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.
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.
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n, Ciudad Universitaria, 28040 Madrid, Spain.
S
S
Rama Iñiguez
M A
MA
Dea-Ayuela
J A
JA
Sanchez-Brunete
J J
JJ
Torrado
J M
JM
Alunda
F
F
Bolas-Fernández
D000666:Amphotericin B / Q000008:administration & dosage*; D000818:Animals; D001483:Base Sequence; D002626:Chemistry, Pharmaceutical; D006224:Cricetinae; D016207:Cytokines / Q000235:genetics*; D007371:Interferon-gamma / Q000235:genetics; D018314:Leishmania infantum; D007898:Leishmaniasis, Visceral / Q000188:drug therapy* / Q000276:immunology; D008297:Male; D008647:Mesocricetus; D008863:Microspheres; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012709:Serum Albumin / Q000008:administration & dosage*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016207:Cytokines; D012333:RNA, Messenger; D012709:Serum Albumin; D014409:Tumor Necrosis Factor-alpha; D000666:Amphotericin B; D007371:Interferon-gamma