Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Natural Infection by Leishmania infantum in domestic cats (Felis catus) in a municipality of moderate transmission in the Brazilian semi-arid region / Infecção natural por Leishmania infantum em gatos domésticos (Felis catus) em município de transmissão moderada no semiárido brasileiro

Abstract The main clinical, anatomopathological, and molecular aspects of the infection by Leishmania infantum are described in two cats with multicentric cutaneous, nodular, and ulcerated lesions. The animals were submitted to a clinical examination, followed by serological, molecular and parasitological exams, with culture and isolation of the parasite, and subsequent isoenzymatic characterization. The animals were euthanized and necropsied. Case 1 was an adult, female, mixed-bred stray cat. Case 2 was an adult, male, mixed-bred and domiciled cat. Both were positive for the presence of anti-L. infantum antibodies. In the cytology of the cutaneous nodules and lymph nodes, amastigote forms of Leishmania spp. could be visualized, free and in the interior of the macrophages. In the histopathology, the lesions were characterized by nodular granulomatous and/or ulcerative dermatitis, associated to amastigote forms of Leishmania spp. By means of the polymerase chain reaction, the sequence of the L. infantum kDNA minicircle was amplified. It is concluded that the infection by L. infantum occurs in cats in the State of Paraíba, Northeast region of Brazil and the need to understand the immunological profile of the visceral leishmaniasis in the feline population is highlighted with aimed at the control measures in public health.

Resumo Descrevem-se os principais aspectos clínicos, anatomopatológicos e moleculares da infecção por Leishmania infantum em dois gatos, cuja queixa era de lesões cutâneas multicêntricas, nodulares e ulceradas. Os animais foram submetidos à avaliação clínica, seguida de exames sorológicos, molecular e parasitológico, com cultura e isolamento do parasita e posterior caracterização isoenzimática. Os animais foram eutanasiados e encaminhados para a necropsia. O caso 1 era uma gata adulta, sem raça definida e errante. O caso 2 era um gato adulto, sem raça definida e domiciliado. Ambos foram positivos para a presença de anticorpos anti-L. infantum. Na citologia dos nódulos cutâneos e linfonodos, puderam ser visualizadas formas amastigotas de Leishmania spp. livres e no interior de macrófagos. Na histopatologia, as lesões se caracterizavam por dermatite granulomatosa nodular e/ou ulcerativa, associadas a formas amastigotas de Leishmania spp. Por meio da reação em cadeia da polimerase, amplificou-se a sequência do minicírculo do kDNA de L. infantum. Conclui-se que a infecção por L. infantum ocorre em gatos no estado da Paraíba, região Nordeste do Brasil. Deve-se ressaltar a necessidade de compreender o perfil imunológico e epidemiológico da leishmaniose visceral na população felina, com vistas às medidas de controle em saúde pública.

Analysis of macrophage subtypes and annexin A1 expression in lesions of patients with cutaneous leishmaniasis

Abstract INTRODUCTION: Cutaneous leishmaniasis is caused by protozoa of the genus Leishmania and transmission occurs through the bite of sandflies. It is an infectious disease, which affects skin and mucosa. The aim was to quantify the macrophages M1 and M2 and the annexin A1 expression in the skin lesions of patients with cutaneous leishmaniasis. METHODS: Skin biopsies from patients (n = 50) were analyzed and classified according to the lesion type as: exudative cellular reaction, exudative granulomatous reaction, exudative necrotic reaction, exudative necrotic-granulomatous reaction. Using the immunofluorescence technique, macrophages were identified by CD163 marker, differentiated by anti-MHCII and anti-CD206 antibodies, and annexin A1 expression was determined by arbitrary unit (a.u.) densitometry. RESULTS: In M1 macrophages, a greater expression of this protein was observed in the exudative cellular reaction type lesions (136.3 ± 2.6 a.u., assuming mean and standard derivation) when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (108.0 ± 2.3, 121.6 ± 3.2 and 124.7 ± 2.4 a.u., respectively). Regarding M2 macrophages, it was observed that patients with exudative cellular reaction lesion also had a higher expression of this protein (128.8 ± 2.6 a.u.), when compared to the expression in the lesions of exudative granulomatous reaction, exudative necrotic reaction and exudative necrotic-granulomatous reaction patients (105.6 ± 2, 113.9 ± 2.8, 114.3 ± 2.1 a.u., respectively). CONCLUSIONS: These data suggest that annexin A1 is assisting macrophages in the phagocytosis process of patients with exudative cellular reaction lesion type.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

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.

Expression of annexin A1 in Leishmania-infected skin and its correlation with histopathological features

ABSTRACTINTRODUCTION:The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4) + and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects.METHODS:Infecting species were identified by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofluorescence.RESULTS:All patients (n = 55) were infected with Leishmania braziliensis . Annexin A1 was expressed more abundantly in CD163 + macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4 + cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8 + T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infiltrate in cellular lesions.CONCLUSIONS:Annexin A1 is differentially expressed in CD163 + macrophages and T cells depending on the histopathological features of Leishmania -infected skin, which might affect cell activation.

Consumo de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH): uma revisão sistemática da literatura / Caffeine consumption during pregnancy and attention deficit hyperactivity disorder (ADHD): a systematic literature review / Consumo de cafeína durante el embarazo y trastorno por déficit de atención e hiperactividad (TDAH): una revisión sistemática de la literatura

O objetivo do presente estudo foi avaliar, mediante revisão sistemática da literatura, as evidências acerca da associação entre consumo materno de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH) na infância. A busca na literatura ocorreu de forma sistemática, em múltiplas etapas, nas bases PubMed, LILACS, BIREME e PsycINFO, com limites para artigos publicados em português, inglês e espanhol, realizados em humanos. Foram encontradas 373 referências. Dessas, somente cinco foram mantidas, por atenderem ao objetivo deste estudo. Os cinco trabalhos foram realizados em países desenvolvidos; a maioria utilizou delineamento longitudinal e foi publicada nos últimos cinco anos. Apenas um estudo encontrou associação positiva. Estudos sobre o consumo de cafeína na gestação e TDAH são escassos, com resultados controversos e se deparam com várias dificuldades metodológicas, como falta de padronização na definição do desfecho.

This aim of this study was to conduct a systematic literature review on the association between maternal caffeine intake during pregnancy and attention deficit hyperactivity disorder (ADHD) in childhood. The systematic multiple-stage literature search in PubMed, LILACS, BIREME, and PsycINFO was limited to research in human subjects and published in Portuguese, English, and Spanish. A total of 373 references were retrieved. Of these, only five met the study's objectives and were kept in the review. Most of the studies employed a longitudinal design, were conducted in developed countries, and were published in the last five years. Only one study found a positive association. Studies on caffeine consumption during pregnancy and ADHD are scarce, with conflicting results and several methodological difficulties such as lack of standardized outcome measures.

El objetivo de este estudio fue evaluar, a través de una revisión sistemática de la literatura, evidencias sobre la asociación entre el consumo de cafeína durante el embarazo y el trastorno por déficit de atención e hiperactividad (TDAH) en la infancia. Se realizó una búsqueda sistemática en la literatura, por etapas múltiples, en PubMed, LILACS BIREME y PsycINFO, limitándose a artículos publicados en portugués, inglés y español, realizados en estudios sobre humanos. Fueron localizadas 373 referencias. De ellas, apenas se mantuvieron cinco, por cumplir el objetivo de este estudio. Los estudios se realizaron en países desarrollados; el diseño longitudinal fue el más utilizado y se trata de publicaciones de los últimos cinco años. Sólo un estudio encontró asociación positiva. Los estudios sobre el consumo de cafeína durante el embarazo y el TDAH son escasos, con resultados controvertidos, y enfrentan varias dificultades metodológicas, como la no estandarización de la evaluación del resultado.

Avaliação da resposta inflamatória tecidual de pacientes com leishmaniose cutânea recente / Evaluation of tissue inflammatory response in patients with recent cutaneous leishmaniasis

Introdução: A leishmaniose cutânea (LC) é a forma clínica mais frequente da leishmaniose humana, considerada um importante problema de saúde no Brasil. A infecção por Leishmania braziliensis induz um amplo espectro de lesões que pode se manifestar como uma única lesão cutânea localizada, geralmente em partes descobertas do corpo. Tem início com uma pápula, caracterizando a leishmaniose cutânea recente (LCR) e, na maioria dos casos, tende a desenvolver uma úlcera, representando a leishmaniose cutânea clássica (LCC). Pacientes com LCR apresentam um elevado número de parasitas na lesão e, frequentemente, não respondem positivamente à terapia padrão, desenvolvendo a lesão mesmo após o tratamento. Objetivo: Descrever de modo comparativo os aspectos histopatológicos na Leishmaniose Cutânea Recente e Leishmaniose Cutânea Clássica. Métodos: Secções histológicas obtidas de biópsias de pele de 15 pacientes com LCR e 28 com LCC, foram coradas em HE e mensuradas as áreas de inflamação e necrose nas diferentes fases da doença. Realizamos imunohistoquímica para marcação de células CD3+, CD4+, CD8+, CD20+, CD68+ e CD138+...

Introduction: Cutaneous leishmaniasis (CL) is the most common clinical form of human leishmaniasis induced by L. braziliensis. It is considered a major health problem in Brazil. Leishmania braziliensis infection induces a large spectrum of lesions that can manifest as one localized skin lesion, usually undressed body parts. It starts with a papule in early cutaneous leishmaniasis (ECL) clinical manifestation and, in most cases, tends to develop an ulcer, in the late cutaneous leishmaniasis (LCL). ECL patients have a high number of parasites in the lesion, and often do not respond to standard therapy, developing the lesion even after treatment. Aim: To describe comparative the histopathological aspects of early cutaneous leishmaniasis compared to late ulcerated cutaneous leishmaniasis. Methods: Histological sections of skin biopsies from 15 ECL patients and 28 LCL, were stained with HE and measured areas of inflammation and necrosis in the different stages of the disease. We performed immunohistochemical for CD3+, CD4+, CD8+, CD20+, CD68+ and CD138+...

Ativação da heme oxigenase-1 e via da necroptose como mecanismos imunopatogênicos na infecção de macrófagos por leishmania infantum / heme oxygenase-1 activation and via the necroptose as immunopathogenics mechanisms for the infection of macrophages by Leishmania infantum

A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10...

Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α...

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.

Avaliação do papel do receptor marco na infecção de macrófagos murinos por Leishmania major / Evaluation of the role of the receptor in March infection of murine macrophages with Leishmania major

Camundongos CBA são resistentes à infecção por Leishmania major e permissivos à infecção por L. amazonensis. Adicionalmente, macrófagos de camundongos CBA controlam à infecção por L. major, mas não por L. mazonensis in vitro. Em estudo comparativo realizado por nosso grupo foi demonstrado que o receptor scavenger MARCO teve expressão aumentada em resposta à infecção por L. major, mas não na infecção por L. amazonensis. Ainda, o bloqueio do receptor com o anticorpo específico reduziu a infecção por L. major em 30%, indicando que esta proteína tem participação no reconhecimento de promastigotas de L. major em macrófagos de CBA. Assim, nossa hipótese é que o receptor MARCO participa do reconhecimento e fagocitose de L. major por macrófagos, direcionando o curso da infecção. O objetivo do presente estudo consistiu em evidenciar o papel do receptor MARCO na infecção de macrófagos por L. major. Inicialmente, células J774 foram transfectadas com os vetores pcDNA3.1-MARCO (J774-MARCO) ou pcDNA3.1 (J774-MOCK)...

CBA mice are resistant to Leishmania major yet permissive to L. amazonensis infection. In addition, CBA macrophages control L. major, but not L. amazonensis infection in vitro. In a comparative study performed by our group increase in expression of the scavenger receptor MARCO has been detected in response to L. major, but not to L. amazonensis infection. Moreover, ED31 monoclonal antibody against MARCO reduced by 30% L. major infection in CBA macrophages. These findings indicate that MARCO plays a role in L. major recognition by CBA macrophages. We hypothesized that MARCO receptor participates in the recognition and phagocytosis of L. major by macrophages, directing the outcome of infection. In the present study, we aimed to further disclose the role MARCO plays in L. major infection of murine macrophages. First J774 cells were transfected with pcDNA3.1-MARCO vector (MARCO-J774) or pcDNA3.1 vector (MOCK-J774)...

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.

Efeito da autofagia sobre a capacidade fagocítica e sobre a infecção de macrófagos de camundongos CBA/J por Leishmania amazonensis / Effect of autophagy on phagocytic capacity and the infection of macrophages from CBA /J mice by Leishmania amazonensis

A autofagia vem sendo alvo de estudos que demonstram sua participação em infecções por diversos patógenos intracelulares. A depender do patógeno, a autofagia pode facilitar a sobrevivência intracelular do patógeno ou pode funcionar como controle da infecção pela célula hospedeira. Pouco se sabe sobre a participação da autofagia na infecção por Leishmanía. Foi demonstrado que o vacúolo parasitóforo induzido por L. mexícana adquire nutrientes citosólicos por microautofagia. Além disso, recentemente foi demonstrado que a indução de autofagia promove aumento da carga parasitária de L. amazonensís em macrófagos infectados. Esses dados sugerem a participação do processo autofágico no estabelecimento da infecção por Leishmanía, como um mecanismo que favorece a sobrevivência intracelular do parasito. Assim, o objetivo desse estudo foi determinar a influência da autofagia na infecção, in vitro, de macrófagos de camundongos CBA/J por L. amazonensís. Macrófagos foram induzidos à autofagia por duas formas, fisiológica ou farmacológica, após ou antes da infecção por L. amazonensís ou exposição a partículas de levedo ou zimosan. O percentual de infecção e de fagocitose foi estimado. Os resultados mostram que a indução de autofagia, após a infecção, não altera o percentual de macrófagos infectados, mas promove o aumento na carga parasitária de macrófagos infectados por L. amazonensís. Além disso, a prévia indução de autofagia promove a inibição da capacidade fagocítica do macrófago murino. Estudos adicionais serão realizados no intuito de esclarecer os mecanismos pelos quais a indução de autofagia favorece a infecção por L. amazonensís e altera a capacidade fagocítica do macrófago murino.

Anti-leishmanial Effects of Trinitroglycerin in BALB/C Mice Infected with Leishmania major via Nitric Oxide Pathway

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.

Comparison of treatment regimens of kala-azar based on culture & sensitivity of amastigotes to sodium antimony gluconate.

BACKGROUND & OBJECTIVE: Present treatment strategies for kala-azar (visceral leishmaniasis, VL) include use of first line drug sodium antimony gluconate (SAG) to all patients but a large number of patients do not get relief with this drug. If a patient does not respond to a full course of SAG, a second or third line drug is given. We undertook this study to test whether an improved outcome can be achieved by employing a strategy of treatment based on culture and sensitivity of amastigotes to SAG compared with conventional empirical treatment. METHODS: In a double-blind, randomized, controlled trial done in Balaji Utthan Sansthan, Patna, of the 181 patients screened,140 were finally randomly allocated to two groups A and B; group A patients were treated with SAG if their amastigotes were sensitive to SAG, and all patients in group B were treated with SAG to start with. Primary outcome measured was as no relapse within 6 months of follow up after cure and other outcomes measured were period of stay of patients in hospital, expenditure involved in the treatment, and infectivity periods of two groups, two-third of treatment period and whole of untreated period were taken as infectivity period. SAG was used at a dosage of 20 mg/kg given deep intramuscular injections in buttock for 28 days, amphotericin B (AMB) given at a dose of 1 mg/kg body wt daily for 20 days as a slow intravenous infusion in 5 per cent dextrose. RESULTS: Of the 70 patients in group A, 29 patients whose amastigotes were sensitive to SAG were treated with SAG, 2 patients were withdrawn due to drug toxicity; and 2 relapsed within 6 months of follow up and ultimate cure occurred in 25 (86.2%) patients only. Of the 70 patients in group B treated with SAG, 5 (7.1%) patients withdrew due to drug toxicity, 35 patients (50%) did not respond to treatment, 5 (7.1%) relapsed during 6 months of follow up and thus only 25 patients (35.7%) were ultimately cured. The difference between the two groups was significant (P<0.001). No patient died during treatment due to any toxicity because of early withdrawal of patients from treatment apprehending toxicity. Patients whose amastigotes were resistant to SAG, withdrawn from the study due to SAG toxicity, relapsed after cure with SAG, and who did not respond to SAG in both the groups were treated with AMB and all were cured. Groups B and A patients spent 3065 and 2340 days respectively in hospital, group B 1.3 times more than group A. The likely period of spread of parasites in society was 1965 days in group B and 1644 days in group A, group B 1.4 times more than group A. The total expenditure on treatment in groups B and A was dollars 65,575 and dollars 50,590 respectively; group B patient had to spend 1.3 times more than group A. INTERPRETATION & CONCLUSION: A new strategy for treatment of kala-azar based on culture and sensitivity of amastigotes improved the cure rate, saved expenditure on the patient's treatment, patients had to stay for shorter periods in hospital and reduced the chance of spread of SAG resistant disease in society. Till the government opts for better drugs, the treatment based on culture and sensitivity of the parasites to SAG may be a better method.

Avaliação da imunidade protetora conferida pela imunização de hamsters com saliva do flebotomíneo Lutzomyia Longipalpis contra desafio com Leishmania Brasiliensis na presença da saliva de diferentes vetores / Assessment of protective immunity conferred by immunization of hamsters with the saliva of Lutzomyia longipalpis phlebotomine against challenge with Leishmania brasiliensis in the presence of saliva of different vectors

Transmitidas por diferentes espécies de Flebótomos, as Leishmanioses apresentam uma grande variedade de manifestações clínicas. Tem sido demonstrada, a possibilidade de imunização contra a infecção por Leishmania utilizando-se a saliva do vetor Flebotomíneo Lutzomyia Longipalpis. O principal vetor da Leishmania Braziliensis é o Flebótomo Lutzomyia Intermedia. O objetivo deste estudo foi avaliar se a imunização de hamsters com a saliva de L. Longipalpis confere proteção contra a infecção por L. Braziliensis na presença da saliva de L. Intermedia. Hamsters machos forma imunizados com o Sonicado de Glândula Salivar (SGS) por três vezes com intervalo de quinze dias. O desafio foi feito na orelha contra-lateral quinze dias após a última imunização 105 (com dez elevado à quinta potência) formas promastigotas de L. Braziliensis na presença do SGS de L. Intermedia. O desenvolvimento da lesão foi acompanhado semanalmente e 3,5 e oito semanas, após o desafio, as orelhas e lifondos drenantes foram retirados para a avaliação da carga parasitária, bem como da produção de citocinas durante a infecção. Quarenta e oito horas após o desafio com SGS, observa-se um infiltrado inflamatório composto predominantemente de células mononucleares nas orelhas dos animais imunizados. Estes animais apresentam redução significativa na carga parasitária dos lifondos drenantes, bem como das orelhas desafiadas. Além disso, produzem significativamente mais anticorpos anti-saliva quando comparados aos não-imunizados e menos anticorpos anti-Leishmania. Os possíveis mecanismos envolvidos nesta proteção são os níveis reduzidos da expressão de IL-10 e TGF- ao longo da infecção. Estes resultados sugerem que a imunização com o SGS de L. Longipalpis confere proteção contra a infecção por L. Braziliensis de modo inespecífico à presença da saliva no momento do desafio. Dessa forma, a possibilidade de vacinação contra diferentes espécies de Leishmania, utilizando proteínas salivares de uma espécie de vetor...

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37 percent when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2 percent formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Leishmania (Viannia) lainsoni: occurrence of intracellular promastigote forms in vivo and in vitro

Experimental chronic (45-day-old) skin lesion in hamster hind foot induced by Leishmania (Viannia) lainsoni infection showed the presence of promastigote forms in the tissue, inside parasitophorous vacuoles, as assessed by transmission electron microscopy. Experimental in vitro interaction (24 and 48 h) between Leishmania (V.)lainsoni and J774-G8 macrophage cells also demonstrated the same profile. This morphological aspect is unusual, since in this parasite genus only amastigote forms have been described as the resistant and obligate intracellular forms.