03100nas a2200469   4500000000100000008004100001653001900042653001200061653002500073653002200098653001400120653003800134653001900172653003000191653001100221653002800232653001600260653000900276653002400285653001500309653002500324653001800349653001700367653003400384653001800418653004000436653002400476653002500500100001500525700001500540700001500555700001200570700001900582700001600601700001100617700001500628245012200643300001300765490000700778520183100785022001402616       2012                            d10aAmphotericin B10aAnimals10aAntiprotozoal Agents10aBlotting, Western10aCytokines10aEnzyme-Linked Immunosorbent Assay10aFlow Cytometry10aGene Expression Profiling10aHumans10aLeishmaniasis, Visceral10aMacrophages10aMice10aMice, Inbred BALB C10aMicroscopy10aMicroscopy, Confocal10aMycobacterium10aNitric Oxide10aNitric Oxide Synthase Type II10aParasite Load10aReal-Time Polymerase Chain Reaction10aSignal Transduction10aToll-Like Receptor 41 aAdhikari A1 aMajumder S1 aBanerjee S1 aGupta G1 aBhattacharya P1 aMajumdar SB1 aSaha B1 aMajumdar S00aMycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis: involvement of TLR4 signalling.  a2892-9020 v673 a<p><strong>OBJECTIVES: </strong>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)].</p>

<p><strong>METHODS: </strong>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.</p>

<p><strong>RESULTS: </strong>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.</p>

<p><strong>CONCLUSIONS: </strong>These results provide a mechanistic understanding of Mw- or Mw + AMB(st)-mediated protection against leishmanial parasites within host macrophages.</p>
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