Chemical composition, in vitro larvicidal and antileishmanial activities of the essential oil from Citrus reticulata Blanco fruit peel / Composição química, atividades larvicida e leishmanicida in vitro do óleo essencial da casca do fruto de Citrus reticulata Blanco

Abstract Numerous studies have investigated the chemical composition and biological activities of essential oils from different Citrus species fruit peel, leaves and flowers. This paper aims to investigate the chemical composition, larvicidal and antileishmanial activities of essential oil from Citrus reticulata fruit peel (CR-EO). CR-EO was obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was analyzed by GC-MS and GC-FID. Limonene (85.7%), ɣ-terpinene (6.7%) and myrcene (2.1%) were identified as its major components. CR-EO showed high activity against promastigote forms of Leishmania amazonensis (IC50 = 8.23 µg/mL). CR-EO also exhibited high larvicidal activity against third instar Aedes aegypti larvae at a lethal concentration (LC50 = 58.35 µg/mL) and 100% mortality at 150 µg/mL. This study suggests, for the first time, the potential use of CR-EO against this important mosquito-borne viral disease caused by the genus Aedes.

Resumo Numerosos estudos têm investigado a composição química e as atividades biológicas de óleos essenciais extraídos de cascas dos frutos, folhas e flores de diferentes espécies de Citrus. Este trabalho tem como objetivo investigar a composição química e as atividades larvicida e leishmanicida in vitro do óleo essencial das cascas dos frutos de Citrus reticulata (CR-EO). CR-EO foi obtido pela técnica de extração em aparelho Clevenger e sua composição química foi determinada por CG-EM e CG-DIC. Limoneno (85,7%), ɣ-terpineno (6,7%) and mirceno (2,1%) foram identificados como os constituintes majoritários. CR-EO mostrou alta atividade contra as formas promastigota de Leishmania amazonensis (CI50 = 8,23 µg/mL). CR-EO também exibiu alta atividade larvicida contra as larvas do terceiro estágio do Aedes aegypti com concentração letal (CL50 = 58,35 µg/mL) e mortalidade de 100% em 150 µg/mL. Este estudo sugere, pela primeira vez, o uso potencial de CR-EO contra esta importante doença viral transmitida por mosquitos do gênero Aedes.

DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes

BACKGROUND Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Chemical composition, in vitro larvicidal and antileishmanial activities of the essential oil from Citrus reticulata Blanco fruit peel / Composição química, atividades larvicida e leishmanicida in vitro do óleo essencial da casca do fruto de Citrus reticulata Blanco

Numerous studies have investigated the chemical composition and biological activities of essential oils from different Citrus species fruit peel, leaves and flowers. This paper aims to investigate the chemical composition, larvicidal and antileishmanial activities of essential oil from Citrus reticulata fruit peel (CR-EO). CR-EO was obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was analyzed by GC-MS and GC-FID. Limonene (85.7%), ɣ-terpinene (6.7%) and myrcene (2.1%) were identified as its major components. CR-EO showed high activity against promastigote forms of Leishmania amazonensis (IC50 = 8.23 µg/mL). CR-EO also exhibited high larvicidal activity against third instar Aedes aegypti larvae at a lethal concentration (LC50 = 58.35 µg/mL) and 100% mortality at 150 µg/mL. This study suggests, for the first time, the potential use of CR-EO against this important mosquito-borne viral disease caused by the genus Aedes.

Numerosos estudos têm investigado a composição química e as atividades biológicas de óleos essenciais extraídos de cascas dos frutos, folhas e flores de diferentes espécies de Citrus. Este trabalho tem como objetivo investigar a composição química e as atividades larvicida e leishmanicida in vitro do óleo essencial das cascas dos frutos de Citrus reticulata (CR-EO). CR-EO foi obtido pela técnica de extração em aparelho Clevenger e sua composição química foi determinada por CG-EM e CG-DIC. Limoneno (85,7%), ɣ-terpineno (6,7%) and mirceno (2,1%) foram identificados como os constituintes majoritários. CR-EO mostrou alta atividade contra as formas promastigota de Leishmania amazonensis (CI50 = 8,23 µg/mL). CR-EO também exibiu alta atividade larvicida contra as larvas do terceiro estágio do Aedes aegypti com concentração letal (CL50 = 58,35 µg/mL) e mortalidade de 100% em 150 µg/mL. Este estudo sugere, pela primeira vez, o uso potencial de CR-EO contra esta importante doença viral transmitida por mosquitos do gênero Aedes.

Chemical composition, in vitro larvicidal and antileishmanial activities of the essential oil from Citrus reticulata Blanco fruit peel

Abstract Numerous studies have investigated the chemical composition and biological activities of essential oils from different Citrus species fruit peel, leaves and flowers. This paper aims to investigate the chemical composition, larvicidal and antileishmanial activities of essential oil from Citrus reticulata fruit peel (CR-EO). CR-EO was obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was analyzed by GC-MS and GC-FID. Limonene (85.7%), -terpinene (6.7%) and myrcene (2.1%) were identified as its major components. CR-EO showed high activity against promastigote forms of Leishmania amazonensis (IC50 = 8.23 µg/mL). CR-EO also exhibited high larvicidal activity against third instar Aedes aegypti larvae at a lethal concentration (LC50 = 58.35 µg/mL) and 100% mortality at 150 µg/mL. This study suggests, for the first time, the potential use of CR-EO against this important mosquito-borne viral disease caused by the genus Aedes.

Resumo Numerosos estudos têm investigado a composição química e as atividades biológicas de óleos essenciais extraídos de cascas dos frutos, folhas e flores de diferentes espécies de Citrus. Este trabalho tem como objetivo investigar a composição química e as atividades larvicida e leishmanicida in vitro do óleo essencial das cascas dos frutos de Citrus reticulata (CR-EO). CR-EO foi obtido pela técnica de extração em aparelho Clevenger e sua composição química foi determinada por CG-EM e CG-DIC. Limoneno (85,7%), -terpineno (6,7%) and mirceno (2,1%) foram identificados como os constituintes majoritários. CR-EO mostrou alta atividade contra as formas promastigota de Leishmania amazonensis (CI50 = 8,23 µg/mL). CR-EO também exibiu alta atividade larvicida contra as larvas do terceiro estágio do Aedes aegypti com concentração letal (CL50 = 58,35 µg/mL) e mortalidade de 100% em 150 µg/mL. Este estudo sugere, pela primeira vez, o uso potencial de CR-EO contra esta importante doença viral transmitida por mosquitos do gênero Aedes.

Eficacia de la artemisina en modelos murinos de leishmaniasis cutánea experimental causada por Leishmania amazonensis / Artemisine efficacy in murine models of experimental cutaneous leishmaniasis caused by Leishmania amazonensis

Los limitados tratamientos disponibles para enfrentar la leishmaniasis requieren el desarrollo de investigaciones para buscar nuevos agentes terapéuticos. Una estrategia recomendada es el reposicionamiento farmacológico, en el que la artemisina figura como un posible candidato. El objetivo de este estudio es evaluar las potencialidades de la artemisina en dos modelos murinos de leishmaniasis cutánea experimental. Para ello, se emplearon ratones BALB/c (susceptibles) y C57BL/6 (resistentes) infectados con Leishmania amazonensis. El tratamiento se realizó por vía oral o intralesional con cinco dosis de artemisina a 30 mg/kg cada 4 días. Se determinó el comportamiento del peso, la evolución del tamaño de la lesión y la carga parasitaria. En ambos modelos animales se observó que el tratamiento con artemisina (oral e intralesional) disminuyó el tamaño de la lesión y la carga parasitaria con respecto a los grupos infectados sin tratamiento (p 0,05). Los ratones C57BL/6 tratados por vía oral fueron los únicos capaces de controlar las lesiones hasta el final del experimento. Se demuestra la eficacia in vivo de la artemisina en dos modelos de leishmaniasis cutánea inducida por L. amazonensis y se destaca la administración por vía oral en el control de la enfermedad. Se sugiere el futuro desarrollo de este fármaco para el tratamiento de la leishmaniasis cutánea.

The limited treatments available for leishmaniasis require the development of research for new therapeutic agents. One recommended strategy is the pharmacological repositioning, where artemisinin stands out as a possible candidate. The aim of this study is to evaluate the potential of artemisinin in two murine models of experimental cutaneous leishmaniasis. For this purpose, BALB/c (susceptible) and C57BL/6 (resistant) mice infected with Leishmania amazonensis were used. Oral or intralesional treatment was performed with five doses of artemisinin at 30 mg/kg every four days. Weight behavior, evolution of lesion size, and parasitic load were determined. In both animal models it was observed that treatment with artemisinin (oral and intralesional) decreased lesion size and parasitic load with respect to the untreated infected groups (p 0.05). Orally treated C57BL/6 mice were the only ones able to control lesions until the end of the experiment. The in vivo efficacy of artemisinin in two models of cutaneous leishmaniasis induced by L. amazonensis is demonstrated and oral administration is highlighted in the control of the disease. Further development of this drug for the treatment of cutaneous leishmaniasis is suggested.

Visceral leishmaniasis caused by Leishmania (Leishmania) amazonensis associated with Hodgkin's lymphoma

ABSTRACT Visceral leishmaniasis (VL) is mainly caused by Leishmania (Leishmania) donovani and Leishmania (L.) infantum; however, other Leishmania species have been associated with VL. We report a case of a patient simultaneously diagnosed with VL caused by Leishmania (L.) amazonensis and Hodgkin's lymphoma. After treatment with liposomal amphotericin B and chemotherapy, the patient presented a clinical cure. This case report reinforces the hypothesis that other Leishmania species can cause visceral lesions mainly related to immunosuppression.

Disseminated cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis in human immunodeficiency virus (HIV)-infected patients: A report of two cases

Rationale: Co-infection of human immunodeficiency virus (HIV) and Leishmania spp. has impact on clinical and therapeutic outcomes of leishmaniases. Most studies do not present the identification of Leishmania species causing American tegumentary leishmaniasis in co-infections. In the Americas, Leishmania (L.) Viannia (V.) braziliensis and L. (V.) guyanensis have been identified. Patient concerns: In this study, two cases of American tegumentary leishmaniasis in patients infected with HIV are described. Patients presented several lesions with rapid dissemination and mucosal involvement. Diagnosis: Disseminated cutaneous leishmaniasis caused by L. amazonensis was identified by molecular test. Interventions: The patients were treated with conventional therapies for HIV infection and American tegumentary leishmaniasis. Outcomes: In co-infection, the clinical manifestations are atypical and the treatment response can be impaired. Lessons: These cases show that HIV infection impacts L. amazonensis infection and point to the relevance of identifying Leishmania species, which can lead to a better patient management.

Efficacy of the 7-chloro-4-(3-hydroxy-benzilidenehydrazo)quinoline derivative against infection caused by Leishmania amazonensis

Abstract INTRODUCTION: The drugs currently available for leishmaniasis treatment have major limitations. METHODS: In vitro and in vivo studies were performed to evaluate the effect of a quinoline derivative, Hydraqui (7-chloro-4-(3-hydroxy-benzilidenehydrazo)quinoline, against Leishmania amazonensis. In silico analyses of absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were performed. RESULTS: Hydraqui showed significant in vitro anti-amastigote activity. Also, Hydraqui-treated mice exhibited high efficacy in lesion size (48.3%) and parasitic load (93.8%) reduction, did not cause hepatic and renal toxicity, and showed appropriate ADMET properties. CONCLUSIONS: Hydraqui presents a set of satisfactory criteria for its application as an antileishmanial agent.

Método fluorométrico para identificar agentes naturales citotóxicos y su aplicación en la evaluación de agentes antiparasitarios selectivos / Fluorometric method for the identification of cytotoxic natural agents and its application in the evaluation of selective antiparasitic agents

El ministerio de salud tiene como una de las prioridades en investigación a las enfermedades infecciosas, entre las que incluye a las leishmaniasis y la tripanosomiasis americana. En la Facultad Ciencias Farmacéuticas y Bioquímicas se estudian especies vegetales medicinales utilizadas por la cultura Tacana como fuente de agentes antiparasitarios potenciales. Los laboratorios dedicados al descubrimiento de moléculas naturales con actividad antiparasitaria, tienen el desafío de desarrollar protocolos que permitan detectar biomoléculas selectivas, efectivas y menos tóxicas que las disponibles. Por ende, las evaluaciones antiparasitarias in vitro (CI50), deberían ir acompañadas de evaluaciones de citotoxicidad (DL50), con el fin de calcular un Índice de Selectividad (IS=DL50 / CI50) como parámetro de especificidad biológica. La citotoxicidad fue medida sobre líneas de macrófagos (RAW 264.7, murino), células involucradas en las infecciones por parásitos intracelulares. El protocolo fluorométrico parte con una población de 5x104 células/mL, incubada a 37ºC en DMEM (Dulbecco's Modified Eagle's médium), por 96 horas, con adición de resazurina (2mM) 3 horas antes de las lecturas finales. Bajo estas condiciones se evaluó la citotoxicidad de drogas control y 15 extractos vegetales seleccionados por su actividad anti-kinetoplastida. El extracto de Cosmailu fue el más citotóxico, Ejije bid'u resultó selectivo para T. cruzi y Leishmania amazonensis, mientras que Id'ene eidhue, fue selectivo para L. amazonensis. Finalmente, los otros 12 extractos resultaron ser poco selectivos o citotóxicos.

The Ministry of Health has infectious diseases as one of the research priorities, including leishmaniasis and American trypanosomiasis. The Faculty of Pharmaceutical and Biochemical Sciences develops evaluations of medicinal plant species used by the Tacana culture as a source of potential antiparasitic agents. Laboratories dedicated to the discovery of natural molecules with antiparasitic activity, have the challenge of developing protocols that allow the detection of selective, effective and less toxic biomolecules than those available. Therefore, in vitro antiparasitic evaluations (IC50) should be accompanied by cytotoxicity evaluations (LD50), in order to calculate a Selectivity Index (IS = LD50/ IC50) as a parameter of biological specificity. The cytotoxicity was measured on macrophage line (RAW 264.7, murine), cells engaged on intracellular parasites infections. The fluorometric protocol starts with an initial population of 5x104 cells/mL, incubated at 37ºC, in DMEM (Dulbecco's Modified Eagle's medium) for 96 hours with addition of resazurin (2mM) 3 hours, before final readings. Under these conditions the cytotoxicity of control drugs and 15 plant extracts, selected by their anti-kinetoplastid activity, was evaluated. Cosumailu extract was the most cytotoxic, Ejije bid'u was selective for T. cruzi, and Leishmania amazonensis, while Id'ene eidhue, was selective for L. amazonensis. Finally, the other 12 extracts were little selective or cytotoxic.

Antileishmanial in vitro activity of essential oil from Myrciaria plinioides, a native species from Southern Brazil

In South American folk medicine members of the genus Myrciaria are used for the treatment of malaria, diarrhoea, asthma, inflammation and post-partum uterine cleansing. The aim of this work was to evaluate its antileishmanial properties (in vitro) of essential oil derived from leaves of Myrciaria plinioides D. Legrand, a plant species that is native in South of Brazil. The essential oil was obtained by hydro-distillation using fresh leaves of M. plinioides. The chemical composition of this essential oil (MPEO, M. plinioides essential oil) was determined by gas chromatography coupled to mass spectrometry (GC-MS). MPEO was assayed in vitro for antileishmanial properties against promastigotes of Leishmania amazonensis and Leishmania infantum, and for cytotoxicity against murine peritoneal macrophages. The MPEO comprised 66 components and was rich in oxygenated sesquiterpenes (82.66%) containing spathulenol (21.12%) as its major constituent. The MPEO was effective against L. amazonensis with IC50 value of 14.16 ± 7.40 µg/mL, while against L. infantum the IC50 value was higher with 101.50 ± 5.78 µg/mL. The MPEO showed significant activity against L. amazonensis, and presented a selectivity index (SI) of 6.60. The results suggest that the essential oil from leaves of M. plinioides is a promising source for new antileishmanial agents against L. amazonensis.

Evaluation of triterpenes derivatives in the viability of Leishmania amazonensis and Trichomonas vaginalis

Trichomonas vaginalis and Leishmania spp. are protozoal species responsible for millions of cases of parasitic diseases worldwide. Considering the potential of natural products and the need for more effective and less toxic alternatives to treat trichomoniasis and leishmaniasis, this study aimed to evaluate the effect of two series of triterpenes derivatives with different modifications at C-3 and C-28 positions of the ursolic acid (UA) and betulinic acid (BA) against trophozoites of Trichomonas vaginalis and promastigotes forms of Leishmania (L.) amazonensis. The compounds modified just at C-3 were the most active. The 3β-acetyl betulinic acid (1b) reduced the trophozoites viability of T. vaginalis at 74%, followed by the 3-oxo ursolic acid and 3-oxo betulinic acid (3a and 3b) compounds (55% of reduction). The compound 3β-isobutyl ursolic acid (7a) inhibited the viability of L. amazonensis promastigotes by 55%. Therefore, analyzing the structure-activity relationship and the data of literature, it is possible to suppose that the inclusion of polar groups in the skeletons could improve the antiprotozoal activity. Overall, further studies are necessary to develop triterpenic derivatives with more powerful trichomonicidal and leishmanicidal properties.

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Background: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.

Phenolic Composition and Leishmanicidal Activity of Red Propolis and Dalbergia ecastaphyllum (L.) Taub (Fabaceae) Extracts from Sergipe, Brazil

ABSTRACT Leishmaniasis is a parasitic disease caused by protozoa of the Leishmania genus. It may manifest in visceral and tegumentary forms, and pentavalent antimonials are the first choice drugs used for the treatment. Frequently these drugs show low efficiency and high toxicity to mammalian host. The present study describes the chemical profile and the in vitro leishmanicidal effects of red propolis and Dalbergia ecastaphyllum extracts from Sergipe, Brazil, in Leishmania chagasi and Leishmania amazonensis promastigotes. The phenolic composition of the extracts was evaluated by direct infusion electrospray ionization mass spectrometry (ESI-MS) fingerprinting. The leishmanicidal effect was evaluated by the Resazurin colorimetric method. Similar composition profiles have been found for D. ecastaphyllum and propolis samples. The isoflavones formononetin, biochanin A, daidzein and pinocembrin were identified in both extracts. Propolis extract showed leishmanicidal activity in both L. chagasi and L. amazonensis, with IC50 values of 21.54 and 9.73 µg/mL, respectively. The D. ecastaphyllum extract presented activity only in L. amazonensis, with IC50 of 53.42 µg/mL. These results suggest that red propolis extract from Sergipe has the leguminosae D. ecastaphyllum as botanical origin, and that it presents potential leishmanicidal activity, which may be associated with the presence of the phenolic compounds found in its composition.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

In vitro antiparasitic activity and chemical composition of the essential oil from Protium ovatum leaves (Burceraceae)

ABSTRACT Leishmaniasis and trypanosomiasis are globally widespread parasitic diseases which have been responsible for high mortality rates. Since drugs available for their treatment are highly hepatotoxic, nephrotoxic and cardiotoxic, adherence to therapy has been affected. Thus, the search for new, more effective and safer drugs for the treatment of these diseases is necessary. Natural products have stood out as an alternative to searching for new bioactive molecules with therapeutic potential. In this study, the chemical composition and antiparasitic activity of the essential oil from Protium ovatum leaves against trypomastigote forms of Trypanosoma cruzi and the promastigote forms of Leishmania amazonensis were evaluated. The essential oil was promising against trypomastigote forms of T. cruzi (IC50= 28.55 μg.mL-1) and L. amazonensis promastigotes (IC50 = 2.28 μg.mL-1). Eighteen chemical constituents were identified by Gas Chromatography coupled to Mass Spectrometry (GC-MS) in the essential oil, whose major constituents were spathulenol (17.6 %), caryophyllene oxide (16.4 %), β-caryophyllene (14.0 %) and myrcene (8.4 %). In addition, the essential oil from P. ovatum leaves had moderate cytotoxicity against LLCMK2 adherent epithelial cell at the concentration range under analysis (CC50 = 150.9 μg.mL-1). It should be highlighted that this is the first report of the chemical composition and anti-Trypanosoma cruzi and anti-Leishmania amazonensis activities of the essential oil from Protium ovatum leaves.

The role of gallery forests in maintaining Phlebotominae populations: potential Leishmania spp. vectors in the Brazilian savanna

BACKGROUND Knowledge on synanthropic phlebotomines and their natural infection by Leishmania is necessary for the identification of potential areas for leishmaniasis occurrence. OBJECTIVE To analyse the occurrence of Phlebotominae in gallery forests and household units (HUs) in the city of Palmas and to determine the rate of natural infection by trypanosomatids. METHODS Gallery forests and adjacent household areas were sampled on July (dry season) and November (rainy season) in 2014. The total sampling effort was 960 HP light traps and eight Shannon traps. Trypanosomatids were detected in Phlebotominae females through the amplification of the SSU rDNA region, and the positive samples were used in ITS1-PCR. Trypanosomatid species were identified using sequencing. FINDINGS A total of 1,527 sand flies representing 30 species were captured in which 949 (28 spp.) and 578 (22 spp.) were registered in July and November, respectively. In July, more specimens were captured in the gallery forests than in the HUs, and Nyssomyia whitmani was particularly frequent. In November, most of the specimens were found in the HUs, and again, Ny. whitmani was the predominant species. Lutzomyia longipalpis was commonly found in domestic areas, while Bichromomyia flaviscutellata was most frequent in gallery forests. Molecular analysis of 154 pools of females (752 specimens) identified Leishmania amazonensis, L. infantum, and Crithidia fasciculata in Ny. whitmani, as well as L. amazonensis in Lu. longipalpis, Trypanosoma sp. and L. amazonensis in Pintomyia christenseni, and L. amazonensis in both Psathyromyia hermanlenti and Evandromyia walkeri. MAIN CONCLUSIONS These results show the importance of gallery forests in maintaining Phlebotominae populations in the dry month, as well as their frequent occurrence in household units in the rainy month. This is the first study to identify Leishmania, Trypanosoma, and Crithidia species in Phlebotominae collected in Palmas, Tocantins, Brazil.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Evaluation of Boldine Activity against Intracellular Amastigotes of Leishmania amazonensis

Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate in vitro activity of boldine against Leishmania amazonensis murine cell infection. Boldine ((S)-2,9-dihydroxy-1,10-dimethoxy-aporphine) is an aporphine alkaloid found abundantly in the leaves/bark of boldo (Peumus boldus Molina), a widely distributed tree native to Chile. The in vitro system consisted of murine macrophage infection with amastigotes of L. amazonensis treated with different concentrations from 50 to 600 μg/ml of boldine for 24 hr. Intracellular parasite destruction was assessed by morphological examination and boldine cytotoxicity to macrophages was tested by the MTT viability assay. When cells were treated with 100 μg/ml of boldine the reduction of parasite infection was 81% compared with untreated cultures cells. Interestingly, boldine-treatment caused a concentration-dependent decrease of macrophage infection that culminated with 96% of reduction when cells were submitted to 600 μg/ml of boldine. Cell cultures exposed to 100 μg/ml of boldine and 300 μg/ml of Glucantime® during 24 hr showed a significant reduction of 50% in parasitized cells compared with cell cultures exposed just to Glucantime®. The study showed that treatment with boldine produces a better effect than treatment with the reference antimonial drug, glucantime, in L. amazonensis infected macrophage. Our results suggest that boldine is a potentially useful agent for the treatment of leishmaniasis.