Real-time reverse transcription-PCR quantification of cytokine mRNA expression in golden Syrian hamster infected with Leishmania infantum and treated with a new amphotericin B formulation

Abstract

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamster Mesocricetus auratus infected with Leishmania infantum and treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 10(7) metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10 per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-gamma) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-gamma and TNF-alpha, with no effect on the deactivating cytokine TGF-beta. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-gamma and TNF-alpha and strong suppression of TGF-beta. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment of L. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-beta, which in turn results in up-regulation of the Th1 cytokines IFN-gamma and TNF-alpha.

FIG. 1.

Amplification products of cDNA (1) and DNA (2) control samples were separated in agarose gels and stained with ethidium bromide. Single amplification products corresponding to the HPRT control gene and the TGF-β, TNF-α, IFN-γ, and IL-4 genes were visualized at 91, 57, 81, 68, and 72 bp, respectively.

FIG. 2.

Relative mRNA expression levels of IL-4 (A), IFN-γ (B), TNF-α (C), and TGF-β (D) in spleen lymphocytes from Syrian hamsters infected by L. infantum (group I) or infected and treated with AMB-HSA at 2 mg/kg (group II) or 40 mg/kg (group III). Treatment was given on days 69, 71, and 73 after infection, and spleen cells were collected following sacrifice on day 3 after treatment. Results are expressed as differences between ΔC_T values of test and comparator samples. The horizontal dotted lines represent the ΔC_T of the comparator samples, to which the value of 1 was arbitrarily assigned. Truncated vertical lines represent the standard deviation ranges for mean values. a, mean values for groups I, II, and III are not significantly different from each other (P > 0.05); b, mean values for group II are significantly different from those for groups I and III (P < 0.05); c, mean values for group III are significantly different from those for groups I and II (P < 0.05).

FIG. 3.

Relative mRNA expression levels of IL-4 (A), IFN-γ (B), TNF-α (C), and TGF-β (D) in spleen lymphocytes from noninfected Syrian hamsters treated with AMB-HSA at 2 mg/kg (group IV) or 40 mg/kg (group V). Treatment was given on days corresponding to days 69, 71, and 73 after infection (for infected groups), and spleen cells were collected following sacrifice on day 3 after treatment. Results are expressed as differences between ΔC_T values of test and comparator samples. The horizontal dotted lines represent the ΔC_T of the comparator samples, to which the value of 1 was arbitrarily assigned. Truncated vertical lines represent the standard deviation ranges for median values. Treatment did not affect the mRNA cytokine expression patterns of both groups compared to healthy untreated animals (test/comparator ratio increase/decrease of <3).