According to WHO, leishmaniasis is endemic in 98 countries, and has been placed ninth in a global analysis of infectious diseases. Treatment of leishmaniasis is based on pentavalent antimonials but toxicity and developing resistance have been reported. Traditional medicine and scientific studies have shown that the extract of Galipea longiflora Krause (Evanta) exhibits antileishmanial activity. We hypothesized that the healing observed when using this plant might not only be due to the direct action on the parasite, but possibly to a parallel effect on the host immune response. We found that an alkaloid extract of Evanta (AEE) inhibited the growth of Leishmania braziliensis promastigotes while viability of eukaryotic cells was practically not affected. We also found that AEE interfered with polyclonal activation or Leishmania-specific re-stimulation of lymphocytes, as revealed by a reduction of in vitro cellular proliferation and IFN-g production. More important, AEE treatment of mice hosting L. braziliensis showed that AEE is able to control both inflammation and parasite load. Additionally, the healing process was improved when AEE and meglumine antimoniate were administered simultaneously. Dendritic cells (DCs) play a pivotal role in T-cell stimulation and polarization of naïve T cells. Therefore, we investigated if AEE could alter the activation of DCs and if allostimulatory DCs properties were altered if activated in the presence of AEE. DCs activated in the presence of AEE reduced the production of IL-12p40 and IL-23. When we analyzed the allostimulatory capacity of AEE-treated DCs, we found that allogeneic CD4+ T-cells secreted lower levels of IFN-γ. In conclusion, this thesis provides valuable insight into the effects of Evanta derived extract. The dual effect found for AEE, on Leishmania parasite and on the immune response, suggests that AEE may be useful in controlling the parasite burden and preventing over-production of inflammatory mediators and subsequently avoiding tissue damage. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Submitted.</p>

Protozoa of the Leishmania genus are transmitted between mammalian hosts by the sandfly and cause the neglected tropical disease leishmaniasis. Upon injection into the mammalian host by the sandfly promastigote-form parasites are phagocytosed by macrophages, where they differentiate into amastigotes. Although many virulence factors are known to modulate macrophage signalling pathways to favour infection, the delivery mechanisms are largely unknown. During differentiation to amastigotes the promastigote flagellum shortens dramatically and the fate of the excess flagellar membrane is unknown. Here we investigate the possibility that during Leishmania mexicana differentiation, shedding of the flagellar membrane is a source of extracellular vesicles (EVs) which provide a virulence factor delivery mechanism. The kinetics and structural mechanisms of EV release from promastigotes were investigated by live cell imaging and by measuring the concentration of shed EVs. Isolated EVs from a differentiating parasite culture or a control promastigote parasite culture were analysed by fluorescence and electron microscopy and mass spectrometry. To study the biological effects of EVs, macrophages were exposed to isolated EVs or live promastigotes and cytokine secretion was quantified by ELISA. An LPG1 null mutant was used to assess the contribution of virulence factor lipophosphoglycan (LPG) to the observed effects. Known protein virulence factors and LPG are present in L. mexicana EV fractions as well as known flagellar proteins. We show that there is a link between L. mexicana flagellar shortening and EV release, which is a recently discovered phenomenon in Chlamydomonas and mammalian cell research. We find that isolated EVs and live promastigotes induce changes in secreted cytokine levels from human and murine macrophages, including a substantial and previously unreported suppression of CXCL10, a chemokine which plays a protective role in Leishmania infection. LPG contributes to the effects observed on cytokine production, and EVs may be an important delivery mechanism for LPG.