Project description:Monastrol treatment of Leishmania donovani infected macrophages Macrophages were infected with Leishmania donovani and treated with monastrol to look for signalling molecules
Project description:Murine bone marrow derived macrophages were infected with Leishmania major or Leishmania donovania promastigotes, allowed to phagocytose latex beads or not treated. Gene expression profiles were compared to identify i) the effect of Leishmania infection; ii) the differences in effects between L. major and L. donovani; and iii) the effect of pahgocytosis of latex beads.
Project description:Infection with antimony resistant (SbR) but not with sentitive (SbS) Leishmania donovani (LD) gives rise to aggressive pathology in mammalian hosts, the cause of which is far from clear. Some intracellular pathogens exploit autophagy for their own benefit. Here we show that induction of autophagy in normal macrophages (MF) by pharmacological mediators prior to infection with SbRLD (SbRLD-MF) enhanced their growth as compared to untreated MF, unlike SbSLD-MF. Autophagy was evident in SbRLD-MF from electron microscopical studies showing double membrane-bound compartment around amastigote. In SbRLD-MF there is induction of beclin 1, which forms the platform to recruit other interacting molecules to initiate autophagy. Knocking down the beclin 1 transcription factor Nrf2 and subsequent infection with SbRLD showed significantly lower organ parasites as compared to wild type BALB/c mice. Cessation of autophagy in SbRLD-MF at the later stage of infection is coupled with induction of miR-30a, whose binding to 3'UTR of beclin 1 leads to its post-transcriptional attenuation followed by rise in intracellular Ca++ and apoptosis. SbRLD mediated translocation of AP-1 transcription factor to the nucleus induce pri-miR-30a over-expression. Rise in Ca++ causes caspase 8 activtion leading to the cleavage of beclin 1 and initiation of apoptosis in SbRLD-MF. Apoptosis may favor parasite egress for cell to cell transmission. We also found that beclin 1 expression is present in splenocytes of kala-azar patients harbouring SbRLD but not SbSLD. Our results suggest that SbRLD has evolved a unique mechanism for its own benefit which explains, in part, the cause of aggressive pathology. Peritoneal exudate macrophages were isolated from mouse, grown in 60mm plates and infected with Leishmania donovani and total RNA was isolated from cells at 12, 18 and 24 hrs post infection. Leishmania infected macrophage miRNA expression signature was generated. Cells grown on 60mm plates and infected with Leishmania. The main objective of the microarray analysis of mmu-miRNA in antimony resistant and antimony sensitive Leishmania donovani infected macrophages are as follows: 1. To study how the expression of miRNA varies in either antimony resistant or antimony sensitive Leishmania infected macrophages as compared to the normal macrophages as a function of time. LPS was used as control. 2. To study the expression of those miRNAs which are differentially expressed in antimony resistant and antimony sensitive Leishmania infected macrophages at each time point post infection. 3. To identify those miRNAs which are responsible for degradation of autophagy initiating protein beclin 1 mRNA
Project description:Mouse models were infected with Leishmania donovani and the effects were observed over multiple passages to observe the macrophage-parasite interaction
Project description:Monocyte derived dendritic cells (MDDC) were infected with Leishmania major or Leishmania donovani parasites and collected at 4, 8, and 24 hours post-infection to analyze the differential effects those parasite species have on human host cell gene expression over time. Monocyte derived dendritic cells (MDDC) were generated from blood buffy coats collected from five anonymous healthy human donors and infected 10:1 (parasite to host cell) with Leishmania major Friedlin V1 strain or Leishmania donovani 1S strain parasites, where after 4, 8, or 24 hours total RNA was harvested from cells, cDNA generated, and hybridized to human gene transcipt expression arrays to assess differential host cell gene transcriptional expression differences relative to uninfected cells.
