IL-10 na patogênese da infecção por Leishmania infantum

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Infecção natural de Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) por Leishmania infantum chagasi em flebotomíneos capturados no município de Janaúba, Estado de Minas Gerais, Brasil

INTRODUÇÃO: A leishmaniose visceral tem sido notificada em quase todos os estados do Brasil, e principalmente no norte de Minas Gerais, onde a doença é endêmica. Este estudo visou detectar a infecção natural de Lutzomyia longipalpis e identificar através da técnica de PCR/RFLP a espécie de Leishmania encontrada nos flebotomíneos do município de Janaúba. MÉTODOS: Utilizando-se armadilhas luminosas, foram capturadas 1.550 fêmeas de L. longipalpis, que agrupadas em pool de 10 exemplares foram submetidas à extração e amplificação de DNA, através das técnicas de PCR genérico e cacofonia. RESULTADOS: Dos 155 pools, seis apresentaram-se positivos para Leishmania sp., sendo a taxa de infecção do município de 3,9%. Através da PCR/RFLP determinou-se que o padrão de digestão das amostras positivas foi semelhante ao da cepa referência Leishmania chagasi (MHOM/BR/74/PP75). CONCLUSÕES: A detecção de infecção natural associada a estudos sobre a epidemiologia da LV sugere que L. longipalpis esteja envolvida na transmissão de L. infantum chagasi em Janaúba, principalmente nas áreas de intensa transmissão de LV.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs

A leishmaniose visceral canina (LVC) é reconhecida pelas características clínicas da doença e é altamente letal. A infecção, entretanto, pode ser totalmente assintomática em alguns cães soropositivos, o que tem levantado questão polêmica sobre a possibilidade desses animais, serem ou não uma fonte importante da infecção para o flebotomíneo, Lutzomyia longipalpis, o principal vetor da leishmaniose visceral americana (LVA). Neste estudo foram examinados 51 cães com LVC aguda, provenientes de área endêmica de LVA no Estado do Pará, Brasil, e a carga parasitária, formas amastigotas de, na pele, linfonodo poplíteo e vísceras (fígado e baço) foi comparada com a de nove cães assintomáticos soropositivos (IFAT-IgG). Fragmentos de biópsia desses tecidos obtidos post-mortem foram processados para análise através de imunohistoquímica, usando um anticorpo policlonal contra Leishmania sp. Os testes do Qui-quadrado (X²) e Mann Whitney foram usados para avaliar as médias da densidade de macrófagos infectados (p < 0,05). Os resultados mostraram que não houve diferença (p > 0,05) na densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²) e do linfonodo (6,3/mm² x 8,3/mm²) entre cães assintomáticos e sintomáticos. Entretanto, a densidade de macrófagos infectados da víscera de cães sintomáticos (5,3/mm²) foi maior (p < 0,05) que a de cães assintomáticos (1,4/mm²). Estes resultados sugerem, fortemente, que cães naturalmente infectados por L. (L.) i. chagasi, assintomáticos ou sintomáticos, podem servir como fonte de infecção, principalmente, considerando-se que a densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²), local onde o flebotomíneo vetor Lu. longipalpis realiza a hematofagia, foi maior (p < 0,05) que as do linfonodo (6,3/mm² x 8.3/mm²) e vísceras (1,4/mm2x 5,3/mm2).

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

Conhecimento de escolares sobre leishmaniose visceral

Pós-graduação em Ciência Animal - FMVA

Análise da expressão de MMP-2 e MMP-9 na pele de cães com Leishmaniose visceral

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Detection of Leishmania infantum in naturally infected Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) and Canis familiaris in Misiones, Argentina: the first report of a PCR-RFLP and sequencing-based confirmation assay

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Alterações eletromiográficas e histopatológicas da musculatura de cães naturalmente infectados por Leishmania infantum

The aim of this study was to evaluate the electromyographic and histopathological changes in skeletal muscles of dogs naturally infected by L. infantum. Twenty five mixed breed adult dogs with parasitological, molecular and serological diagnosis were selected. The evaluated muscles were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. One dog had locomotor clinical signs with hind limbs paresis associated with severe muscle atrophy. Twenty-three (92%) had some type of muscular change, and in 22 (88%) such changes were directly identified by electromyography. Even without any clinical signs of the disease, 10 (40%) dogs had electromyographic and histopathological changes. Leishmania antigens were detected in muscles of four (16%) dogs. The electromyographic evaluation indicated the occurrence of chronic polymyositis in 13 (52%) dogs, the presence of both acute and chronic muscle inflammation four (16%), acute myopathy in two (8%) and absence of electromyographic abnormalities in three (12%) dogs. The most frequently observed histopathological changes were degeneration and necrosis of myofibers and inflammatory infiltration observed in 12 (48%) dogs. Other changes were decreased diameter of muscle fibers in 15 (60%) and peri or endomysial fibrosis in 14 (56%) animals. The changes observed in the present study showed that even in the absence of clinical signs, most dogs infected by Leishmania infantum have chronic polymyositis.

Caracterização genotípica de amostras de Leishmania spp. isoladas de área endêmica para Leishmaniose Visceral Canina no estado de São Paulo

Pós-graduação em Doenças Tropicais - FMB

Ocorrência de leishmaniose visceral canina em assentamento agrícola no Estado de Mato Grosso do Sul, Brasil

Durante estudos sobre a fauna flebotomínea em assentamento do Instituto Nacional de Reforma Agrária (INCRA) na Serra da Bodoquena, Estado de Mato Grosso do Sul, Brasil, foram observados cães com manifestações clínicas sugestivas de leishmaniose visceral. Inquérito sorológico para leishmaniose em 97 cães, utilizando reação de imunofluorescência indireta, mostrou 23 (23,7%) soros reagentes. Amostras do parasita foram identificadas como Leishmania (Leishmania) chagasi.

Characterization of Leishmania infantum species in dogs from the urban area of Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Visceral leishmaniasis presents urban behavior in some Brazilian cities, with domestic dogs as the main infection source. In Cuiabá, MT, canine visceral leishmaniasis was diagnosed and characterized as recommended by the Ministry of Health. METHODS: Biological samples from suspected canine carriers were analyzed by the isoenzyme electrophoresis technique. The 6PGDH enzyme and reference strain IOC/L0566 (MHOM/BR/1975/M2903) of Leishmania (Leishmania) infantum was used as one of the controls. RESULTS: Electrophoresis analysis revealed that the canine isolates belonged to the species L. (L.) infantum. CONCLUSIONS: The authors emphasize the importance of species characterization, particularly in areas of mixed infection like Cuiabá.

Expressão gênica das interleucinas IL-4, IL-10, IL-12 e de interferon gama no baço de gatos infectados por Leishmania infantum chagasi

Pós-graduação em Ciência Animal - FMVA

Estado nutricional e efeito da vitamina A na resposta imune frente à infecção por Leishmania Infantum

Nutritional status is an important determinant to the response against Leishmania infection, although few studies have characterized the molecular basis for the association found between malnutrition and the disease. Vitamin A supplementation has long been used in developing countries to prevent mortality by diarrheal and respiratory diseases, but there are no studies on the role of vitamin A in Leishmania infection, although we and others have found vitamin A deficiency in visceral Leishmaniasis (VL). Regulatory T cells are induced in vitro by vitamin A metabolites and are considered important cells implicated T CD4+ cell suppression in human VL. This work aimed to examine the correlation of nutritional status and the effect of vitamin A in the response against Leishmania infantum infection. A total of 179 children were studied: 31 had active VL, 33 VL history, 44 were DTH+ and 71 were DTH- and had negative antibody to Leishmania (DTH-/Ac-). Peripheral blood monuclear cells were isolated in a subgroup of 10 active VL and 16 DTH-/Ac- children and cultivated for 20h under 5 different conditions: 1) Medium, 2) Soluble promastigote L. infantum antigens (SLA), 3) All-trans retinoic acid (ATRA), 4) SLA + ATRA and 5) Concanavalin A. T CD4+CD25highFoxp3+, T CD4+CD25-Foxp3- and CD14+ monocytes were stained and studied by flow cytometry for IL-10, TGF-β and IL-17 production. Nutritional status was compromised in VL children, which presented lower BMI/Age and retinol concentrations when compared to healthy controls. We found a negative correlation between nutritional status (measured by BMI/Age and serum retinol) and anti-Leishmania antibodies and acute phase proteins. There was no correlation between nutritional status and parasite load. ATRA presented a dual effect in Treg cells and monocytes: In healthy children (DTH-/Ac-), it induced a regulatory response, increasing IL-10 and TGF-β production; in VL children it modulated the immune response, preventing increased IL-10 production after SLA stimulation. Furthermore, we found a positive correlation between BMI/Age and IL-17 production and negative correlation between serum retinol and IL-10 and TGF-β production in T CD4+CD25highFoxp3+ cells after SLA stimulus. Our results show a potential dual role of vitamin A in the immune system: improvement of regulatory profile during homeostasis and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might improve recovery from disease status in Leishmania infantum infection

Determinantes envolvidos na resposta imune celular humana à infecção por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients