Full text
| |
| Author(s): |
Maira Cegatti Bosetto
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2007-04-05 |
| Examining board members: |
Selma Giorgio;
Wirla Maria da Silva Cunha Tamashiro;
Marcelo Brocchi;
Hiro Goto;
Lourdes Isaac
|
| Advisor: | Selma Giorgio |
| Abstract | |
Dendritic cells (DCs) are antigen-presenting cells involved in the initiation of primary immune response. Human DC were obtained from peripheral blood monocytes after culturing with GM-CSF and IL-4. Leishmania parasites are intracellular protozoan that replicate in Mononuclear Phagocytic System cells, mainly macrophages and DCs. Leishmania amazonensis, a species used in our studies is associated with cutaneous infection that can become chronical lesions with a inflammatory process and small regions of hypoxia. In this work we evaluated the role of hypoxia in a in vitro model of intracellular infection, leishmaniasis. Human DCs infection with infected by L. amazonensis amastigotes were performed under normoxic (21% O2) and hypoxic (6% O2) conditions. The phenotypic analysis of DC cultured under hypoxic condition showed a reduced expression of CD80, CD86 and CD1a compared with normoxic control. In these cell cultures, viability was not affected. Hypoxia did not affect the amastigote entrance into DCs although it modulated their functional activity reducing 30% the percentage of infected DCs. Furthermore, hypoxia did not act synergistically with IFN-g plus LPS in DCs to induce killing of parasites. DCs activated and infected with amastigotes under hypoxic condition produced low levels of IL-12 compared with normoxic control. Moreover, in the abscence of stimulation, infected DCs did not produce IL-12. We also evaluated the leishmanicidal activity of Amphotericin-B, Glucantime and Miltefosine under normoxic and hypoxic conditions during 48 h. Under hypoxic condition, Amphotericin-B and Glucantime were less efective in L. amazonensis infected DCs. None effect of hypoxia was observed in Miltefosine was observed against L. amazonensis infected DC. Finally, we investigated in vitro mechanisms of entry into human DCs of L. amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein (AU) | |