RESUMO: O objetivo deste trabalho foi avaliar a soroconversão em cães imunizados com as vacinas Leishmune® e Leish tec®, através do teste imunocromatográfico rápido DPP® (Dual Path Platform) (DPP LVC) e do ensaio imunoenzimático (EIE) durante um ano após o protocolo vacinal. Trata-se de um estudo onde 28 cães divididos em dois grupos foram imunizados cada um com uma vacina anti - LVC e acompanhados durante um ano através de avaliação clínica e exames laboratoriais. Foi possível acompanhar 22 (78.5%) cães. Nos exames dos tempos 1, 2 e 3 (respectivamente 30 dias, 6 messes e 1 ano após a vacinação) os resultados de todos os cães também foram negativos para LVC, exceto de um cão que recebeu a vacina Leish tec® e soroconverteu no DPP LVC no T2, após 6 meses a vacina. Os exames posteriores deste cão foram negativos. Os resultados do presente estudo demostraram que, em área não endêmica e mesmo em diferentes tempos de avaliação, cães vacinados contra LVC, independente da vacina utilizada, não foram capazes de soroconverter no protocolo utilizado pelo Ministério da Saúde brasileiro (DPP/EIE).

ABSTRACT: The objective of the current research was to assess seroconversion in dogs immunized with Leishmune® and Leish Tec® vaccines using rapid chromatographic immunoassay DPP® (Dual Path Platform) (DPP CVL) and enzyme immunoassay (EIE) up to one year after the vaccination protocol. The study sample comprised 28 dogs divided into two groups, each group immunized with an anti-CVL vaccine and clinically monitored for one year through clinical evaluation and laboratory tests. 22 (78.5%) dog were monitored. During the evaluation time (T1-30 days, T2-6 months, and T3-1 year after vaccination) the results for all dogs were negative for CVL, except for one animal vaccinated with Leish tec® that seroconverted in the DPP CVL test at T2. Subsequent examinations of this dog were negative. Our results showed that in a non-endemic area, even at different evaluation times, dogs vaccinated against CVL with Leishmune® or Leish tec® did not seroconvert in the serological protocol used by the Brazilian Ministry of Health (DPP/EIE).

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

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.

Tumor necrosis factor-alpha (TNF-α) is a cytokine produced by activated macrophages and other cells. In order to verify whether the serum levels of TNF-α in American tegumentary leishmaniasis patients are associated with the process of cure or aggravation of the disease, 41 patients were studied: 26 cases of cutaneous leishmaniasis (CL) and 15 of mucocutaneous leishmaniasis (MCL). During active disease the serum levels of TNF-α of MCL patients were significantly higher than those of CL patients and control subjects (healthy individuals and cutaneous lesions from other etiologies). The MCL patients had serum titers of TNF-α significantly lower at the end of antimonial therapy than before therapy. After a six-month follow-up, the MCL patients had serum levels of TNF-α similar to those observed at the end of the therapy as well as to those of CL patients and control subjects. No significant variation in the serum levels of TNF-α was observed in CL patients throughout the study period (before, at the end of therapy and after a six-month follow-up). The possible relationship between the high TNF-α serum levels and severity of the disease is discussed.