Treatment of Leishmania donovani-infected hamsters with miltefosine: analysis of cytokine mRNA expression by real-time PCR, lymphoproliferation, nitrite production and antibody responses.

OBJECTIVES: Miltefosine, an orally effective antileishmanial drug, works directly on the parasite by impairing membrane synthesis and subsequent apoptosis of the parasite and has also been reported to have macrophage-activating functions that aid parasite killing. We investigated the type of immunological responses generated in miltefosine-treated Leishmania donovani-infected hamsters, which simulate the clinical situation of human kala-azar. METHODS: Twenty-five-day-old infected hamsters, treated with miltefosine at 40 mg/kg for 5 consecutive days, were euthanized on days 30 and 45 post treatment (p.t.) and checked for parasite clearance and for real-time analysis of mRNAs of the Th1/Th2 cytokines interferon-γ (IFN-γ), interleukin-12 (IL-12), tumour necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-4, IL-10 and transforming growth factor-ß (TGF-ß), nitric oxide (NO) production, the lymphocyte transformation test (LTT) and antibody responses. Responses were compared with the normal and Leishmania-infected groups at the same time points. RESULTS: By day 45 p.t. there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α, whereas there were significant decreases in IL-4, IL-10 and TGF-ß in cured hamsters as compared with their infected counterparts. In vitro stimulation of lymphocytes with concanavalin A and soluble Leishmania donovani antigen showed a maximum LTT response and there was a gradual increase in the NO level (∼7-fold compared with infected counterparts). Anti-Leishmania IgG and IgG1 levels, found to be elevated in the infected group, decreased significantly after treatment but there was a significant increase in IgG2 isotype. CONCLUSIONS: Treatment of Leishmania-infected hamsters with miltefosine reverses the Th2-type response into a strong Th1-type immune response.