title: Effectiveness of miltefosine treatment in targeting anti-leishmanial HO-1/Nrf-2-mediated oxidative responses in visceral leishmaniasis patients.
abstract: OBJECTIVE
  Miltefosine, an alkylphosphocholine anti-cancer drug, exhibits direct activity against Leishmania donovani and also promotes anti-leishmanial host immunomodulatory functions. Nuclear factor-erythroid 2-related factor 2 (Nrf-2), a redox-sensitive transcription factor, regulates the host stress responses leading to effective microbial clearance by a positive effect on haem oxygenase-1 (HO-1) enzyme expression/activity. We aimed to investigate the role of miltefosine in regulating HO-1/Nrf-2-mediated oxidative responses in visceral leishmaniasis (VL) patients in vivo and in vitro.
  
  
  METHODS
  Splenic aspirate and bone marrow aspirate cells of VL patients (n = 23) were used in the study. RT-PCR of HO-1, Nrf-2 translocation analysis and HO-1 ELISA were used to investigate the HO-1/Nrf-2-mediated modulation of oxidative responses by miltefosine in vivo. Fluorometric measurement of reactive oxygen species (ROS) was performed, determination of glutathione peroxidase (GPx) activity was performed, and bilirubin and superoxide dismutase (SOD) levels were determined. The in vitro HO-1/Nrf-2-dependent anti-leishmanial effect of miltefosine was assessed by the use of specific inhibitors/inducers and subsequent microscopic measurement of parasite killing and Th1/Th2 cytokine regulation by ELISA.
  
  
  RESULTS
  Increased levels of transcript and serum HO-1, Nrf-2 nuclear translocation, serum bilirubin, GPx and SOD activity in untreated VL patients were reversed after miltefosine chemotherapy. The effectiveness of miltefosine for positive induction of ROS via NADPH correlated with a decrease in HO-1/ERK/Nrf-2-dependent parasite load. Furthermore, HO-1 blockade by miltefosine led to suppression of interleukin-10 and transforming growth factor-β, but enhanced interleukin-12 and tumour necrosis factor-α production, in VL patients.
  
  
  CONCLUSIONS
  The antioxidant promoting property of L. donovani is crucial for protection against the mounting redox threat in the host. Therefore, these findings provide direct evidence for targeting HO-1/Nrf-2 as an anti-leishmanial approach for chemotherapy in human VL.
authors.affiliation: Department of Molecular Parasitology, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research (ICMR), Agamkuan, Patna, India.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Das
authors.forename: Krishna
authors.initials: K
authors.lastname: Pandey
authors.forename: Vidya N
authors.initials: VN
authors.lastname: Rabidas
authors.forename: Abhishek
authors.initials: A
authors.lastname: Mandal
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
mesh_terms: D000293:Adolescent; D000328:Adult; D002478:Cells, Cultured; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005470:Fluorometry; D020869:Gene Expression Profiling; D005979:Glutathione Peroxidase / Q000378:metabolism; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007898:Leishmaniasis, Visceral / Q000188:drug therapy*; D008297:Male; D010767:Phosphorylcholine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D017382:Reactive Oxygen Species / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D013482:Superoxide Dismutase / Q000378:metabolism; D055815:Young Adult
chemical_list: C111027:NKRF protein, human; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D010767:Phosphorylcholine; C039128:miltefosine; D005979:Glutathione Peroxidase; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D013482:Superoxide Dismutase
keywords: NADPH; SOD; bilirubin; glutathione peroxidase; haem oxygenase; stress response