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Antimicrobial Agents and Chemotherapy, April 2006, p. 1195-1201, Vol. 50, No. 4
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.4.1195-1201.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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
S. Rama Iñiguez,1,
M. A. Dea-Ayuela,1,
J. A. Sanchez-Brunete,2
J. J. Torrado,2
J. M. Alunda,3 and
F. Bolas-Fernández1*
Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Madrid, Spain,1 Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain,2 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain3
Received 1 January 2006/ Returned for modification 10 January 2006/ Accepted 28 January 2006
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 107 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-
) and, to a lesser extent, tumor necrosis factor alpha (TNF-
) and transforming growth factor beta (TGF-ß) 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- and TNF-, with no effect on the deactivating cytokine TGF-ß. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN- and TNF- and strong suppression of TGF-ß. 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-ß, which in turn results in up-regulation of the Th1 cytokines IFN- and TNF-.
* Corresponding author. Mailing address: Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n, Ciudad Universitaria, 28040 Madrid, Spain. Phone: 34 91 394 18 18. Fax: 34 91 394 18 15. E-mail: francisb{at}farm.ucm.es.
S.R.I. and M.A.D.-A. participated equally in this paper.
Antimicrobial Agents and Chemotherapy, April 2006, p. 1195-1201, Vol. 50, No. 4
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.4.1195-1201.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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