Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

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

Histological evaluation of the lesion induced by inoculation of Leishmania mexicana in the cheek pouch of the hamster

No presente estudo, investigamos o papel da resposta imune na morfologia do granuloma leishmaniótico induzido na bolsa jugal do hamster, um local imunologicamente privilegiado, após inoculação de 3x10(5) Leishmania mexicana. Os animais foram avaliados histológica e imunologicamente até os 120 dias da inoculação. Independente da época do sacrifício, os animais foram sempre não reatores ao teste do coxim plantar. Histologicamente, a inoculação de Leishmania mexicana na bolsa jugal resultou na formação de abcesso que evoluiu para reação granulomatosa rica em formas amastigotas e, posteriormente, para resolução. Esses resultados sugerem que o desenvolvimento da resposta imune não é preponderante no controle da infecção induzida pela Leishmania mexicana inoculada subcutaneamente na bolsa jugal do hamster. Sugerem ainda que os macrófagos que compõe os granulomas leishmanióticos são capazes de eliminar esse parasita, independente da presença de resposta imune avaliável pelo teste do coxim plantar.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune((R)) vaccine

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune (R) vaccine, formulated with an increased adjuvant concentration (1 mg of saponin rather than 0.5 mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune (R) vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune (R)-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p < 0.0001), a higher and stable IgG2 and a decreasing IgG I response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune (R) immumotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p < 0.0001), the parasitological evidence (p = 0.038) and a decrease in Leishinania-specific CD4+ lymphocyte proportions (p = 0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune (R) vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune (R) vaccine was subjected to a safety analysis and found to be well tolerated and safe. (c) 2007 Elsevier Ltd. All rights reserved.

Immunotherapy with the saponin enriched-Leishmune (R) vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD(+)-dependent deacetylase

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

Avaliação do efeito antiparasitário do omeprazol na prevenção do desenvolvimento de lesões cutâneas em hamsters infectados por Leishmania brasiliensis

FUNDAMENTOS: A leishmaniose tegumentar americana permanece doença endêmica em diversas regiões brasileiras. A sobrevivência do parasita no interior dos macrófagos se deve, em parte, pela atividade de uma K+/H+-ATPase de membrana que pode ser inibida pelo omeprazol. OBJETIVOS: Avaliar a eficácia do omeprazol na prevenção do desenvolvimento de lesões de leishmaniose em hamsters. MÉTODOS: Empregaram-se 18 hamsters, divididos em três grupos: o grupo L recebeu apenas a inoculação de L. brasiliensis na pata anterior direita, o grupo O recebeu apenas doses diárias de 0,4mg de omeprazol subcutâneo, e o grupo L+O recebeu o inóculo de leishmanias e o tratamento com omeprazol desde o dia da inoculação. O estudo foi conduzido por 42 dias, realizaram-se medidas dos diâmetros das patas semanalmente, e, ao final do estudo, foram realizados esfregaços das lesões para verificação dos parasitas. RESULTADOS: Os hamsters dos grupos L e L+O desenvolveram lesões de leishmaniose tegumentar havendo ulceração em duas patas do grupo L e uma do grupo L+O. Ao final do estudo, a mobilidade e vitalidade no grupo L foram menores que em L+O, e estas menores que no grupo O. Os diâmetros das patas inoculadas nos grupos L e L+O foram significativamente maiores que no início do estudo (p<0.05). Não houve diferença significativa entre os diâmetros das patas dos grupos L e L+O ao final do estudo (p0,05), sendo detectados parasitas no esfregaço das lesões dos dois grupos. CONCLUSÕES: Omeprazol, no protocolo utilizado, não evitou o desenvolvimento de lesões de leishmaniose tegumentar em hamsters.

Saponins, IL12 and BCG adjuvant in the FML-vaccine formulation against murine visceral leishmaniasis

The FML antigen of Leishmania donovani, in combination with either Riedel de Haen (R), QuilA, QS21 saponins, IL12 or BCG, was used in vaccination of an outbred murine model against visceral leishmaniasis (VL). Significant and specific increases in anti-FML IgG and IgM responses were detected for all adjuvants, and in anti-FML IgG1, IgG2a and IgG2b and delayed type of hypersensitivity to L. donovani lysate (DTH), only for all saponins and IL12. The QS21-FML and QuilA-FML groups achieved the highest IgG2a response. QuilA-FML developed the strongest DTH and QS21-FML animals showed the highest serum IFN-gamma concentrations. The reduction of parasitic load in the liver in response to each FML-vaccine formulation was: 52% (P < 0.025) for BCG-FML, 73% (P < 0.005) for R-FML, 93% (P < 0.005) for QuilA-FML and 79.2% (P < 0.025) for QS21-FML treated animals, respectively. Protection was specific for R-FML and QS21-FML while the QuilA saponin treatment itself induced 69% of LDU reduction. The FML-saponin vaccines promote significant, specific and strong protective effects against murine visceral leishmaniasis. BCG-FML induced minor and non-specific protection while IL 12-FML, although enhancing the specific antibody and IDR response, failed to reduce the parasitic load of infected animals. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Immunotherapy against murine experimental visceral leishmaniasis with the FML-vaccine

The fucose mannose ligand (Leishmania donovani FML)-saponin vaccine has earlier shown its immunoprophylactic potential against visceral leishmaniasis in the CB hamster (87.7% of parasite load reduction), Balb/c (84.4%) and Swiss albino mouse (85-93%) models. In this investigation its specific immunotherapeutic efficacy against L. donovani infection in Balb/c mice was studied. The effects of vaccine treatment on the Immoral response, delayed type of hypersensitivity to promastigote lysate (DTH), cytokine levels in sera and reduction of the liver parasitic load of L. donovani infected mice, were examined. The types and subtypes of anti-FML antibodies increased significantly in the vaccinees over the saline and saponin controls. As expected for a saponin vaccine, the highest ratios were found in relation to IgG1, IgG2a and IgG2b (4.4, 5 and 2.5, respectively). The DTH response and the in vitro ganglion cell proliferative response against FML antigen were also significantly higher than controls (P < 0.005). Concomitantly, an impressive and specific decrease of liver parasitic burden was detected only in vaccine-treated animals (94.7%). Our results indicate that the therapeutic FML-vaccine has a potent effect on modulation of the murine infection leading to the reduction of parasitic load and signs of disease, being a new potential tool in the therapy and control of visceral leishmaniasis. (C) 2003 Elsevier Ltd. All rights reserved.

Canine cerebral leishmaniasis: Potential role of matrix metalloproteinase-2 in the development of neurological disease

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

IgG subclass profile of serum antibodies to Leishmania chagasi in naturally infected and vaccinated dogs

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

Natural Products from Garcinia brasiliensis as Leishmania Protease Inhibitors

The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (+/- SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0 +/- 1.3 mu g/mL and 3.2 +/- 0.5 mu M/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases.

Metacyclogenesis modulates the ability of Leishmania promastigotes to induce IL-12 production in human mononuclear cells

Immunity against the intracellular protozoan Leishmania species is highly dependent on Th1 development. We have previously shown that IL-12 is a powerful and probably obligatory factor for Th1 cell generation and proliferation. We also observed that the experimental infection of C3H and BALB/c mice with Leishmania major is associated with IL-12 production in vivo. Now we demonstrate that metacyclic L. major promastigotes are poor inducers of IL-12 in vitro in fresh human PBMC and monocytes. In addition, we show that the ability of this pathogen to induce IL-12 and other cytokines is modulated by the metacyclogenic process, which had previously not been recognized. In contrast to the infective parasites (metacyclic promastigotes), the procyclic promastigotes collected at the logarithmic phase of the culture displayed a striking ability to induce IL-12, IFN-γ, TNF-α, and IL-10. Despite this differential effect of procyclic and metacyclic parasites in terms of IL-12 induction, both stages were inhibitory for IL-12 production induced by Staphylococcus aureus. The ability of procyclic promastigotes and, to a much lesser extent, that of metacyclic promastigotes to induce IL-12 were enhanced by pretreatment of monocytes in a cytokine milieu containing IFN-γ, IL-4, IL-13, or granulocyte-macrophage CSF or. by neutralization of endogenous IL-10. Our results suggest the development of an evasion mechanism as the Leishmania promastigotes mature to infectious forms and the possi-bility of using Ags derived from procyclic promastigotes for immunization procedures. Copyright © 1997 by The American Association of Immunologists.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Subclinical muscle injuries in dogs infected with Leishmania (Leishmania) infantum chagasi

Although canine visceral leishmaniasis (CVL) has been extensively studied, muscular damage due to Leishmania (Leishmania) infantum chagasi infection remains to be fully established. The aim of this study was to describe the electromyographic and histological changes, as well as search for the presence of amastigote forms of Leishmania spp, CD3+ T-lymphocytes, macrophages and IgG in skeletal muscles of dogs with visceral leishmaniasis (VL). Four muscles (triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius) from a total of 17 naturally infected and six healthy dogs were used in this study. Electromyographic alterations such as fibrillation potentials, positive sharp waves and complex repetitive discharges were observed in, at least, three muscles from all infected dogs. Myocyte necrosis and degeneration were the most frequent muscular injury seen, followed by inflammatory reaction, fibrosis and variation in muscle fibers size. Immunohistochemistry in muscle samples revealed amastigote forms in 4/17 (23. 53%), IgG in 12/17 (70. 58%), CD3+ T-lymphocytes in 16/17 (94. 12%) and macrophages in 17/17 (100%) dogs. Statistically positive correlation was observed between: inflammatory infiltrate (p=0. 0305) and CD3+ immunoreaction (p=0. 0307) in relation to the number of amastigote forms; inflammatory infiltrate (p=0. 0101) and macrophage immunoreaction (p=0. 0127) in relation to the amount of CD3+; and inflammatory infiltrate (p=0. 0044) and degeneration/necrosis (p<0. 0001) in relation to the presence of macrophages. Our results suggest that different mechanisms contribute to the development of myocytotoxicity, including celular and humoral immune responses and direct muscular injury by the parasite. Nevertheless, the catabolic nature of the disease can probably interact with other factors, but cannot be incriminated as the only responsible for myositis.