: 08/04/19 Accepted: 20/12/19

A leishmaniose é uma doença que pode afetar órgãos viscerais (leishmaniose visceral; LV) ou as mucosas e a pele, provocando lesões de diferentes formas e gravidades (leishmaniose tegumentar; LT). Como várias outras moléstias, a leishmaniose é uma doença negligenciada, já que a indústria farmacêutica parece mostrar pouco ou nenhum interesse em desenvolver novos medicamentos direcionados à enfermidade. O estudo teve como objetivo traçar o perfil epidemiológico da leishmaniose no município de Patrocínio, estado de Minas Gerais, ao longo de um período de tempo. Dados secundários de casos registrados no período de 2000 a 2017 foram analisados, conforme fornecido pelo Departamento de Saúde de Patrocínio. Como não foi encontrada literatura sobre a situação dessa doença em Patrocínio, é importante traçar o perfil epidemiológico da leishmaniose na região. Os achados apontaram que a doença afetou predominantemente o sexo masculino da população economicamente ativa, principalmente da área urbana, e que não tinha relação com a atividade profissional. Foram notificados 22 casos de leishmaniose (15 de LT e 7 de LV), todos tratados e curados. Cinco casos de LT e 1 de LV foram considerados autóctones, e houve casos confirmados de infecção canina nos anos de 2011, 2016 e 2017.

Increased ROS generation causes apoptosis-like death: Mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex.

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm = bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(η2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(η2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(η2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

Increased ROS generation causes apoptosis-like death: mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm=bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(n2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(n2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(n2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

Increased ROS generation causes apoptosis-like death: mechanistic insights into the anti-Leishmania activity of a potent ruthenium(II) complex

Some metallodrugs that exhibit interesting biological activity contain transition metals such as ruthenium, and have been extensively exploited because of their antiparasitic potential. In previous study, we reported the remarkable anti-Leishmania activity of precursor cis-[RuIICl2(dppm)2], where dppm=bis(diphenylphosphino)methane, and new ruthenium(II) complexes, cis-[RuII(n2-O2CC10H13)(dppm)2]PF6 (bbato), cis-[RuII(n2-O2CC7H7S)(dppm)2]PF6 (mtbato) and cis-[RuII(n2-O2CC7H7O2)(dppm)2]PF6 (hmxbato) against some Leishmania species. In view of the promising activity of the hmxbato complex against Leishmania (Leishmania) amazonensis promastigotes, the present work investigated the possible parasite death mechanism involved in the action of this hmxbato and its precursor. We report, for the first time, that hmxbato and precursor promoted an increase in reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, formation of a pre-apoptotic peak, alterations in parasite morphology and formation of autophagic vacuoles. Taken together, our results suggest that these ruthenium complexes cause parasite death by apoptosis. Thus, this work provides relevant knowledge on the activity of ruthenium(II) complexes against L. (L.) amazonensis. Such information will be essential for the exploitation of these complexes as future candidates for cutaneous leishmaniasis treatment.

A fourteen-year retrospective of clinic-epidemiological aspects of cutaneous and visceral leishmaniasis in Uberlândia, Minas Gerais, Brazil / Uma retrospectiva de 14 anos dos aspectos clínico-epidemiológicos da leishmaniose cutânea e visceral em Uberlândia, Minas Gerais, Brasil

The purpose of this study was to describe clinic-epidemiological characteristics of leishmaniasis cases attended at the Clinical Hospital of Federal University of Uberlândia (CHU), state of Minas Gerais, from January 2000 to December 2013. This is a descriptive and retrospective review of medical records of patients diagnosed with leishmaniasis and treated at the CHU. 168 cases of leishmaniasis were analyzed and most patients were male, aged 23 to 60 years. For cutaneous and mucosal leishmaniasis, single lesions, located mainly in lower limbs and head (CL) and nasal mucosa (ML), were the most common clinical presentation. Regarding the diagnosis, the most performed methods for CL and ML were biopsy plus histopathology and biopsy plus immunohistochemistry; biopsy plus direct parasitological examination methods were the most frequent for VL. Most patients (84%) received treatment, mainly glucantime for both CL and ML; VL treatment was based on amphotericin B or glucantime. According to data from Sistema de Informações de Agravos de Notificação (SINAN), the Brazilian information system to notify and investigate cases of diseases and their aggravations, no case has been confirmed as autochthonous. It is noteworthy the underreporting of cases and the lack of complete information in medical records. Our results may contribute to a better understanding of the real situation in Uberlândia region concerning to leishmaniasis.

O propósito desse estudo foi descrever as características clínico-epidemiológicas dos casos de leishmaniose atendidos no Hospital de Clínicas da Unversidade Federal de Uberlândia (HCU), Minas Gerais, de janeiro de 2000 a dezembro de 2013. Trata-se de uma revisão descritiva e retrospectiva de registros médicos de pacientes diagnosticados com leishmaniose e tratados no HCU. 168 casos diagnosticados com leishmaniose foram analisados e os pacientes, em sua marioria, foram homens, com idade entre 23 e 60 anos. Para a leishmaniose cutânea e mucosa, lesões únicas, localizadas principalmente nos membros inferiores e na cabeça (LC) e mucosa nasal (LM), foram as apresentações clínicas mais comuns. Em relação ao diagnóstico, os métodos mais realizados para LC e LM foram biópsia mais histopatologia e biópsia mais imunohistoquímica; biópsia mais parasitológico direto foi o mais frequente para LV. A maioria dos pacientes (84%) recebeu tratamento, principalmente glucantime tanto para LC quanto para LM; o tratamento para LV foi atribuido à anfotericina B ou glucantime. De acordo com os dados do Sistema de Informação de Agravos de Notificação (SINAN), o sistema de informação brasileiro para notificar e investigar casos de doenças e seus agravamentos, nenhum caso foi confirmado como autóctone. Ressalta-se a subnotificação dos casos e a informação incompleta nos prontuários médicos. Nossos resultados podem contribuir para um melhor entendimento da real situação da leishmaniose na região de Uberlândia.

In vitro additive interaction between ketoconazole and antimony against intramacrophage Leishmania (Leishmania) amazonensis amastigotes.

Leishmaniasis is a group of diseases caused by protozoa of Leishmania genus. The currently available treatments for this disease are expensive, present high toxicity and are associated to difficulties of healing and parasite resistance. Therefore, the development of strategies for leishmaniasis treatment is indispensable and includes reposition of existing drugs, as well as drug combination therapy. The aim of this study was to assess the nature of ketoconazole and antimony association on the cytotoxic effect against Leishmania (Leishmania) amazonensis amastigotes. The calculated mean sum of fractional 50% inhibitory concentration ([Formula: see text]ΣFIC50) was 2.54 and 1.43 for free and intracellular amastigotes, respectively, values that suggest an additive interaction between ketoconazole and antimony concerning to Leishmania toxicity only in the intramacrophage parasite form. Despite the clinical efficacy of ketoconazole-antimony combination has been shown in the literature, our study is the first to describe the nature of ketoconazole-antimony interaction against L. (L.) amazonensis amastigotes. Moreover, our results point out the need for future in vivo studies to confirm the nature of ketoconazole-antimony interaction and also to determine possible effective dosage regimens related to ketoconazole administration in association with the optimal lower dose of antimony.

Clinical-epidemiologic aspects of ophidian accidents occurred in Triângulo Mineiro region, Minas Gerais State, Brazil: retrospective case series / Aspectos clínico-epidemiológicos dos acidentes ofídicos ocorridos na região do Triângulo Mineiro, Minas Gerais, Brasil: estudo retrospectivo

Ophidian accidents constitute a serious problem of public health in the tropical countries. In Central and South America, most of the accidents are caused by Bothrops (90.5%), followed by the Crotalus (7.7%), Lachesis (1.4%) and Micrurus (0.4%) genus. The aim of this work was to evaluate clinical-epidemiological aspects of ophidian accidents reported and treated at the Clinical Hospital at Federal University of Uberlândia, in the central region of Brazil. In this study, 641 medical records from January 1999 to December 2013 were analyzed. The results showed that the accidents were more common in the afternoon, from October to April. The major bite occurrence frequency was attributed to the Bothrops (54.76%), followed by Crotalus (30.58%) and Micrurus (1.40%) snakes. Most of the victims were males (80.34%). The main anatomical regions bitten were the lower and upper limbs, 65.67% and 30.58%, respectively. Approximately 80% of the victims were treated in the first 6 hours after the accident.

Os acidentes ofídicos constituem um sério problema de saúde pública em países tropicais. Nas Américas Central e do Sul, a maioria dos acidentes são causados pelo gênero Bothrops (90,5%), seguido por Crotalus (7,7%), Lachesis (1,4%) e Micrurus (0,4%). O objetivo deste trabalho foi avaliar os aspectos clínico-epidemiológicos dos acidentes ofídicos registrados e tratados no Hospital de Clínicas da Universidade Federal de Uberlândia, na região central do Brasil. Neste estudo, foram analisados 641 prontuários médicos de janeiro de 1999 a dezembro de 2013. Os resultados mostraram que os acidentes ofídicos foram mais comuns durante o período da tarde, de outubro a abril. A maior frequência de ocorrência das picadas foi atribuída às serpentes do gênero Bothrops (54,76%), seguido por Crotalus (30,58%) e Micrurus (1,40%). A maioria das vítimas foi do sexo masculino (80.34%). As principais regiões anatômicas acometidas foram os membros inferiores e superiores, 65,67% e 30,58%, respectivamente. Aproximadamente 80% das vítimas foram tratadas nas primeiras 6 horas após o acidente.

Biochemical and functional characterization of a C-type lectin (BpLec) from Bothrops pauloensis snake venom.

In the present work, we report the isolation and partial biochemical characterization of BpLec, a C-type lectin purified from Bothrops pauloensis venom by one chromatographic step on an affinity agarose column immobilized with d-galactose. This protein was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to be a 33.6 kDa homodimer by MALDI TOF analysis. BpLec presented an isoeletric point of 5.36. Its partial sequence of 132 amino acids for each subunit, determined by Edman degradation, revealed high identity (between 86% and 95%) when aligned with sequences of other related proteins. BpLec was capable of agglutinating native dog and cat erythrocytes and this activity was inhibited by ß-galactosides and EDTA. Its hemagglutinating activity was abolished at high temperatures and stable in any pH range. BpLec was effective in inhibiting Gram-positive but not Gram-negative bacteria. In addition, BpLec agglutinated promastigote forms of Leishmania (Leishmania) amazonensis.