title: Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis: involvement of TLR4 signalling.
abstract: OBJECTIVE
  The aim of this study was to characterize the antileishmanial activity of heat-killed Mycobacterium indicus pranii (Mw) alone or in combination with a subtoxic dose of amphotericin B [AMB(st)].
  
  
  METHODS
  Mw- and Mw + AMB(st)-mediated antileishmanial activity was evaluated by microscopic counting of intracellular amastigotes in Giemsa-stained macrophages and real-time PCR analysis of inducible nitric oxide synthase (iNOS) expression and measurement of nitric oxide generation by Griess reagent. The relationship between Mw and Toll-like receptor 4 (TLR4) signalling was studied by fluorescence-activated cell sorting, western blot and confocal microscopy. The effect of Mw alone or in combination with AMB(st) on the expression and production of interleukin (IL)-12, tumour necrosis factor-α, IL-10 and transforming growth factor-β was analysed by real-time PCR and ELISA, respectively.
  
  
  RESULTS
  Mw treatment alone or with AMB(st) caused a significant increase in TLR4 expression of L. donovani-infected macrophages along with the activation of TLR4 downstream signalling, facilitating active nuclear translocation of nuclear factor κB (NF-κB). These events culminated in the up-regulation of the proinflammatory response, which was abrogated by treatment with TLR4-specific small-interfering RNA. In addition, this study demonstrates that this chemoimmunotherapeutic strategy confers protection against leishmanial pathogenesis via TLR4-dependent counter-regulation of inducible nitric oxide synthase (iNOS) and arginase1 activity.
  
  
  CONCLUSIONS
  These results provide a mechanistic understanding of Mw- or Mw + AMB(st)-mediated protection against leishmanial parasites within host macrophages.
authors.affiliation: Division of Molecular Medicine,  Bose Institute, Kolkata 700054,  India.
authors.forename: Anupam
authors.initials: A
authors.lastname: Adhikari
authors.forename: Saikat
authors.initials: S
authors.lastname: Majumder
authors.forename: Sayantan
authors.initials: S
authors.lastname: Banerjee
authors.forename: Gaurav
authors.initials: G
authors.lastname: Gupta
authors.forename: Parna
authors.initials: P
authors.lastname: Bhattacharya
authors.forename: Suchandra Bhattacharyya
authors.initials: SB
authors.lastname: Majumdar
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Saha
authors.forename: Subrata
authors.initials: S
authors.lastname: Majumdar
mesh_terms: D000666:Amphotericin B / Q000008:administration & dosage; D000818:Animals; D000981:Antiprotozoal Agents / Q000008:administration & dosage; D015153:Blotting, Western; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D007898:Leishmaniasis, Visceral / Q000276:immunology* / Q000517:prevention & control*; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D008853:Microscopy; D018613:Microscopy, Confocal; D009161:Mycobacterium / Q000276:immunology*; D009569:Nitric Oxide / Q000032:analysis; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D059208:Parasite Load; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000276:immunology*
chemical_list: D000981:Antiprotozoal Agents; D016207:Cytokines; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D000666:Amphotericin B; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse