Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis.

BACKGROUND: Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania. RESULTS: Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods. CONCLUSIONS: These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

IFN-γ-Dependent Recruitment of CD4(+) T Cells and Macrophages Contributes to Pathogenesis During Leishmania amazonensis Infection.

Interferon gamma (IFN-γ) is a key factor in the protection of hosts against intracellular parasites. This cytokine induces parasite killing through nitric oxide and reactive oxygen species production by phagocytes. Surprisingly, during Leishmania amazonensis infection, IFN-γ plays controversial roles. During in vitro infections, IFN-γ induces the proliferation of the amastigote forms of L. amazonensis. However, this cytokine is not essential at the beginning of an in vivo infection. It is not clear why IFN-γ does not mediate protection during the early stages of infection. Thus, the aim of our study was to investigate the role of IFN-γ during L. amazonensis infection. We infected IFN-γ(-/-) mice in the footpad and followed the development of leishmaniasis in these mice compared with that in WT mice. CD4(+) T lymphocytes and macrophages migrated earlier to the site of infection in the WT mice, and the earlier migration of these 2 cell types was associated with lesion development and parasite growth, respectively. These differences in the infiltrate populations were explained by the increased expression of chemokines in the lesions of the WT mice. Thus, we propose that IFN-γ plays a dual role during L. amazonensis infection; it is an important inducer of effector mechanisms, particularly through inducible nitric oxide synthase expression, and conversely, it is a mediator of inflammation and pathogenesis through the induction of the expression of chemokines. Our data provided evidence for a pathogenic effect of IFN-γ production during leishmaniasis that was previously unknown.

Short-term protection conferred by Leishvacin® against experimental Leishmania amazonensis infection in C57BL/6 mice.

To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.

Identification of virulence factors in Leishmania infantum strains by a proteomic approach.

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.