title: Infrared fluorescent imaging as a potent tool for in vitro, ex vivo and in vivo models of visceral leishmaniasis.
abstract: BACKGROUND
  Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.
  
  
  RESULTS
  We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.
  
  
  CONCLUSIONS
  The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Estefanía
authors.initials: E
authors.lastname: Calvo-Álvarez
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Kostantinos
authors.initials: K
authors.lastname: Stamatakis
authors.affiliation: Diomune, Parque Cientifico de Madrid, Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Punzón
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Álvarez-Velilla
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Tejería
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Escudero-Martínez
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Pérez-Pertejo
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Fresno
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Balaña-Fouce
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, Campus de Vegazana, León, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Reguera
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D055808:Drug Discovery / Q000379:methods*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007259:Infrared Rays; D018314:Leishmania infantum / Q000235:genetics*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D061848:Optical Imaging / Q000379:methods*
references.citation: Immunol Today. 2000 Feb;21(2):73-8
references.pmid: 10652464
references.citation: Mol Biochem Parasitol. 2000 Apr 15;107(2):251-61
references.pmid: 10779601
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Mol Biochem Parasitol. 2000 Oct;110(2):195-206
references.pmid: 11071276
references.citation: Antimicrob Agents Chemother. 2002 Mar;46(3):828-33
references.pmid: 11850268
references.citation: J Biol Chem. 2002 May 31;277(22):19511-20
references.pmid: 11912202
references.citation: Parasitol Today. 1986 Mar;2(3):64-9
references.pmid: 15462773
references.citation: Infect Immun. 2005 Feb;73(2):695-702
references.pmid: 15664907
references.citation: Cell Microbiol. 2005 Mar;7(3):383-92
references.pmid: 15679841
references.citation: J Biol Chem. 2005 Oct 21;280(42):35172-83
references.pmid: 16105831
references.citation: Nat Biotechnol. 2006 Feb;24(2):167-75
references.pmid: 16465162
references.citation: Nat Protoc. 2006;1(1):476-85
references.pmid: 17406270
references.citation: Parasitol Res. 2007 Oct;101(5):1449-52
references.pmid: 17676404
references.citation: Nat Methods. 2007 Sep;4(9):741-6
references.pmid: 17721542
references.citation: N Engl J Med. 2007 Sep 6;357(10):1018-27
references.pmid: 17804846
references.citation: Clin Vaccine Immunol. 2008 Dec;15(12):1764-70
references.pmid: 18945882
references.citation: Science. 2009 May 8;324(5928):804-7
references.pmid: 19423828
references.citation: J Antimicrob Chemother. 2009 Aug;64(2):370-4
references.pmid: 19525291
references.citation: PLoS Negl Trop Dis. 2009 Jul 21;3(7):e486
references.pmid: 19621071
references.citation: Trends Parasitol. 2009 Sep;25(9):432-9
references.pmid: 19720564
references.citation: J Antimicrob Chemother. 2010 Mar;65(3):508-11
references.pmid: 20089542
references.citation: Exp Parasitol. 2010 Nov;126(3):310-7
references.pmid: 20501336
references.citation: Exp Parasitol. 2011 Mar;127(3):637-45
references.pmid: 21187086
references.citation: PLoS Negl Trop Dis. 2011 Feb 15;5(2):e962
references.pmid: 21358812
references.citation: Cell Microbiol. 2011 Jul;13(7):978-91
references.pmid: 21501362
references.citation: Nat Biotechnol. 2011 Jul 17;29(8):757-61
references.pmid: 21765402
references.citation: J Trop Med. 2012;2012:892817
references.pmid: 21912561
references.citation: Acta Trop. 2012 Apr;122(1):36-45
references.pmid: 22155571
references.citation: PLoS Negl Trop Dis. 2012;6(6):e1671
references.pmid: 22720099
references.citation: PLoS Negl Trop Dis. 2012;6(11):e1927
references.pmid: 23209866
references.citation: J Vis Exp. 2012 Dec 30;(70):null
references.pmid: 23299097
references.citation: Diagn Microbiol Infect Dis. 2013 Mar;75(3):282-91
references.pmid: 23312610
references.citation: Antimicrob Agents Chemother. 2013 Jul;57(7):2913-22
references.pmid: 23571538
references.citation: Antimicrob Agents Chemother. 2013 Oct;57(10):4608-14
references.pmid: 23796924
references.citation: Nature. 2014 Jan 9;505(7482):142
references.pmid: 24402261
references.citation: Int J Parasitol Drugs Drug Resist. 2012 Jan 28;2:11-9
references.pmid: 24533267
references.citation: Trop Parasitol. 2014 Jan;4(1):4-9
references.pmid: 24754020
references.citation: J Trop Med. 2014;2014:646932
references.pmid: 24876851
references.citation: Drug Discov Today. 2015 Jan;20(1):114-21
references.pmid: 25256779
references.citation: Int J Parasitol Drugs Drug Resist. 2014 May 22;4(3):355-7
references.pmid: 25516847
references.citation: Parasite Immunol. 1985 Sep;7(5):545-55
references.pmid: 3877902
references.citation: Clin Exp Immunol. 1977 Oct;30(1):119-29
references.pmid: 606433
references.citation: Science. 1984 Mar 30;223(4643):1417-9
references.pmid: 6701528
references.citation: J Biol Chem. 1996 Jul 19;271(29):17081-90
references.pmid: 8663340
references.citation: Infect Immun. 1998 Jan;66(1):18-27
references.pmid: 9423834