Saikat Majumder

Mannosylated solid lipid nanoparticles as vectors for site-specific delivery of an anti-cancer drug

The purpose of the present study was to investigate the tumor targeting potential of surface tailored solid lipid nanoparticles (SLNs) loaded with an anti-cancer drug doxorubicin HCl (DOX). DOX encapsulating SLNs were prepared, characterized and further mannosylated. The developed formulations were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), particle size/polydispersity index and zeta-potential analysis. The formulations were evaluated for in vitro drug release and hemolytic toxicity. The ex vivo cytotoxicity and cellular uptake studies were performed on A549 cell lines. In vivo studies were conducted to determine pharmacokinetics, tissue distribution pattern and nephrotoxic/hepatotoxic effect of mannosylated SLNs. In vitro, the formulations exhibited a biphasic pattern characterized by initial rapid release of the drug followed by rather slow and prolonged release. Further, the in vitro studies depicted mannose-conjugated SLNs to be least hemolytic and suitable for sustained drug delivery. Mannosylated SLNs were most cytotoxic and were preferably taken up A549 tumor cells as evaluated against uncoated SLNs and plain DOX. Pharmacokinetic studies revealed improved bioavailability, half life and mean residence time of DOX upon mannose conjugation. The biodistribution pattern exhibited that mannosylated SLNs were able to deliver a higher concentration of DOX in the tumor mass. They were also proficient to circumvent damage to renal as well as hepatic tissues. It may therefore be interpreted that mannosylated SLNs are capable to ferry bioactives selectively and specifically to the tumor sites with the interception of minimal side effects, thereby suggesting their potential application in cancer chemotherapy.

Glycyrrhizic acid suppresses Cox-2-mediated anti-inflammatory responses during Leishmania donovani infection

OBJECTIVES The aim of the present study was to characterize glycyrrhizic acid (GA) and assess its immunomodulatory potential in a model of experimental visceral leishmaniasis. METHODS The antileishmanial activity of GA was tested in an amastigote-macrophage model and its non-cytotoxic dose was measured by a cell viability assay. To understand the effector mechanism of GA-treated macrophages against leishmanial parasites, real-time PCR analysis of inducible nitric oxide synthase 2 (iNOS2) was carried out followed by measurement of nitric oxide generation by Griess reagent. The effect of GA on the production of cytokines, such as interleukin (IL)-12, tumour necrosis factor (TNF)-α, IL-10 and transforming growth factor (TGF)-β, was measured by ELISA (protein) and real-time PCR. The expression of iNOS2 and cyclooxygenase-2 (Cox-2) was studied by western blotting. The parasite burden of the liver and spleen following GA treatment was determined by the stamp-smear method, and T cell proliferation was assessed via [³H]thymidine uptake, measured by a liquid scintillation counter. RESULTS Results showed that GA treatment caused an enhanced expression of iNOS2 along with inhibition of Cox-2 in Leishmania donovani-infected macrophages. GA treatment in infected macrophages enhanced the expression of IL-12 and TNF-α, concomitant with a down-regulation of IL-10 and TGF-β. GA increased macrophage effector responses via inhibition of Cox-2-mediated prostaglandin E2 release in L. donovani-infected macrophages. GA also decreased hepatic and splenic parasite burden and increased T cell proliferation in Leishmania-infected BALB/c mice. CONCLUSIONS These results provide a mechanistic understanding of GA-mediated protection against leishmanial parasites within the host.

Arabinosylated Lipoarabinomannan—Mediated Protection in Visceral Leishmaniasis through Up-Regulation of Toll-Like Receptor 2 Signaling: An Immunoprophylactic Approach

Visceral leishmaniasis is characterized by severe immunosuppression of the host cell, resulting in loss of the proinflammatory response. Toll-like receptor 2 (TLR2) is involved in myriad disease forms, including visceral leishmaniasis. During Leishmania donovani infection, the parasite modulates TLR2 to suppress interleukin 12 production, indicating the possible involvement of TLR2 in regulation of the immune response against L. donovani infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immunomodulatory properties and induces proinflammatory responses via induction of TLR2-mediated signaling. Here, we found that pretreatment of L. donovani-infected macrophages with Ara-LAM caused a significant increase in TLR2 expression along with the activation of TLR2-mediated downstream signaling, facilitating active nuclear translocation of nuclear factor kappaB. These events culminated in up-regulation of the proinflammatory response, which was abrogated by treatment with TLR2-specific small interfering RNA. In vivo experiments were also suggestive of Ara-LAM playing a long-term protective role. This study demonstrates that Ara-LAM confers protection against leishmanial pathogenesis via TLR2 signaling-mediated induction of the proinflammatory response.

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.

Leishmania-Induced Biphasic Ceramide Generation in Macrophages Is Crucial for Uptake and Survival of the Parasite

The initial macrophage-Leishmania donovani interaction results in the formation of membrane platforms, termed lipid rafts, that help in the entry of the parasite. Therefore, it is imperative that the parasite designs a strategy to modulate its uptake and survival within the macrophages. Herein, we report Leishmania-triggered biphasic ceramide generation. In the first phase, L. donovani promastigotes induce activation of acid sphingomyelinase (ASMase), which catalyzes the formation of ceramide from sphingomyelin. Inhibition of ASMase resulted in reduced uptake and infection with the parasite. In the second phase, de novo synthesis generates ceramide that reduces the cellular cholesterol level and displaces the cholesterol from the membrane, leading to enhanced membrane fluidity, disruption of rafts, and impaired antigen-presentation to the T cells. The results reveal a novel role for ceramide in the perspective of L. donovani infection and help formulate an antileishmanial strategy that can possibly be applied to other intracellular infections as well.

Arabinosylated Lipoarabinomannan Skews Th2 Phenotype towards Th1 during Leishmania Infection by Chromatin Modification: Involvement of MAPK Signaling

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal–regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.

CXCL10 Is Critical for the Generation of Protective CD8 T Cell Response Induced by Antigen Pulsed CpG-ODN Activated Dendritic Cells

The visceral form of leishmaniasis is the most severe form of the disease and of particular concern due to the emerging problem of HIV/visceral leishmaniasis (VL) co-infection in the tropics. Till date miltefosine, amphotericin B and pentavalent antimony compounds remain the main treatment regimens for leishmaniasis. However, because of severe side effects, there is an urgent need for alternative improved therapies to combat this dreaded disease. In the present study, we have used the murine model of leishmaniasis to evaluate the potential role played by soluble leishmanial antigen (SLA) pulsed-CpG-ODN stimulated dendritic cells (SLA-CpG-DCs) in restricting the intracellular leishmanial growth. We found that mice vaccinated with a single dose of SLA-pulsed DC stimulated by CpG-ODN were protected against a subsequent leishmanial challenge and had a dramatic reduction in parasite burden along with the generation of parasite specific cytotoxic T lymphocytes. Moreover, we demonstrate that the induction of protective immunity conferred by SLA-CpG-DCs depends entirely on the CXC chemokine IFN-γ-inducible protein 10 (CXCL10; IP-10). CXCL10 is directly involved in the generation of a parasite specific CD8+ T cell-mediated immune response. We observed significant reduction of CD8+ T cells in mice depleted of CXCL10 suggesting a direct role of CXCL10 in the generation of CD8+ T cells in SLA-CpG-DCs vaccinated mice. CXCL10 also contributed towards the generation of perforin and granzyme B, two important cytolytic mediators of CD8+ T cells, following SLA-CpG-DCs vaccination. Together, these findings strongly demonstrate that CXCL10 is critical for rendering a protective cellular immunity during SLA-CpG-DC vaccination that confers protection against Leishmania donovani infection.

Mycobacterium indicus pranii (Mw) re-establishes host protective immune response in Leishmania donovani infected macrophages: critical role of IL-12.

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.

Immune Subversion by Mycobacterium tuberculosis through CCR5 Mediated Signaling: Involvement of IL-10

Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.

Antigen-Pulsed CpG-ODN-Activated Dendritic Cells Induce Host-Protective Immune Response by Regulating the T Regulatory Cell Functioning in Leishmania donovani-Infected Mice: Critical Role of CXCL10

Visceral leishmaniasis (VL), caused by Leishmania donovani, is a systemic infection of reticulo-endothelial system. There is currently no protective vaccine against VL and chemotherapy is increasingly limited due to appearance of drug resistance to first line drugs such as antimonials and amphotericin B. In the present study, by using a murine model of leishmaniasis we evaluated the function played by soluble leishmanial antigen (SLA)-pulsed CpG-ODN-stimulated dendritic cells (SLA–CpG–DCs) in restricting the intracellular parasitic growth. We establish that a single dose of SLA–CpG–DC vaccination is sufficient in rendering complete protection against L. donovani infection. In probing the possible mechanism, we observe that SLA–CpG–DCs vaccination results in the significant decrease in Foxp3+GITR+CTLA4+CD4+CD25+ regulatory T cells (Treg) cell population in Leishmania-infected mice. Vaccination with these antigen-stimulated dendritic cells results in the decrease in the secretion of TGF-β by these Treg cells by possible regulation of the SMAD signaling. Moreover, we demonstrate that a CXC chemokine, IFN-γ-inducible protein 10 (IP-10; CXCL10), has a direct role in the regulation of CD4+CD25+ Treg cells in SLA–CpG–DC-vaccinated parasitized mice as Treg cells isolated from IP-10-depleted vaccinated mice showed significantly increased TGF-β production and suppressive activity.