Reproduction de : Thèse de doctorat : Chimie, biologie, santé : Toulouse, INPT : 2007. / Titre provenant de l'écran-titre. Bibliogr. 163 réf.

Abstract Visceral leishmaniasis is a devastating neglected parasitic disease caused by infection with Leishmania donovani. It life cycle has two stages with promastigote (insect stage) and amastigote (animal stage) morphologies. Miltefosine is currently the only commercially available oral drug available to treat leishmaniasis and recent evidence suggests clinical resistance has emerged. Due to the importance of this drug and the scarcity of new drugs in the pipeline, work has been done on understanding the mechanism(s) of miltefosine, yet the mechanism of action for resistance is still not known. In previous studies investigators generated miltefosine resistance on the insect vector stage (promastigotes), yet there is an important gap in understanding miltefosine resistance in the human infectious stage (amastigotes) of the disease. Before we could accomplish this goal a L. donovani cell line was converted into a stable, continuously cultured axenic amastigote cell line and characterized by disease burden generated in vitro and in vivo. The axenic line of L. donovani (MHOM/SD/75/1246/130) retains characteristics of amastigotes infecting macrophages and proliferated better than metacyclic promastigotes in macrophages in vitro and hamsters. The axenic amastigote line was used to induce miltefosine resistance by using discontinuous stepwise increasing drug pressure. Stable high-grade resistance was established (62-fold) and characterized in vitro and in vivo. Lastly, the fitness of miltefosine resistant versus susceptible parasite was established for the first time. This work adds the first efforts characterizing and understanding the complex problem of miltefosine resistance in amastigotes.

The ability of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) to stimulate gene expression in bone marrow-derived macrophages (BMM) was first compared. It is demonstrated that they stimulated gene expression through distinct signal transduction pathways and that TNF stimulated gene expression through a protein kinase C (PKC)-dependent signal transduction pathway. The effect of the intracellular parasite of macrophages Leishmania donovani in BMM was then investigated. It is demonstrated that L. donovani impaired c-fos and TNF gene expression through two distinct mechanisms. The first one is indomethacin-reversible, and the second one involves the inhibition of diacylglycerol-induced PKC-dependent gene expression. A purified cell surface glycoconjugate of the parasite, termed lipophosphoglycan, selectively inhibited PKC-dependent gene expression in BMM. While the translocation of PKC from the cytosol to the membrane was normal, total cellular PKC enzyme activity was inhibited in the U937 human monocyte cell line pretreated with lipophosphoglycan.

The role of a parasite-produced or -induced soluble immunosuppressor in experimental kala azar was examined. It was found that in vivo infections with Leishmania donovani in the hamster (Mesocricetus auratus) produce a soluble immunosuppressor, which appears in the serum of the host and which reduces the proliferation of responding populations of murine splenocytes in a one-way mixed leukocyte reaction (MLR). The production in vitro infections of murine splenic macrophages from C57BL/6J ($Lsh sp{ rm s}$), C57L/J ($Lsh sp{ rm R}$) and BALB/c strains, the suppressive activity was not contained in either parasite-conditioned culture medium or in parasite extracts or from macrophages which have internalized killed parasites or inert particles and it is not blocked by the action of 2-mercaptoethanol or indomethacin in the culture medium. The suppressor was found to be able to selectively inhibit or reduce the proliferation of splenocytes of both the C57BL/6J and C57LN strains in a one way MLR, with the level of suppression being significantly greater upon splenocytes of the susceptible $Lsh sp{ rm s}$ strain. The suppression was dependent upon the genotype of the macrophages present in the responding population. The suppressor was also able to significantly inhibit the processing of human serum albumin by macrophages, to reduce the number of Ia ligands on the surfaces of macrophages and the production by these cells of IL-1 upon silica stimulation. / Significant reduction was also seen in the production of IL2 and in the expression of its receptor by PHA-stimulated T cells exposed to the suppressor. Partial purification and identification of the suppressor demonstrated that the suppressive activity was present in fractions between 30 and 50 kDa in size; the suppressor was also heat labile and freeze-thaw sensitive. The suppressive molecule(s) may therefore play a significant role in the establishment and pathology of L. donovani infections.

If used early in infection, prophylactic treatment with the immunomodulatory drug cyclosporin A of Leishmania ma'or infected Balb/c mice has been shown to enhance resistance of these mice to serious disease. It is thought that CsA treatment affects disease progression by altering the balance of specific T lymphocyte populations as well as the secretion of various cytokines. We have followed the levels of L3T4+ T cells, Ly-2+ T cells, and total T and B lymphocytes, as well as IL-4 in susceptible Balb/c mice, CsA-treated Balb/c mice, and naturally resistant C57B1/6 mice during the course of L. ma'or infection. The CsA-treated mice displayed a disease pattern similar to that of the C57B1/6 group throughout infection. Most importantly, CsA treatment appeared to inhibit IL-4 production early post infection in both spleen and lymph node, and also appeared to inhibit the dramatic early increase of L3T4+ (CD4+) T cells which is characteristic of the susceptible Balb/c mice. / Department of Biology

Leishmania protozoans are the etiologic agents of the disease leishmaniasis. The parasite exists in two morphological forms: promastigote and amastigote. Promastigotes are found in the gut of the sandfly vector while amastigotes reside inside the vertebrate macrophage. Leishmania, an obligate intracellular parasite, resists toxic reactive oxygen species (ROS) from both endogenous and exogenous sources. Like other protozoa, Leishmania lacks some of the antioxidant defence enzymes such as catalase and glutathione peroxidase (Gpx) that are usually found in aerobic cells. Instead they possess the antioxidant thiol compound trypanothione, in association with specific trypanothione linked antioxidant enzymes such as peroxidoxins. The transformation from promastigote to amastigote is a crucial step for parasite infection and survival. The molecular basis for this transformation is not clearly understood. Recently it was shown that the peroxidoxin gene is present in multiple copies in Leishmania. In the present study we examined the potential of antisense RNA and double stranded RNA (dsRNA) to perform functional knockout of the peroxidoxin gene. Towards that end antisense RNA and dsRNA expressing plasmids, targeting the peroxidoxin gene, were constructed. Leishmania promastigotes were subsequently transfected with these plasmids and the levels of peroxidoxin gene expression were studied. The results from this study suggest that there is no apparent reduction in either the levels of peroxidoxin mRNA or the protein in the transfected promastigotes as compared to the non-transfected cells.

Thèse (Ph. D.)--Université Laval, 2004. / Bibliogr.: f. 125-142. Publié aussi en version électronique.

Thèse (Ph.D.)--Université Laval, 2004. / Titre de l'écran-titre (visionné le 29 novembre 2004). Bibliogr.

Thèse (Ph. D.) -- Université Laval, 1998. / Bibliogr.: f. 80-101. Publié aussi en version électronique.

Thèse (M.Sc.) -- Université Laval, 1997. / Bibliogr.: f. 67-83. Publ. aussi en version électronique.