Effector functions of Th17 cells are regulated by IL-35 and TGF-ß in visceral leishmaniasis.

Visceral leishmaniasis (VL) is a debilitating human pathogenesis in which the body's immune functions are severely compromised. Various subsets of T cells, including Th17 cells are important regulators of immune responses observed in various pathologies. The role of Th17 cells and its correlation with immuno-regulatory cytokines are however not well understood in human VL. Herein we studied how IL-17 is associated with the progression of Leishmania donovani infection using murine model of VL. We found induction of a strong IL-17 response at the early phase of infection which progressively reduced to basal level during chronic VL. The mechanistic study of this behavior was found to be linked with the role of regulatory T cells (CD4+ CD25+ T cells) that suppresses the proliferation of the Th17 cell population. Moreover, TGF-ß and IL-35 derived from CD4+ CD25+ T cells are the key mediators for the downregulation of IL-17 during chronic VL. Thus, this study points to an antagonistic effect of Tregs and Th17 cells that can be used for designing better therapeutic and preventive strategies against leishmaniasis.

A Novel Therapeutic Strategy for Cancer Using Phosphatidylserine Targeting Stearylamine-Bearing Cationic Liposomes.

There is a pressing need for a ubiquitously expressed antigen or receptor on the tumor surface for successful mitigation of the deleterious side effects of chemotherapy. Phosphatidylserine (PS), normally constrained to the intracellular surface, is exposed on the external surface of tumors and most tumorigenic cell lines. Here we report that a novel PS-targeting liposome, phosphatidylcholine-stearylamine (PC-SA), induced apoptosis and showed potent anticancer effects as a single agent against a majority of cancer cell lines. We experimentally proved that this was due to a strong affinity for and direct interaction of these liposomes with PS. Complexation of the chemotherapeutic drugs doxorubicin and camptothecin in these vesicles demonstrated a manyfold enhancement in the efficacies of the drugs both in vitro and across three advanced tumor models without any signs of toxicity. Both free and drug-loaded liposomes were maximally confined to the tumor site with low tissue concentration. These data indicate that PC-SA is a unique and promising liposome that, alone and as a combination therapy, has anticancer potential across a wide range of cancer types.

Attenuation of nociceptive pain and inflammatory disorders by total steroid and terpenoid fraction of Euphorbia tirucalli Linn root in experimental in vitro and in vivo model.

The plant Euphorbia tirucalli Linn has been successfully used as a tribal folk medicine in India and Africa for the management of acute inflammatory, arthritic, nociceptive pain and asthmatic symptoms. The present study was conducted to assess the anti-inflammatory, analgesic, anti-asthmatic and anti-arthritic role of the total steroid and terpenoid rich fractions of the hydro-alcoholic extract of E. tirucalli root (STF-HAETR). STF-HAETR fraction demonstrated 71.25 ± 2.5 and 74.25 ± 5.1% protection against acetic acid-induced pain and central neuropathic pain at 75 and 100 mg/kg doses, respectively. It showed 96.97% protection against acute inflammation at 100 mg/kg with 1.6-fold better activity than the standard drug. The fraction exhibited such efficacy via inhibition of proinflammatory cytokines TNF-α, IFN-γ, by 61.12 and 65.18%, respectively, at 100 µg/mL. Inhibition of cyclooxygenase and Nitric oxide synthase in a dose-dependent manner affirms its analgesic and anti-inflammatory activity. The spectrophotometric analysis reveals that STF-HAETR induces ameliorative effect against heat-induced denaturation of Bovine serum albumin (BSA) and exhibits significant anti-proteinase activity. The plant fraction also demonstrated anti-asthmatic activity by displaying 62.45% protection against histamine induced bronchoconstriction or dyspnoea. Our findings suggest that STF-HAETR could be an effective safe therapeutic agent to treat nociceptive pain, acute inflammation, asthma, and arthritis which may authenticate its traditional use.

Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B.

OBJECTIVE: The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. METHODOLOGY/PRINCIPAL FINDINGS: We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2- (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. CONCLUSIONS: KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function.

Alternative to Chemotherapy-The Unmet Demand against Leishmaniasis.

Leishmaniasis is a neglected protozoan disease that mainly affects the tropical as well as subtropical countries of the world. The primary option to control the disease still relies on chemotherapy. However, a hindrance to treatments owing to the emergence of drug-resistant parasites, enormous side effects of the drugs, their high cost, and requirement of long course hospitalization has added to the existing problems of leishmaniasis containment program. This review highlights the prospects of immunotherapy and/or immunochemotherapy to address the limitations for current treatment measures for leishmaniasis. In addition to the progress in alternate therapeutic strategies, the possibility and advances in developing preventive measures against the disease have been pointed. The review highlights our recent understandings of the protective immunology that can be exploited to develop an effective vaccine against leishmaniasis. Moreover, an update on the approaches that have evolved over the recent years are predominantly focused to overcome the current challenges in developing immunotherapeutic as well as prophylactic antileishmanial vaccines is discussed.

Recent Advances in Non-Invasive Delivery of Macromolecules using Nanoparticulate Carriers System.

BACKGROUND: The drug delivery of macromolecules such as proteins and peptides has become an important area of research and represents the fastest expanding share of the market for human medicines. The most common method for delivering macromolecules is parenterally. However parenteral administration of some therapeutic macromolecules has not been effective because of their rapid clearance from the body. As a result, most macromolecules are only therapeutically useful after multiple injections, which causes poor compliance and systemic side effects. METHOD: Therefore, there is a need to improve delivery of therapeutic macromolecules to enable non-invasive delivery routes, less frequent dosing through controlled-release drug delivery, and improved drug targeting to increase efficacy and reduce side effects. RESULT: Non-invasive administration routes such as intranasal, pulmonary, transdermal, ocular and oral delivery have been attempted intensively by formulating macromolecules into nanoparticulate carriers system such as polymeric and lipidic nanoparticles. CONCLUSION: This review discusses barriers to drug delivery and current formulation technologies to overcome the unfavorable properties of macromolecules via non-invasive delivery (mainly intranasal, pulmonary, transdermal oral and ocular) with a focus on nanoparticulate carrier systems. This review also provided a summary and discussion of recent data on non-invasive delivery of macromolecules using nanoparticulate formulations.

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Leishmania donovani Aurora kinase: A promising therapeutic target against visceral leishmaniasis.

BACKGROUND: Aurora kinases are key mitotic kinases executing multiple aspects of eukaryotic cell-division. The apicomplexan homologs being essential for survival, suggest that the Leishmania homolog, annotated LdAIRK, may be equally important. METHODS: Bioinformatics, stage-specific immunofluorescence microscopy, immunoblotting, RT-PCR, molecular docking, in-vitro kinase assay, anti-leishmanial activity assays, flow cytometry, fluorescence microscopy. RESULTS: Ldairk expression is seen to vary as the cell-cycle progresses from G1 through S and finally G2M and cytokinesis. Kinetic studies demonstrate their enzymatic activity exhibiting a Km and Vmax of 6.12µM and 82.9pmoles·min(-1)mg(-1) respectively against ATP using recombinant Leishmania donovani H3, its physiological substrate. Due to the failure of LdAIRK-/+ knock-out parasites to survive, we adopted a chemical knock-down approach. Based on the conservation of key active site residues, three mammalian Aurora kinase inhibitors were investigated to evaluate their potential as inhibitors of LdAIRK activity. Interestingly, the cell-cycle progressed unhindered, despite treatment with GSK-1070916 or Barasertib, inhibitors with greater potencies for the ATP-binding pocket compared to Hesperadin, which at nanomolar concentrations, severely compromised viability at IC50s 105.9 and 36.4nM for promastigotes and amastigotes, respectively. Cell-cycle and morphological studies implicated their role in both mitosis and cytokinesis. CONCLUSION: We identified an Aurora kinase homolog in L. donovani implicated in cell-cycle progression, whose inhibition led to aberrant changes in cell-cycle progression and reduced viability. GENERAL SIGNIFICANCE: Human homologs being actively pursued drug targets and the observations with LdAIRK in both promastigotes and amastigotes suggest their potential as therapeutic-targets. Importantly, our results encourage the exploration of other proteins identified herein as potential novel drug targets.

Production, Characterization and Evaluation of Kaempferol Nanosuspension for Improving Oral Bioavailability.

CONTEXT: Kaempferol has a large particle size and poor water solubility, leading to poor oral bioavailability. The present work aimed to develop a kaempferol nanosuspension (KNS) to improve pharmacokinetics and absolute bioavailability. METHODS: A nanosuspension was prepared using high pressure homogenization (HPH) techniques. The physico-chemical properties of the kaempferol nanosuspension (KNS) were characterized using photon correlation spectroscopy (PCS), transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR) and x-ray diffractometry (XRD). A reversephase high performance liquid chromatography (RP-HPLC) method for the analysis of the drug in rat plasma was developed and validated as per ICH guidelines. In vivo pharmacokinetic parameters of oral pure kaempferol solution, oral kaempferol nanosuspension and intravenous pure kaempferol were assessed in rats. RESULTS AND DISCUSSION: The kaempferol nanosuspension had a greatly reduced particle size (426.3 ± 5.8 nm), compared to that of pure kaempferol (1737 ± 129 nm). The nanosuspension was stable under refrigerated conditions. No changes in physico-chemical characteristics were observed. In comparison to pure kaempferol, kaempferol nanosuspension exhibited a significantly (P<0.05) increased in Cmax and AUC(0-∞) following oral administration and a significant improvement in absolute bioavailability (38.17%) compared with 13.03% for pure kaempferol. CONCLUSION: These results demonstrate enhanced oral bioavailability of kaempferol when formulated as a nanosuspension.

Synthesis of Unsymmetrical Sulfides and Their Oxidation to Sulfones to Discover Potent Antileishmanial Agents.

Unsymmetrical sulfides were first synthesized using combinations of a 1,3-dicarbonyl, an aromatic aldehyde and a thiol in the presence of 10 mol % ethanolic piperidine. These sulfides derivatives were subsequently converted into corresponding sulfones via oxidation in the presence of m-chloroperoxybenzoic acid (m-CPBA) at ice-bath to room temperature. The former reaction was achieved at room temperature through one-pot three-component. The later was obtained in good yields using mild reaction conditions with flexibility in choice from a range of substrates. The antimicrobial properties of the newly synthesized sulfone derivatives were investigated against the protozoan parasite, Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Nine sulfone derivatives were found to be efficacious and exhibited significant antimicrobial activity. Further, these compounds were nontoxic on murine peritoneal macrophages thus eliminating potential cytoxicity in the host cells. These compounds may be indicated as potential leads in the treatment of visceral leishmaniasis.

Evasion of Host Defence by Leishmania donovani: Subversion of Signaling Pathways.

Protozoan parasites of the genus Leishmania are responsible for causing a variety of human diseases known as leishmaniasis, which range from self-healing skin lesions to severe infection of visceral organs that are often fatal if left untreated. Leishmania donovani (L. donovani), the causative agent of visceral leishmaniasis, exemplifys a devious organism that has developed the ability to invade and replicate within host macrophage. In fact, the parasite has evolved strategies to interfere with a broad range of signaling processes in macrophage that includes Protein Kinase C, the JAK2/STAT1 cascade, and the MAP Kinase pathway. This paper focuses on how L. donovani modulates these signaling pathways that favour its survival and persistence in host cells.