title: Appraisal of a Leishmania major strain stably expressing mCherry fluorescent protein for both in vitro and in vivo studies of potential drugs and vaccine against cutaneous leishmaniasis.
abstract: BACKGROUND
  Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease.
  
  
  METHODS
  We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice.
  
  
  RESULTS
  In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC(50) values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo.
  
  
  CONCLUSIONS
  A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.
authors.affiliation: Departamento de Ciencias Biomédicas, Universidad de León, León, Spain.
authors.forename: Estefania
authors.initials: E
authors.lastname: Calvo-Álvarez
authors.forename: Nestor Adrian
authors.initials: NA
authors.lastname: Guerrero
authors.forename: Raquel
authors.initials: R
authors.lastname: Alvarez-Velilla
authors.forename: Christopher Fernández
authors.initials: CF
authors.lastname: Prada
authors.forename: Jose María
authors.initials: JM
authors.lastname: Requena
authors.forename: Carmen
authors.initials: C
authors.lastname: Punzón
authors.forename: Miguel Ángel
authors.initials: MÁ
authors.lastname: Llamas
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Arévalo
authors.forename: Luis
authors.initials: L
authors.lastname: Rivas
authors.forename: Manuel
authors.initials: M
authors.lastname: Fresno
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Pérez-Pertejo
authors.forename: Rafael
authors.initials: R
authors.lastname: Balaña-Fouce
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Reguera
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000008:administration & dosage / Q000494:pharmacology*; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D018320:Leishmania major / Q000187:drug effects* / Q000235:genetics / Q000276:immunology*; D054332:Leishmaniasis Vaccines / Q000008:administration & dosage / Q000276:immunology*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology*; D035002:Lower Extremity / Q000469:parasitology; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D059208:Parasite Load / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D000981:Antiprotozoal Agents; D054332:Leishmaniasis Vaccines; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein