Stachys lavandulifolia Vahl. exhibits promising in vitro and in vivo antileishmanial activity against Leishmania major infection

@#This study aimed to consider the in vitro and in vivo effects of the Stachys lavandulifolia methanolic extract (SLME) (2.5, 5, 10, 25, 50, 100 µg/mL) against Leishmania major infection. The in vitro antileishmanial effects of SLME was studies on promastigote and amastigote forms of L. major. The effect of SLME on the nitric oxide (NO) and apoptosis, secretion of Th1/2 cytokines, and infectivity rate in macrophages cells were also studies. The cytotoxicity of SLME on human (THP-1) and murine (J774-A1 cell) macrophage cells was investigated through the measuring the 50% cytotoxic concentrations (CC50). Moreover, the in vivo effects of SLME for healing the cutaneous leishmaniasis (CL) lesions in infected BALB/c mice studied by assessing the lesions size and the parasite load during four weeks of treatment. The calculated 50% inhibitory concentration (IC50) valuesfor SLME and meglumine antimoniate (MA) against the promastigote stage were 23.4 and 71.1 µg/mL, respectively. For amastigote stage, the IC50 values for SLME and MA were 39.3 µg/mL and 44.3 µg/mL, respectively. Followed by 28 days’ topically therapy with SLME at doses of 50 and 100 mg/kg/day, the CL lesions size as well as parasite load were significantly (p<0.001) reduced; such that the recovery percentage of the infected mice was 80% and 97% after treatment with SLME at the dose of 50 and 100 mg/kg, respectively. SLME also markedly induced the NO production and apoptosis; whereas decreased infection rate in macrophage cells. After incubation of infected macrophages with SLME, the level interferon gamma was meaningfully (p<0.001) elevated as a dose-dependent response; in contrast, release of interleukin 10 (IL-10) and IL-4 markedly (p<0.001) decreased. The CC50 value for SLME against THP-1 and J774-A1 cell was 996.4 µg/mL and 741.3 µg/mL, respectively. The calculated selectivity index of >10 for SLME and MA confirmed their specificity to amastigotes and the low toxicity for macrophages. Our results showed the potent effects of SLME in eliminating and controlling Leishmania parasites in both in vitro and in vivo assays. Based on the current experimental study, SLME can be suggested as an alternative medicine for the isolation and production of a new agent for treating CL caused by L. major. Although, we found some cellular mechanisms of SLME against Leishmania parasites, but, additional surveys are necessary to specify the accurate mechanisms of action, toxicity, and its efficacy mainly in human subjects.

Detection of Leishmania infantum amastigotes in neutrophil from peripheral blood in a naturally infected dog / Detecção de formas amastigotas de Leishmania infantum em neutrófilos do sangue periférico de cão naturalmente infectado

Abstract Canine visceral leishmaniasis (CVL) is a zoonotic disease of high lethality caused by Leishmania infantum in the Americas. In the infected dog, the amastigotes are scarce in blood, especially in the late phase of the disease. This study aimed to report a rare case of L. infantum amastigotes found in neutrophils from peripheral blood of a naturally infected dog in terminal phase of CVL, also describing its clinical status before and after treatment with miltefosine 2%. The dog, which presented as polysymptomatic and with classical signs and symptoms of CVL was submitted to the following tests: Dual Path Platform (DPP) rapid test, ELISA and parasitological examination of peripheral blood. Hematological and biochemical parameters were obtained before and after treatment. All diagnostic tests were positive for CVL. The identification of L. infantum amastigotes inside neutrophils from peripheral blood was confirmed through microscopy, and the species was confirmed by molecular analysis. At the end of the treatment, peripheral parasitemia was not detected, and improvements were observed in clinical and laboratorial parameters. Finally, this atypical finding can be used as example to raise discussions about the real immunological role of neutrophils in late phases of CVL and its clinical/therapeutic implications.

Resumo A leishmaniose visceral canina (LVC) é uma doença zoonótica de alta letalidade causada por Leishmania infantum nas Américas. No cão infectado, as formas amastigotas são escassas no sangue, principalmente na fase tardia da doença. Este estudo teve como objetivo relatar um caso raro de amastigotas de L. infantum encontradas em neutrófilos do sangue periférico de um cão naturalmente infectado e terminal da LVC, descrevendo também seu estado clínico antes e após o tratamento com miltefosina a 2%. O cão, que se apresentou como polissintomático e com sinais e sintomas clássicos da LVC foi submetido aos seguintes testes: teste rápido Dual Path Platform (DPP), ELISA e exame parasitológico de sangue periférico. Os parâmetros hematológicos e bioquímicos foram obtidos antes e após o tratamento. Todos os testes diagnósticos foram positivos para LVC. A identificação de formas amastigotas de L. infantum, dentro de neutrófilos do sangue periférico foi confirmada por microscopia, e a espécie foi confirmada por análise molecular. Ao final do tratamento, não foi detectada parasitemia periférica, observando-se melhora dos parâmetros clínicos e laboratoriais. Por fim, esse achado atípico pode ser usado como exemplo para levantar discussões sobre o real papel imunológico dos neutrófilos nas fases tardias da LVC e suas implicações clínicas/terapêuticas.

Are Aedes aegypti mosquitoes potential vectors for leishmaniasis? ­ Case report / Mosquitos Aedes aegypti são vetores potenciais de leishmaniose? ­ Relato de caso

In Brazil dipters of the Lutzomyia genus are the main vectors of leishmaniasis for humans and animals. However, other hematophagous insects such as ticks, fleas, and horse flies may also be considered potential vectors of this protozoon. This paper, regarding an endemic area for visceral leishmaniasis, is the the first description of the Leishmania spp. presence in Aedes aegypti mosquitoes. Two A. aegypti mosquitoes were captured: one of them was feeding on a polysymptomatic dog with leishmaniasis, confirmed by parasitic demonstration and positive PCR for Leishmania spp., and the other was collected in the environment where the dog was isolated. The mosquito engorged with dog's blood was crushed between two microscopic slides and the other one was processed by the polymerase chain reaction assay (PCR) searching for the presence of Leishmania spp. DNA. Amastigote forms of Leishmania sp, were observed in the smear prepared from one mosquito by microscopic examination, as well as other protozoa's flagellated forms. In the other insect it was observed Leishmania DNA amplification. This observation reinforces the role of dogs as sources of infection of Leishmania spp. even to other potential vector species.(AU)

No Brasil, os dípteros do gênero Lutzomyia são os principais vetores da leishmaniose para humanos e animais. No entanto, tem sido constatado que outras espécies de invertebrados hematófagos, como carrapatos, pulgas e mutucas, também podem ser vetores desse protozoário. Este trabalho, realizado em uma área endêmica de leishmaniose visceral, é a primeira descrição da presença de Leishmania spp. em mosquitos da espécie A. aegypti. Dois mosquitos A. aegypti foram capturados no local onde estava isolado um cão polissintomático acometido por leishmaniose visceral, confirmada pela demonstração do parasita em biópsias de órgãos e por resultado positivo na prova de PCR para Leishmania spp. Um dos mosquitos estava sugando o sangue do cão e o outro estava livre no ambiente. O mosquito ingurgitado com o sangue do animal foi esmagado entre duas lâminas de microscopia e o outro foi processado por meio da reação em cadeia pela polimerase (PCR) aplicada à pesquisa do ADN de Leishmania spp. Ao exame microscópico do esfregaço preparado com o mosquito que estava parasitando o cão foram observadas formas amastigotas de Leishmania spp., bem como formas flageladas de outra espécie de protozoário. No outro inseto foi detectada amplificação de ADN do gênero Leishmania. Esta constatação reforça o papel dos cães como fontes de infecção de Leishmania spp. até mesmo para outras espécies de vetores potenciais.(AU)

Bone marrow smear VS Blood smear in diagnosis of Kala-azar

Background  and Objectives:  Bone marrow specimen is considered as superior to the blood in the laboratory diagnosis of Kala-azar. The  main objective of this study is to compare these two methods of diagnosis and determine the usefulness of the diagnostic techniques.Material and Methods:  This  prospective  cross sectional  study  was  conducted at Janakpur Zonal Hospital, Janakpur which was  aimed  to  determine  the usefulness of the bone marrow specimen and blood specimen in the  laboratory diagnosis of Kala-azar. Bone marrow aspirate and venous blood  was  collected  aseptically  from  the  cases  were  processed  simultaneously.  The  results  of these two cultures were compared. Results:  Total 60 cases  of Kala-azar were included in the study of which 32 were male  and 28 were  female.   Amastigote  form  of  Leishmania  donovani  were  detected  in  56  (93.33%)  samples with  high  titre  of  parasitemiae  and  119  (18%)  in  the  blood  sample  with  low  parasitemiae. Sensitivity and Specificity of the test was calculated of the bone marrow sample test have more sensitivity (98%) and specificity (100%) over the sensitivity (90%) and specificity (96%) of blood smear test.Conclusion:  Bone marrow specimens were  found to be more useful than the blood sample in the  laboratory diagnosis of Kala-azar.

Sensibilidade parasitológica de diferentes locais de colheita para o diagnóstico de leishmaniose em cães / Sensitivity parasitological of different places of harvest for the diagnosis of leishmaniose in dogs / Sensibilidad parasitológica de diferentes sitios de recogida para diagnóstico de leishmaniosis en perros

A leishmaniose é uma zoonose de grande importância para a saúde pública. São grandes os estudos para que se conheçam diferentes técnicas para diagnóstico e que estes testes tenham alta sensibilidade. Este estudo teve como objetivo comparar a sensibilidade do teste parasitológico frente a diferentes locais de colheita de material biológico. As amostras colhidas do linfonodo e medula óssea se mostraram igualmente os mais sensíveis, na ordem de 44% dos casos. Estes valores elevaram-se ao realizar a pesquisa do parasito simultaneamente em duas técnicas.

Leishmaniasis is a zoonosis of great importance to public health. They are great studies to get to know different techniques for diagnosis and these tests have high sensitivity. This study aimed to compare the sensitivity of parasitological testing against different biological material collection sites. Samples taken from the lymph node and bone marrow also showed the most sensitive in the order of 44% of the cases. These figures amounted to perform the search of the parasite simultaneously in both techniques.

La leishmaniosis es una zoonosis de gran importancia para la salud pública. Son grandes los estudios para que se conozcan diferentes técnicas de diagnóstico y que estas pruebas tengan alta sensibilidad. Este estudio tuvo como objetivo comparar la sensibilidad de prueba parasitológica frente a diferentes sitios de recolección de material biológico. Las muestras tomadas de los ganglios linfáticos y médula ósea se demostraron, igualmente, los más sensibles, en el orden de 44% de los casos. Estas cifras se elevaron al realizar la investigación del parásito de forma simultánea en dos técnicas.

Planejamento de fármacos anti-T. cruzi: Síntese de compostos nitrofurânicos, avaliação da atividade biológica in vitro e estudos de relações estrutura-atividade / Design of anti-T. cruzi compounds: Synthesis of 5-nitrofuran derivatives, in vitro biological activity and study of structure-activity relationships

A doença de Chagas afeta cerca de 6 a 7 millhões de pessoas no mundo, principalmente América Latina. A busca de alternativas terapêuticas para esta enfermidade tem grande relevância para a sociedade, já que as opções atuais são limitadas, sendo disponível apenas o benznidazol (BZD) e nifurtimox. Os derivados nitroheterocíclicos são considerados compostos bioativos com número crescente de estudos na comunidade científica contra seu agente etiológico, o Trypanosoma cruzi. Neste sentido, o presente trabalho tem por objetivo a identificação de derivados do 5-nitrofurano com atividade frente a diferentes cepas do T. cruzi, assim como estudar possíveis modo de ação desta classe de compostos. Esta investigação envolve estudos computacionais com o propósito de construir modelos quantitativos de relações estrutura-atividade (QSAR multivariado) que possam auxiliar na previsão de novas estruturas com perfil farmacológico otimizado. No presente trabalho foram realizadas as etapas de planejamento, síntese e identificação de 36 compostos com resultados satisfatórios quanto à identificação estrutural, pureza e rendimento, que foi da ordem de 70%. A determinação da atividade anti-T. cruzi in vitro dos compostos obtidos foi realizada frente às cepas Silvio X10 cl1, Y, Bug 2149 cl10 e Colombiana na forma epimastigota do parasito. A maioria dos compostos analisados apresentou maior capacidade de inibição de crescimento do parasito, comparado ao BZD: Silvio X10 cl1 - IC50 = 29,16 ±2,90 µM, Y - IC50 = 40,40 ±3,37µM, Bug 2149 cl10 - IC50 = 30,63 ±3,21 µM, Colombiana - IC50 = 47,91 ±4,96 µM. O composto mais ativo (BSF-35) apresentou os seguintes valores: Silvio X10 cl1 - IC50 = 3,17 ±0,32 µM, Y - IC50 = 1,17 ±0,12 µM, Bug 2149 cl10 - IC50 = 1,81 ±0,18 µM e Colombiana - IC50 = 3,06 ±0,23 µM. Foram realizados cálculos de propriedades moleculares das estruturas tridimensionais dos compostos, seguido pela análise exploratória de dados por análise de agrupamentos hierárquicos (HCA) e análise de componentes principais (PCA), possibilitando o reconhecimento de padrões do conjunto. Considerando esta análise prévia, foram obtidos modelos QSAR com abordagem multivariada, aplicando algorítmo OPS e método de regressão por quadrados mínimos parciais, PLS. Os melhores modelos gerados foram obtidos considerando os compostos benzenos substituídos para as quatro cepas estudadas. Os descritores que mais influenciaram na análise foram o ClogP (coeficiente de partição) e cargas CHELPG. Considerando as informações obtidas, foram planejados e sintetizados quatro novos compostos com objetivo de obter compostos mais ativos e validar os modelos QSAR. Estes compostos apresentaram alta atividade frente a forma epimastigota das quatro cepas estudadas. Os compostos mais ativos foram avaliados quanto a citotoxicidade frente células LLC-MK2 e apresentaram seletividade até 25 vezes superior ao BZD. Estudos in vitro frente a forma amastigota da cepa Y em células U2OS foram realizados com metodologia fenotípica de análise de alto conteúdo (HCA') e os compostos apresentaram atividade até 64 vezes superior ao BZD e com seletividade de até 50 vezes superior a este fármaco. Quanto à determinação da atividade dos compostos frente às enzimas tripanotiona redutase (TcTR) e glutationa redutase (GR), os compostos analisados não apresentaram atividade relevante, indicando não ser este o mecanismo desta classe de compostos. Com finalidade de explorar outro possível mecanismo de ação dos compostos 5-nitrofurânicos, foi realizada a análise de potencial de redução da membrana mitocondrial, porém a morte parasitária não foi atribuída à despolarização da membrana em estudos simultâneos com iodeto de propídio

Chagas disease affects approximately 6-7 millions people worldwide, especially Latin America. The search for therapeutic alternatives for this disease is of great relevance to society, as current options are limited and there are only two available drugs: benznidazole (BZD) and nifurtimox. The nitroheterocyclic derivatives are considered bioactive compounds with increasing number of studies in the scientific community against its etiologic agent, Trypanosoma cruzi. In this sense, this work aims to identify derivatives of 5-nitrofuran with activity against different strains of T. cruzi, and to study possible mode of action of this compounds. This research involves computational studies to obtain models of quantitative structure-activity relationships (QSAR multivariate) that can help predict new structures with optimized pharmacological profile. In this work were carried out the design, synthesis and identification of 36 compounds with satisfactory results regarding the structural identification, purity and yield (approximately 70%). The determination of anti-T. cruzi activity in vitro of the compounds obtained was carried out with Silvio X10 cl1, Y, Bug 2149 CL10 and Colombiana strains of epimastigote form of the parasite. Most of the compounds examined showed greater capacity of growth inhibition of the parasite compared to the BZD (Silvio X10 CL1 - IC 50 = 29.16 ± 2.90 µM, Y - IC50 = 40.40 ± 3,37µM, 2149 CL10 Bug - IC 50 = 30.63 ± 3.21 µM, Colombiana - IC 50 = 47.91 ± 4.96 µM). The most active compound (BSF-35) showed the following values: Silvio X10 cl1 - IC 50 = 3.17 ± 0.32 uM, Y - IC 50 = 1.17 ± 0.12 µM, Bug 2149 CL10 - IC50 = 1, 81 ± 0.18 µM and Colombiana - IC 50 = 3.06 ± 0.23 µM. Calculations were performed for the molecular properties of three-dimensional structures of the compounds, followed by exploratory data analysis by hierarchical cluster analysis (HCA) and principal component analysis (PCA), allowing the recognition of the set. Considering this preliminary analysis were obtained QSAR models with multivariate approach, using OPS algorithm and regression method of partial least squares, PLS. The best generated models were obtained considering the benzyl substituted compounds for the four strains. The descriptors that most influenced the analysis were ClogP (partition coefficient) and CHELPG charges. Considering the information obtained, four new compounds were designed and synthesized to obtain more active compounds and validate QSAR models. These compounds showed high activity against epimastigote form of the four strains studied. The most active compounds were evaluated for cytotoxicity against LLC-MK2 cells and the compounds selectivity values were up to 25 times higher than BZD. In vitro studies against amastigote form of the Y strain in U2OS cells were performed with phenotypic method of high content analysis (HCA') and the compounds showed activity to 64 times higher than BZD and selectivity of up to 50 times. The activity of the compounds against trypanothione reductase enzymes (TcTR) and glutathione reductase (GR) showed no significant activity, indicating that this is not the mechanism of this class of compounds. In order to exploit another possible mechanism of action of 5-nitrofuran derivatives, analysis reduction of mitochondrial membrane potential was held, however the cell death was not attributed to membrane depolarization in simultaneous studies with propidium iodide

Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 µg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 µg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

Antileishmanial and Cytotoxic Effects of Essential Oil and Methanolic Extract of Myrtus communis L

Plants used for traditional medicine contain a wide range of substances that can be used to treat various diseases such as infectious diseases. The present study was designed to evaluate the antileishmanial effects of the essential oil and methanolic extract of Myrtus communis against Leishmania tropica on an in vitro model. Antileishmanial effects of essential oil and methanolic extract of M. communis on promastigote forms and their cytotoxic activities against J774 cells were evaluated using MTT assay for 72 hr. In addition, their leishmanicidal activity against amastigote forms was determined in a macrophage model, for 72 hr. Findings showed that the main components of essential oil were alpha-pinene (24.7%), 1,8-cineole (19.6%), and linalool (12.6%). Findings demonstrated that M. communis, particularly its essential oil, significantly (P<0.05) inhibited the growth rate of promastigote and amastigote forms of L. tropica based on a dose-dependent response. The IC50 values for essential oil and methanolic extract was 8.4 and 28.9 mug/ml against promastigotes, respectively. These values were 11.6 and 40.8 mug/ml against amastigote forms, respectively. Glucantime as control drug also revealed IC50 values of 88.3 and 44.6 mug/ml for promastigotes and amastigotes of L. tropica, respectively. The in vitro assay demonstrated no significant cytotoxicity in J774 cells. However, essential oil indicated a more cytotoxic effect as compared with the methanolic extract of M. communis. The findings of the present study demonstrated that M. communis might be a natural source for production of a new leishmanicidal agent.

In vitro anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves / 中国天然药物

AIM@#The anti-leishmanial activity of methanolic extracts of Calendula officinalis flowers, Datura stramonium seeds, and Salvia officinalis leaves against extracellular (promastigote) and intracellular (amastigote) forms of Leishmania major were evaluated in this study.@*METHOD@#In the first stage, promastigote forms of L. major, were treated with different doses of the plant extracts in a 96-well tissue-culture microplate and IC50 values for each extract were measured with colorimetric MTT assay. In the second stage, macrophage cells were infected with L. major promastigotes. Infected macrophages were treated with plant extracts. Then the macrophages were stained with Gimsa and the number of infected macrophages and amastigotes were counted with a light microscope.@*RESULTS@#The results indicated that the plant extracts inhibited the growth of promastigotes and amastigotes of L. major. Inhibitory concentrations (IC50) for promastigote assay were 108.19, 155.15, and 184.32 μgmL(-1) for C. officinalis flowers, D. stramonium seeds and S. officinalis, respectively. The extracts also reduced the number of amastigotes in macrophage cells from 264 for control group to 88, 97, and 102 for test groups. Although the anti-leishmanial activity of the extracts were not comparable with the standard drug, miltefosine; but they showed significant efficiency in reducing the number of amastigotes in macrophages, in comparison with the control group (P < 0.001). These plant extracts had lower toxicity compared with miltefosine.@*CONCLUSION@#This study demonstrates the potential efficacy of the methanolic extracts of C. officinalis flowers, D. stramonium seeds, and S. officinalis leaves to control of cutaneous leishmaniasis.

El estadio amastigota precede la evolución del epimastigota durante la epimastigogénesis in vitro de Trypanosoma cruzi / The amastigote stadium precedes epimastigote evolution during in vitro Trypasnosoma cruzi epimastigogenesis

La transformación de tripomastigotas sanguíneos de Trypanosoma cruzi en epimastigotas, ocurre naturalmente en el intestino del insecto y se identifica como epimastigogénesis. Aquí reportamos que durante la epimastigogénesis in vitro, los tripomastigotas se transforman en formas redondeadas, biológica y antigénicamente equivalentes al estadio amastigota; con un tiempo de persistencia dependiente de la altura del medio de cultivo. Formas tripomastigotas procedentes de cultivo de tejidos infectados se incubaron en envases con diferentes alturas de medio LITB (3 y 83 mm), y se compararon las cinéticas de transformación hacia epimastigotas. Para la obtención de antígenos se colectaron masas de parásitos a diferentes tiempos de diferenciación. Los cambios morfológicos, incremento del inóculo y resistencia a complemento, se estudiaron por microscopía de contraste de fases y extendidos coloreados con Giemsa. Los cambios antigénicos se analizaron mediante Western Blot usando anticuerpo IgY anti-amastigotas. Los resultados mostraron que las formas redondeadas resisten la lisis por complemento, expresan antígenos amastigota-específicos y la velocidad de transformación hacia epimastigotas depende de la altura del medio sobrenadante en el cultivo. Las evidencias sugieren que además de caída de la temperatura, una baja tensión de oxígeno disuelto y una alta densidad de parásitos por área, aceleran el proceso de diferenciación.

Transformation of blood Trypanosoma cruzi tripomastigotes into epimastigotes occurs naturally at the insect gut and is identified as epimastigogenesis. Here we report that during in vitro epimastigogenesis, tripomastigotes transform into rounded forms, biologically and antigenically equivalent to the amastigote stadium, with a persistance time which depends on the height of the culture medium. Tripomastigote forms from cultures of infected tissues were incubated in flasks with different LITB medium heights (3 and 83 mm), and kinetics of transformation to epimastigotes were compared. Masses of parasites at different differentiation times were collected for antigen production. Morphological changes, inoculum increase and resistance to complement were studied by phase contrast microscopy and Giemsa stained smears. Antigenic changes were analyzed through Western Blot using an IgY anti-amastigote antibody. Results showed that rounded forms resist lysis by complement, express amastigote-specific antigens, and that the speed of transformation to epimastigotes depends on the height of the supernatant medium in the culture. Evidences suggest that apart from a temperature fall, a low dissolved oxygen tension and a high parasite per area density accelerate the differentiation process.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

A Case of Post Kala-Azar Dermal Leishmaniasis in India

Post kala-azar dermal leishmaniasis (PKDL) is a rare disease. This is a solitary case report from Orissa, India. We describe a case of PKDL in a 55-year-old male who presented with multiple nodular lesions over face, trunk, and extremities. The patient had been to an endemic area of kala-azar and had a previous history of leishmaniasis. Fine needle aspiration cytology samples from skin nodules revealed Leishmania amastigotes.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Actividad in vitro de la mezcla de alcaloides de Ervatamia coronaria (Jacq) Staff. Apocynaceae sobre amastigotes de Leishmania braziliensis / Atividade in vitro da mistura de alcalóides de Ervatamia coronaria (Jacq) Staff. Apocynaceae sobre amastigotas de Leishmania braziliensis / In vitro activity of the alkaloids mixture of Ervatamia coronaria (Jacq) Staff. Apocynaceae on Leishmania braziliensis amastigotes

A leishmaniose é considerada uma importante causa de morbidade e mortalidade a nível mundial, principalmente nos países tropicais. As formas cutânea e mucocutânea são causadas, entre outras espécies, por Leishmania braziliensis. Na procura de compostos leishmanicidas de origem natural, foi estudada a atividade da mistura de alcalóides de Ervatamia coronaria (Apocynaceae) contra amastigotas de L. braziliensis em 6 concentrações diferentes (1, 10, 20, 25, 50 e 100 µg/mL). Foram tratados macrófagos de ratos da linha J774, infectados com promastigotas de L. braziliensis, com a mistura de alcalóides 1 hora após-infecção e diariamente por 3 dias sem mudança de meio. As experiências de citotoxicidade foram efetuadas sobre os macrófagos com azul tripam. Todos os cultivos foram feitos de forma triplicada e os grupos de controle não foram submetidos à mistura de alcalóides. Foi obtido que o composto adicionado exerce atividade doses/dependente sobre a parasita. No entanto, as concentrações mais altas (50 e 100 µg/mL), adicionado durante 3 dias, mostraram os maiores índices de infecção, provavelmente devido a diminuição no número de macrófagos, sobre os quais não foi observado efeito tóxico do tratamento durante 24 horas DL50/24h = 233,52 µg/mL. Os resultados dessa pesquisa revelaram uma nova atividade farmacológica de alcalóides da espécie Ervatamia coronaria sobre a forma amastigota de Leishmania braziliensis, com IC50 = 2,6 e 12,4 µg/mL sem mostrar toxicidade sobre a célula hospedeira.

Leishmaniasis, caused by Leishmania sp., is one of the mean reason of considerable mortality and morbidity throughout the world, especially in the tropics. Cutaneous and mucocutaneous manifestations are caused by Leishmania braziliensis, and the cutaneous form is the most common one in Colombia. In the search for antileishmanial compounds from natural sources, we studied the alkaloids mixture from Ervatamia coronaria against L. braziliensis at six different concentrations (1.0, 10, 20, 25, 50 and 100 µg/mL). Macrophages J774 infected with L. braziliensis were treated with alkaloids one hour, and once a day for three days, after parasitic infection and preserving the same culture medium. Cytotoxicity with trypan blue was undertaken in macrophages J774 by using the same concentrations. Three different cultures samples were carried out. As a control we used medium alone. The alkaloids mix showed a dose/dependent activity on amastigote, but by increasing concentrations from 50 to 100 µg/mL for three days, we saw a high index of infection, probably caused by cellular death. We did not see any toxic effect on macrophages J774 at 100 µg/mL, LD50/24h= 233.52 µg/mL. These results revealed a novel pharmacological activity of alkaloids from E. coronaria against amastigotes of L. braziliensis IC50 = 2.6 and 12.4 µg/mL without toxicity on host cells.

Population Polymorphism of Trypanosoma cruzi in Latin America indicated by Proteome analysis and by in vitro amastigote proliferation

Nineteen stocks of <I>Trypanosoma cruzi</I> originating from several endemic countries for Chagas‘ disease in Central and South America were subjected to two-dimensional protein electrophoresis analysis. The presence or absence of a total of492polypeptide spots among19gel profiles was determined. The stocks were classified into three major distinctive groups derived from (I) Central America and the northern part of South America; (IIa) Central America and the northern part of South America; and (IIb) central and southern parts of South America, which showed perfect concordance with the previously reported classification based on isozyme and DNA sequence analyses. Late log phase of each epimastigote was inoculated to human cell lines WI-38and Hs224.T originating from the lung and muscle, respectively, and the number of trypomastigotes released was counted. The number of trypomastigotes from <I>T</I>. <I>cruzi</I> in group I released from the two cell lines was significantly higher than that in group III (p&It;0.05). The findings suggested that the phenetic distance appearing within the <I>T</I>. <I>cruzi</I> may, to some extent, be associated with the intracellular growth of <I>T</I>. <I>cruzi</I>, one of the characteristic features of growth found in the species.

Detection of P-4 and GP-46 Expression in Leishmania amazonensis Amastigotes and Promastigotes by RT-PCR / 中国寄生虫学与寄生虫病杂志

Objective To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. Methods Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions,infected J774.G8 macrophages,and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction(PCR)was used to amplify the specific fragments of amastigote-specific nuclease(P-4)and promastigote-specific membrane glycoprotein(GP-46). PCR products were analyzed in 1.5% agarose gel. Results A P-4-specific band(273 bp)was observed in all three types of amastigotes with similar density,but it was almost undetectable in promastigotes. In contract,a GP-46-specific band(325 bp)was expressed at a higher level in promastigotes than in all three types of amastigotes. Conclusion Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.

Comparative Proteomic Analysis of the Promastigotes and Amastigotes of Leishmania donovani / 中国寄生虫学与寄生虫病杂志

Objective To explore the protein profile and identify developmentally regulated proteins of the promastigotes and axenic amastigotes with comparative proteomics technique.Methods The total proteins of promas-tigotes and axenic amastigotes of Leishmania donovani SC6 strain were separated by two-dimensional electrophoresis(2-DE) in a broad pH range(3-10) , and the gel was stained with Coomassie blue.The images were analyzed by PDQuest 1.0 software, and the major developmentally regulated proteins were identified by electrospray mass spectrometry.Results Approximately 700 protein spots were revealed in equivalent proteins of the promastigotes and axenic amastigotes separated by 2-DE, among which more than 90% protein spots showed equivalent quantity and distribution, with 6 proteins up-regulated and 3 proteins down-regulated in axenic amastigotes compared with promastigotes.Five of the 6 up-regulated proteins were with known function, respectively ascribed as Reiske iron-sulfur protein precursor, ?-tubulin, peroxidoxin 1, dihydrolipoamide acetyltransferase precursor, and mannose-1-phosphate guanyltransferase.Two of the 3 down-regulated proteins were identified as heat shock protein 70 and ?-tubulin.The functions of the developmentally regulated proteins were related to the carbohydrate/energy metabolism, stress response, or formation of cell membrane/cytoskeleton.Conclusion The findings demonstrate the differences in protein expression profiles between promastigotes and amastigotes.