L-myo-inositol-1-phosphate synthase expressed in developing organ: isolation and characterisation of the enzyme from human fetal liver.

L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0-60 % ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46 % of MIPS could be recovered. The human fetal liver MIPS specifically utilised D-glucose-6-phosphte and NAD(+) as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40 °C. The enzyme activity was remarkably stimulated by NH (4) (+) , slightly stimulated by K(+) and Ca(2+) and highly inhibited by Zn(2+), Cu(2+) and Hg(2+). The K (m) values of MIPS for D-glucose-6-phosphate and NAD(+) were found to be as 1.15 and 0.12 mM respectively while the V (max) values were 280 nM and 252 nM for D-glucose-6-phosphate and NAD(+) correspondingly. The apparent molecular weight of the native enzyme was determined to be 170 kDa.

Miltefosine triggers a strong proinflammatory cytokine response during visceral leishmaniasis: role of TLR4 and TLR9.

Visceral leishmaniasis (VL) caused by the protozoan parasite, Leishmania donovani, is associated with irregular fever, weight loss, hepatosplenomegaly and anemia. The therapeutic arsenal against VL is limited and the recent advent of a novel immunomodulatory drug, Miltefosine has shown promising results for effective treatment of VL but its dependence on Toll like receptors (TLR) has not been explored. In this study, we have shown that the non-cytotoxic dose (5 µM) of Miltefosine could render significant protection corresponding to 88% and 95% reduction in intracellular parasite load at 24 h and 48 h in L. donovani infected THP1 cells. This was accompanied by a strong proinflammatory cytokine response in the form of IFN-γ, IL-12 and TNF-α as evident by enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR). This Miltefosine induced proinflammatory cytokine response in infected THP1 cells was also accompanied by simultaneous 10- and 12-fold increase in TLR4 mRNA and TLR9 mRNA. These changes in cytokine response and TLR expression were also studied in peripheral blood mononuclear cells (PBMC) of VL patients treated with Miltefosine by RT-PCR which showed similar results as in THP1 cells. Thereby, suggesting a probable dependence of Miltefosine on TLR4 and TLR9 in triggering a proinflammatory response.

Treatment with IP-10 induces host-protective immune response by regulating the T regulatory cell functioning in Leishmania donovani-infected mice.

Visceral leishmaniasis (VL), caused by the protozoan parasite, Leishmania donovani, is characterized by an infection in the liver and spleen. The failure of the first-line drugs has led to the development of new strategies for combating VL. Recently, our group has shown that interferon-γ-inducible protein (IP)-10, a CXC chemokine, renders protection against VL. In the present study, we have elucidated the mechanism by which IP-10 renders protection in in vivo L. donovani infection. We observed that IP-10-treated parasitized BALB/c mice showed a strong host-protective T helper cell (Th) 1 immune response along with marked decrease in immunosuppressive cytokines, tumor growth factor (TGF)-ß, and interleukin (IL)-10 secreting CD4(+) T cells. This IP-10-mediated decrease in immunosuppressive cytokines was correlated with the reduction in the elevated frequency of CD4(+)CD25(+) T regulatory (Treg) cells along with the reduced TFG-ß production from these Treg cells in Leishmania-infected mice. This reduction in TGF-ß production was due to effective modulation of TGF-ß signaling by IP-10, which reduced the immunosuppressive activity of Treg cells. Thus, these findings put forward a detailed mechanistic insight into IP-10-mediated regulation of the Treg cell functioning during experimental VL, which might be helpful in combating Leishmania-induced pathogenesis.

Amphotericin B regulates the host immune response in visceral leishmaniasis: reciprocal regulation of protein kinase C isoforms.

OBJECTIVES: Treatment of visceral leishmaniasis (VL) is marked by the failure of pentavalent antimonials which has brought amphotericin B (AmpB) to the forefront. In this study we have focused on signaling pathway regulating AmpB triggered effector response. METHODS: AmpB triggered effector response in the form of free radicals and proinflammatory cytokines was determined by FACS, colorimetric estimation or Real-Time PCR (RT-PCR). Specific peptide inhibitors for classical and atypical protein kinase C (PKC) were used to investigate the role of PKC isoforms in the functioning of AmpB during VL. RESULTS: In vitro studies with THP1 cells showed that 2 microg/ml dose of AmpB could mediate effective parasite clearance due to strong induction of free radicals and proinflammatory cytokines. This induction of proinflammatory response paralleled with antagonistic regulation of classical and atypical PKC. Further confirmation was provided by RT-PCR of (peripheral blood mononuclear cells) PBMC isolated from VL infected patients undergoing AmpB treatment. CONCLUSIONS: Overall, our results suggest that classical and atypical PKC signaling pathways are involved in the modulation of proinflammatory response triggered by AmpB against Leishmania donovani. These observations may contribute to the understanding of the mechanism responsible for the initiation of protective response induced by AmpB during VL.