Search

Muzamil Yaqub Want,1,2 Priya Yadav,1,3 Rakin Khan,1 Garima Chouhan,1,4 Mohammad Islamuddin,1,5 Sheka Yagub Aloyouni,6 Asoke P Chattopadhyay,7 Suliman Yousef AlOmar,8 Farhat Afrin1,9 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi, 110062, India; 2Roswell Park Comprehensive Cancer Center, Buffalo, NY, 14263, USA; 3Department of Microbiology, University of Kalyani, Kalyani, West Bengal, 741235, India; 4Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, Uttar Pradesh, 201306, India; 5Molecular Virology and Vaccinology Laboratory, Department of Biotechnology, Jamia Millia Islamia, New Delhi, 110025, India; 6Health Sciences Research Center, Princess Nourah Bint Abdulrahman University, Riyadh, 11671, Saudi Arabia; 7Department of Chemistry, University of Kalyani, Kalyani, West Bengal, 741235, India; 8Doping Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia; 9Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Taibah University, Madina, 41477, Saudi ArabiaCorrespondence: Farhat Afrin; Suliman Yousef AlOmar Tel +91-7596846639; +966-500767717Email farhatafrin@gmail.com; syalomar@ksu.edu.saIntroduction: The current therapeutic armory for visceral leishmaniasis (VL) caused by Leishmania donovani complex is inadequate, coupled with serious limitations. Combination therapy has proved ineffective due to mounting resistance; however, the search for safe and effective drugs is desirable, in the absence of any vaccine. There is a growing interest in the application of nanoparticles for the therapeutic effectiveness of leishmaniasis. Aimed in this direction, we assessed the antileishmanial effect of gold nanoparticles (GNP) against L. donovani in vitro.Methods: GNP were synthesized and characterized for particle size by dynamic light scattering (DLS) and atomic force microscopy (AFM) and for optical properties by UV-visible spectroscopy. Cytotoxicity of GNP was measured by the MTT proliferation assay. The antileishmanial activity of the nanoparticles was evaluated against L. donovani promastigotes and macrophage-infected amastigotes in vitro.Results: GNP showed a strong SPR peak at 520 nm and mean particle size, polydispersity index (PDI), and zeta potential of 56.0 ± 10 nm, 0.3 ± 0.1 and − 27.0 ± 3 mV, respectively. The GNPs were smooth and spherical with a mean particle diameter of 20 ± 5 nm. Nanoparticles [1.2– 100 μM] did not reveal any cytotoxicity on RAW 264.7 murine macrophage cell line, but exerted significant activity against both promastigotes and amastigote stages of L. donovani with 50% inhibitory concentrations (IC50) of 18.4 ± 0.4 μM and 5.0 ± 0.3 μM, respectively. GNP showed significant antileishmanial activity with deformed morphology of parasites and the least number of surviving promastigotes after growth reversibility analysis.Conclusion: GNP may provide a platform to conjugate antileishmanial drugs onto the surface of nanoparticles to enhance their therapeutic effectiveness against VL. Further work is warranted, involving more in-depth mechanistic studies and in vivo investigations.Keywords: gold nanoparticles, surface plasmon resonance, visceral leishmaniasis L. donovani, promastigotes, amastigotes

Muzamil Y Want,1 Mohammad Islammudin,1 Garima Chouhan,1 Hani A Ozbak,2 Hassan A Hemeg,2 Asoke P Chattopadhyay,3 Farhat Afrin2 1Parasite Immunology Laboratory, Department of Biotechnology, Jamia Hamdard, Hamdard University, New Delhi, India; 2Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia; 3Department of Chemistry, University of Kalyani, Kalyani, India Abstract: Visceral leishmaniasis (VL) is a fatal, vector-borne disease caused by the intracellular protozoa of the genus Leishmania. Most of the therapeutics for VL are toxic, expensive, or ineffective. Sesquiterpenes are a new class of drugs with proven antimicrobial and antiviral activities. Artemisinin is a sesquiterpene lactone with potent antileishmanial activity, but with limited access to infected cells, being a highly lipophilic molecule. Association of artemisinin with liposome is a desirable strategy to circumvent the problem of poor accessibility, thereby improving its efficacy, as demonstrated in a murine model of experimental VL. Nanoliposomal artemisinin(NLA) was prepared by thin-film hydration method and optimized using Box–Behnken design with a mean particle diameter of 83±16nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7mV, and drug loading of 33.2%±2.1%. Morphological study of these nanoliposomes by microscopy showed a smooth and spherical surface. The mechanism of release of artemisinin from the liposomes followed the Higuchi model in vitro. NLA was free from concomitant signs of toxicity, both ex vivo in murine macrophages and in vivo in healthy BALB/c mice. NLA significantly denigrated the intracellular infection of Leishmania donovani amastigotes and the number of infected macrophages ex vivo with an IC50 of 6.0±1.4µg/mL and 5.1±0.9µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared toartemisinin with a percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen atthe highest dose of 20mg/kg body weight with modulation of cell-mediated immunity towards protective Th1 type. This study is the first report on the use of a liposomal drug delivery system for artemisinin as a promising alternative intervention against VL. Keywords: visceral leishmaniasis, Box–Behnken, nanoliposomes, drug delivery, artemisinin, Leishmania&nbsp

Leishmaniasis is one of the major neglected tropical diseases, for which no vaccines exist. Chemotherapy is hampered by limited efficacy coupled with development of resistance and other side effects. Leishmania parasites elude the host defensive mechanisms by modulating their surface proteins as well as dampening the host's immune responses. The parasites use the conventional RNA polymerases peculiarly under different environmental cues or pressures such as the host's milieu or the drugs. The mechanisms that restructure post-translational modifications are poorly understood but altered epigenetic histone modifications are believed to be instrumental in influencing the chromatin remodeling in the parasite. Interestingly, the parasite also modulates gene expression of the hosts, thereby hijacking or dampening the host immune response. Epigenetic factor such as DNA methylation of cytosine residues has been incriminated in silencing of macrophage-specific genes responsible for defense against these parasites. Although there is dearth of information regarding the epigenetic alterations-mediated pathogenesis in these parasites and the host, the unique epigenetic marks may represent targets for potential anti-leishmanial drug candidates. This review circumscribes the epigenetic changes during Leishmania infection, and the epigenetic modifications they enforce upon the host cells to ensure a safe haven. The non-coding micro RNAs as post-transcriptional regulators and correlates of wound healing and toll-like receptor signaling, as well as prognostic biomarkers of therapeutic failure and healing time are also explored. Finally, we highlight the recent advances on how the epigenetic perturbations may impact leishmaniasis vaccine development as biomarkers of safety and immunogenicity.

Visceral leishmaniasis (VL) or kala-azar is a vector borne infectious disease caused by the protozoan parasites of the genus Leishmania. VL is endemic in more than 85 countries with an estimated 0.2-0.5 million people at risk, causing high morbidity and mortality across the globe. In the absence of effective vaccines, treatment solely relies on chemotherapy and can be 100% fatal within two years, if left untreated. However, the present chemotherapeutics is limited by toxicity, non-compliance, location of parasites within the lysosomal vacuoles of macrophages, impairing the accession of many potential antileishmanial drugs, prolonged and cumbersome regimen that is unaffordable by rural population with alarming increase in unresponsiveness, complications of post kala-azar dermal leishmaniasis (PKDL) and HIV co-infections. Nanotechnology offers promising approach in the treatment of VL as it reduces toxicity, improves the therapeutic index of drugs, and can selectively deliver the antileishmanial cargos to the intracellular pathogens. In addition, nanoparticles can interact with the host immune system, modulating the immune response in a way that may favor the elimination of the Leishmania parasites. In this review, we give an overview of the strategies and delivery systems employed for the antileishmanial drugs towards the riddance of deadly VL.

In continuance with earlier reported work, an extension has been carried out by the same research group. Mulling over the ongoing condition of resistance to existing antimalarial agents, we had reported synthesis and antimalarial activity of certain pyrazole-1,3,4-oxadiazole hybrid compounds. Bearing previous results in mind, our research group ideated to design and synthesize some more derivatives with varied substitutions of acetophenone and hydrazide. Following this, derivatives 5a–r were synthesized and tested for antimalarial efficacy by schizont maturation inhibition assay. Further, depending on the literature support and results of our previous series, certain potent compounds (5f, 5n and 5r) were subjected to Falcipain-2 inhibitory assay. Results obtained for these particular compounds further strengthened our hypothesis. Here, in this series, compound 5f having unsubstituted acetophenone part and a furan moiety linked to oxadiazole ring emerged as the most potent compound and results were found to be comparable to that of the most potent compound (indole bearing) of previous series. Additionally, depending on the available literature, compounds (5a–r) were tested for their antileishmanial potential. Compounds 5a, 5c and 5r demonstrated dose-dependent killing of the promastigotes. Their IC50 values were found to be 33.3 ± 1.68, 40.1 ± 1.0 and 19.0 ± 1.47 μg/mL respectively. These compounds (5a, 5c and 5r) also had effects on amastigote infectivity with IC50 of 44.2 ± 2.72, 66.8 ± 2.05 and 73.1 ± 1.69 μg/mL respectively. Further target validation was done using molecular docking studies. Acute oral toxicity studies for most active compounds were also performed.

Hypothesis Artemisinin, a potential antileishmanial compound with poor bioavailability and stability has limited efficacy in visceral leishmaniasis. Encapsulating artemisinin into poly lactic-co glycolic nanoparticles may improve its effectiveness and reduce toxicity. Experiments Artemisinin-loaded nanoparticles were prepared, optimized (using Box–Behnken design) and characterized by dynamic light scattering technique, Atomic force microscopy (AFM), Transmission electron microscopy (TEM) and Fourier Transform-Infra Red spectroscopy. Release kinetics of artemisinin from optimized nanoformulation was studied by dialysis method at pH 7.4 and 5.5. Cytotoxicity and antileishmanial activity of these nanoparticles was tested on murine macrophages by MTT assay and macrophage-infested Leishmania donovani amastigotes ex vivo , respectively. Findings Artemisinin-loaded nanoparticles were 221 ± 14 nm in diameter, with polydispersity index, zeta potential, drug loading and entrapment efficiency of 0.1 ± 0.015, −9.07 ± 0.69 mV, 28.03 ± 1.14 and 68.48 ± 1.97, respectively. AFM and TEM studies indicated that the particles were spherical in shape. These colloidal particles showed a sustained release pattern in vitro . Treatment with artemisinin-loaded nanoparticles significantly reduced the number of amastigotes per macrophage and percent infected macrophages ex vivo compared to free artemisinin. These nanoparticles were also non-toxic to macrophages compared to artemisinin alone.

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.

Visceral leishmaniasis (VL) is a pestilent form of leishmaniasis that chiefly impinges the poorest sections of the society. The prototypical therapeutic interventions in vogue are handicapped due to toxicity and alarming increase in drug resistance. In the absence of vaccines, progressive emer- gence of HIV-VL co-infection and relapse in the form of post kala-azar dermal leishmaniasis, have fuelled the quest for alternative therapies. Herein, we report antileishmanial activity of Piper ni- grum, which is endowed with multifarous medicinal properties. Hexane (PNH) and ethanolic (PNE) extracts of P. nigrum substantially inhibited the growth of Leishmania donovani promastigotes with 50% inhibitory concentration (IC50) of 31.6 and 37.8 µg·ml−1, respectively. Growth reversibil- ity analysis revealed the leishmanicidal effect of PNH which caused cell shrinkage and flagellar disruption. In contrast, PNE treated promastigotes showed partial effect. PNH and PNE also abro- gated the growth of intra-macrophagic Leishmania amastigotes with IC50 of 14.6 and 18.3 µg·ml −1 , respectively. Anti-amastigote efficacy of PNH was accompanied by higher selectivity over host macrophages than PNE. Gas-Chromatography-Mass Spectrometry showed the presence of several secondary metabolites such as trans-β-caryophyllene, piperine, β-bisabolene and other sesquiter- penes in PNH and piperine, δ-(sup 9)-cis oleic acid and piperyline in PNE. Conclusively, our work revealed discernible antileishmanial activity of P. nigrum extracts.

Background There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. Methodology Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). Principal findings CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 μg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 μg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. Conclusions Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial compounds., Author summary Leishmaniasis encompasses a broad spectrum of vector-borne neglected tropical diseases with significant worldwide health impact, ranging from the self-healing cutaneous lesions to stigmatizing and disfiguring skin ulcers (mucocutaneous), and the systemic visceral manisfestations (kala azar or visceral leishmaniasis, VL). A poverty-stricken disease, VL is fatal, if left untreated and resurfaces as post-kala azar dermal leishmaniasis after several years of apparent cure. Moreover, there is an upward trend in development of resistance to most of the currently available chemotherapeutic arsenal. Absence of vaccines, progressive emergence of HIV-Leishmania co-infection delineate the gravity of VL affliction. Natural products from medicinal plants have shown leishmanicidal effect, which in some cases, is potentiated by immunomodulation. Here, we elucidate the antileishmanial efficacy of Cinnamomum cassia bark fraction (CBD) with no adverse side effects. The parasites were eliminated by apoptosis in vitro while the protection in vivo was complemented by partial immunomodulation. CBD may be used in synergy with known or pipeline drugs for effective maintenance of VL. Our study represents an important step in the regional drive towards VL elimination.

Background The therapy of visceral leishmaniasis (VL) is limited by resistance, toxicity and decreased bioavailability of the existing drugs coupled with dramatic increase in HIV-co-infection, non-availability of vaccines and down regulation of cell-mediated immunity (CMI). Thus, we envisaged combating the problem with plant-derived antileishmanial drug that could concomitantly mitigate the immune suppression of the infected hosts. Several plant-derived compounds have been found to exert leishmanicidal activity via immunomodulation. In this direction, we investigated the antileishmanial activity of eugenol emulsion (EE), complemented with its immunomodulatory and therapeutic efficacy in murine model of VL. Methodology/Principal Findings Oil-in-water emulsion of eugenol (EE) was prepared and size measured by dynamic light scattering (DLS). EE exhibited significant leishmanicidal activity with 50% inhibitory concentration of 8.43±0.96 μg ml-1 and 5.05±1.72 μg ml─1, respectively against the promastigotes and intracellular amastigotes of Leishmania donovani. For in vivo effectiveness, EE was administered intraperitoneally (25, 50 and 75 mg/kg b.w./day for 10 days) to 8 week-infected BALB/c mice. The cytotoxicity of EE was assessed in RAW 264.7 macrophages as well as in naive mice. EE induced a significant drop in hepatic and splenic parasite burdens as well as diminution in spleen and liver weights 10 days post-treatment, with augmentation of 24h-delayed type hypersensitivity (DTH) response and high IgG2a:IgG1, mirroring induction of CMI. Enhanced IFN-γ and IL-2 levels, with fall in disease-associated Th2 cytokines (IL-4 and IL-10) detected by flow cytometric bead-based array, substantiated the Th1 immune signature. Lymphoproliferation and nitric oxide release were significantly elevated upon antigen revoke in vitro. The immune-stimulatory activity of EE was further corroborated by expansion of IFN-γ producing CD4+ and CD8+ splenic T lymphocytes and up-regulation of CD80 and CD86 on peritoneal macrophages. EE treated groups exhibited induction of CD8+ central memory T cells as evidenced from CD62L and CD44 expression. No biochemical alterations in hepatic and renal enzymes were observed. Conclusions Our results demonstrate antileishmanial activity of EE, potentiated by Th1 immunostimulation without adverse side effects. The Th1 immune polarizing effect may help to alleviate the depressed CMI and hence complement the leishmanicidal activity., Author Summary In the absence of vaccines and escalating resistance to the available chemotherapeutic drugs, alternate therapeutic options are urgently needed for visceral leishmaniasis (VL) or kala-azar, a systemic and potentially fatal, vector-borne neglected tropical disease, caused by Leishmania donovani. In the present study, we explored the two-prong effect of eugenol emulsion (EE) in eliminating the parasites with synergistic boosting of the dampened immune system, characteristic of active disease. Infected BALB/c mice upon intraperitoneal administration of EE, exhibited a significant decline in liver and spleen parasite load with concomitant drop in splenomegaly. Protection in treated mice coincided with Th1 immunopotentiation as was evident from substantial DTH and lymphoproliferative responses, elevated levels of IgG2a over IgG1 isotypes, significant enhancement in IFN-γ producing CD4+ and CD8+ T cells, waning levels of IL-4 and IL-10, augmented nitrite levels, induction of immunological memory and stimulation of antigen presenting capacity of macrophages, compared to infected mice. The dual antileishmanial and immunostimulatory potential of EE with no adverse toxic effects validates it as an adjunct to chemotherapy that may aid in leishmanicidal activity via ameliorating the depressed cellular immunity.

Background Exploration of immunomodulatory antileishmanials of plant origin is now being strongly recommended to overcome the immune suppression evident during visceral leishmaniasis (VL) and high cost and toxicity associated with conventional chemotherapeutics. In accordance, we assessed the in vitro and in vivo antileishmanial and immunomodulatory potential of ethanolic fractions of Azadirachta indica leaves (ALE) and seeds (ASE). Methods A. indica fractions were prepared by sequential extraction of the powdered plant parts in hexane, ethanol and water. Erythrosin B staining was employed to appraise the anti-promastigote potential of ALE and ASE. Cytostatic or cytocidal mode of action was ascertained and alterations in parasite morphology were depicted under oil immersion light microscopy. Study of apoptotic correlates was performed to deduce the mechanism of induced cell death and anti-amastigote potential was assessed in Leishmania parasitized RAW 264.7 macrophages. In vivo antileishmanial effectiveness was evaluated in L. donovani infected BALB/c mice, accompanied by investigation of immunomodulatory potential of ALE and ASE. Adverse toxicity of the bioactive fractions against RAW macrophages was studied by MTT assay. In vivo side effects on the liver and kidney functions were also determined. Plant secondary metabolites present in ALE and ASE were analysed by Gas chromatography–mass spectrometry (GC-MS). Results ALE and ASE (500 μg ml−1) exhibited leishmanicidal activity in a time- and dose-dependent manner (IC50 34 and 77.66 μg ml−1, respectively) with alterations in promastigote morphology and induction of apoptosis. ALE and ASE exerted appreciable anti-amastigote potency (IC50 17.66 and 24.66 μg ml−1, respectively) that was coupled with profound in vivo therapeutic efficacy (87.76% and 85.54% protection in liver and 85.55% and 83.62% in spleen, respectively). ALE exhibited minimal toxicity with selectivity index of 26.10 whereas ASE was observed to be non-toxic. The bioactive fractions revealed no hepato- and nephro-toxicity. ALE and ASE potentiated Th1-biased cell-mediated immunity along with upregulation of INF-γ, TNF-α and IL-2 and decline in IL-4 and IL-10 levels. GC-MS analysis revealed several compounds that may have contributed to the observed antileishmanial effect. Conclusion Dual antileishmanial and immunostimulatory efficacy exhibited by the bioactive fractions merits their use alone or as adjunct therapy for VL. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0788-3) contains supplementary material, which is available to authorized users.

Leishmaniasis is one of the major tropical parasitic diseases, and the condition ranges in severity from self-healing cutaneous lesions to fatal visceral manifestations. There is no vaccine available against visceral leishmaniasis (VL) (also known as kala-azar in India), and current antileishmanial drugs face major drawbacks, including drug resistance, variable efficacy, toxicity and parenteral administration. We report here that n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania donovani promastigotes, with GI(50) of 14.4 and 14.6 µg ml(-1), respectively, and the IC(50) against intracellular amastigotes was found to be 6.6 and 5.05 µg ml(-1), respectively. Changes in the morphology of promastigotes and growth reversibility analysis following treatment confirmed the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on mammalian cells. The antileishmanial activity was mediated via apoptosis, as evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. High-performance thin-layer chromatography (HPTLC) fingerprinting showed that the content of artemisinin in crude bioactive extracts (~1.4 µg per 100 µg n-hexane fraction) was too low to account for the observed antileishmanial activity. Characterization of the active constituents by GC-MS showed that α-amyrinyl acetate, β-amyrine and derivatives of artemisinin were the major constituents in AAL and cetin, EINECS 211-126-2 and artemisinin derivatives in AAS. Our findings indicate the presence of antileishmanial compounds besides artemisinin in the n-hexane fractions of A. annua leaves and seeds.

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.

This study was undertaken to investigate the synergistic interaction between amphotericin B (AmB) and acteoside, isolated from the aerial parts of the shrub Colebrookea oppositifolia (Lamiaceae). Acteoside alone exhibited no intrinsic antifungal activity but showed a potent synergism in combination with AmB against selected pathogenic species, with fractional inhibitory concentration indices in the range of 0.0312-0.1562. The combination of acteoside at 3.12 and 12.5 µg ml(-1) with subinhibitory concentrations of AmB resulted in a potent fungicidal effect and also exhibited a significantly extended post-antifungal effect. Furthermore, the combination also reduced the minimum biofilm reduction concentration values of AmB (2-16-fold) in preformed biofilms of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. There was decreased viability of the cells, increased uptake of propidium iodide and enhanced leakage of 260 nm-absorbing material by Candida albicans cells when exposed to AmB in the presence of acteoside. The reason for potentiation is likely to be that the subinhibitory concentrations of AmB facilitated the uptake of acteoside, which resulted in increased killing of the fungal cells. Administration of acteoside in mice at up to 2000 mg (kg body weight)(-1) by the intraperitoneal or oral route produced no overt toxicity. The data presented here support synergism between acteoside and AmB, and it is therefore proposed that a prospective new management strategy for therapeutic application of this combination should be explored.

• Introduction • Mesenchymal stem cells and its characteristics • Experimental/preclinical MSC-based studies – MSC transplantation – Genetically modified MSC-based therapy – MSC-based protein therapy – Tissue engineering using MSCs • Clinical studies • Challenges and future prospects Mesenchymal stem cells (MSCs), adherent fibroblastoid cells, present in bone marrow and many other tissues can be easily isolated and expanded in vitro. They are capable of differentiating into different cell types such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes, endothelial cells and neuronal cells. Such immense plasticity coupled with their ability to modulate the activity of immune cells makes them attractive for stem cell-based therapy aimed at treating previously incurable disorders. Preclinical studies have reported successful use of MSCs for delivering therapeutic proteins and repairing defects in a variety of disease models. These studies highlighted the in vivo potential of MSCs and their ability to home to injury sites and modify the microenvironment by secreting paracrine factors to augment tissue repair. Their therapeutic applicability has been widened by genetic modification to enhance differentiation and tissue targeting, and use in tissue engineering. Clinical trials for diseases such as osteogenesis imperfecta, graft-versus-host disease and myocardial infarction have shown some promise, demonstrating the safe use of both allogeneic and autologous cells. However, lack of knowledge of MSC behaviour and responses in vitro and in vivo force the need for basic and animal studies before heading to the clinic. Contrasting reports on immunomodulatory functions and tumorigenicity along with issues such as mode of cell delivery, lack of specific marker, low survival and engraftment require urgent attention to harness the potential of MSC-based therapy in the near future.

The author Muzamil Y. Want was inadvertently missed in the original manuscript and we wish to add his name for contribution to ex vivo experiments.

2-Deoxy-d-glucose (2-DG) has been found to enhance the cytotoxicity of ionizing radiation and chemotherapeutic drugs in several tumor cell lines in vitro. Systemic administration of 2-DG together with localized irradiation of the tumor leads to tumor regression and cure (disease free survival), which correlate with the differential levels of anti-tumor immunity observed in Ehrlich ascites tumor (EAT) bearing mice. Macrophages being a major player of the innate immune system, we investigated the activation status of splenic macrophages during radio-sensitization of EAT in mice as well as in peritoneal macrophages ex vivo and macrophagic cell line (Raw 264.7) in vitro. Results suggest that under in vivo conditions, the combined treatment (2-DG+radiation) restores the M1 phenotype in spleen that correlated with the tumor response. However, 2-DG neither induced significant cytotoxicity nor enhanced radiation-induced cell death in peritoneal macrophages and the macrophage cell line (Raw 264.7). Further, increased arborization and enhanced functional status (expression of MHC class II, CD80, CD86 and phagocytosis) were observed after the combined treatment. Besides this activation, the combined treatment also skewed the macrophages towards M1 phenotype as evidenced by the enhanced secretion of IL-12, IL-2, TNF-α and IFN-γ. These observations suggest that 2-DG not only preserves the survival of normal macrophages during irradiation, but also activates macrophages by polarizing towards M1 phenotype, which is known to be tumoricidal in nature. This study for the first time sheds light on a potential antitumor immune activation by 2-DG involving macrophagic stimulation during in vivo radio-sensitization of tumors, besides the other known antitumor effects of this glucose analogue.

Visceral leishmaniasis (VL) is a fatal vector-borne parasitic syndrome attributable to the protozoa of the Leishmania donovani complex. The available chemotherapeutic options are not ideal due to their potential toxicity, high cost and prolonged treatment schedule. In the present study, we conjectured the use of nano drug delivery systems for plant-derived secondary metabolite; artemisinin as an alternative strategy for the treatment of experimental VL. Artemisinin-loaded poly lactic co-glycolic acid (ALPLGA) nanoparticles prepared were spherical in shape with a particle size of 220.0±15.0 nm, 29.2±2.0% drug loading and 69.0±3.3% encapsulation efficiency. ALPLGA nanoparticles administered at doses of 10 and 20mg/kg body weight showed superior antileishmanial efficacy compared with free artemisinin in BALB/c model of VL. There was a significant reduction in hepatosplenomegaly as well as in parasite load in the liver (85.0±5.4%) and spleen (82.0±2.4%) with ALPLGA nanoparticles treatment at 20mg/kg body weight compared to free artemisinin (70.3±0.6% in liver and 62.7±3.7% in spleen). In addition, ALPLGA nanoparticle treatment restored the defective host immune response in mice with established VL infection. The protection was associated with a Th1-biased immune response as evident from a positive delayed-type hypersensitivity reaction, escalated IgG2a levels, augmented lymphoproliferation and enhancement in proinflammatory cytokines (IFN-γ and IL-2) with significant suppression of Th2 cytokines (IL-10 and IL-4) after in vitro recall, compared to infected control and free artemisinin treatment. In conclusion, our results advocate superior efficacy of ALPLGA nanoparticles over free artemisinin, which was coupled with restoration of suppressed cell-mediated immunity in animal models of VL.

Background In the absence of vaccines and limitations of currently available chemotherapy, development of safe and efficacious drugs is urgently needed for visceral leishmaniasis (VL) that is fatal, if left untreated. Earlier we reported in vitro apoptotic antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) against Leishmania donovani. In the present study, we investigated the immunostimulatory and therapeutic efficacy of AAL and AAS. Methodology/Principal Findings Ten-weeks post infection, BALB/c mice were orally administered AAL and AAS for ten consecutive days. Significant reduction in hepatic (86.67% and 89.12%) and splenic (95.45% and 95.84%) parasite burden with decrease in spleen weight was observed. AAL and AAS treated mice induced the strongest DTH response, as well as three-fold decrease in IgG1 and two-fold increase in IgG2a levels, as compared to infected controls. Cytometric bead array further affirmed the elicitation of Th1 immune response as indicated by increased levels of IFN-γ, and low levels of Th2 cytokines (IL-4 and IL-10) in serum as well as in culture supernatant of lymphocytes from treated mice. Lymphoproliferative response, IFN-γ producing CD4+ and CD8+ T lymphocytes and nitrite levels were significantly enhanced upon antigen recall in vitro. The co-expression of CD80 and CD86 on macrophages was significantly augmented. CD8+ T cells exhibited CD62Llow and CD44hi phenotype, signifying induction of immunological memory in AAL and AAS treated groups. Serum enzyme markers were in the normal range indicating inertness against nephro- and hepato-toxicity. Conclusions/Significance Our results establish the two-prong antileishmanial efficacy of AAL and AAS for cure against L. donovani that is dependent on both the direct leishmanicidal action as well as switching-on of Th1-biased protective cell-mediated immunity with generation of memory. AAL and AAS could represent adjunct therapies for the treatment of leishmaniasis, either alone or in combination with other antileishmanial agents., Author Summary Visceral leishmaniasis (VL) is a fatal, vector-borne tropical disease that affects the poorest sections of the society. The currently available drugs are toxic, expensive and have severe side effects. The problem is further compounded by emergence of VL-HIV co-infection and occurence of PKDL after apparent cure. Thus, alternate therapeutic interventions are needed in the absence of vaccines and mounting drug resistance. VL is also characterized by severe depression of cell-mediated immunity that complicates the efficiency of chemotherapeutic drugs. Restoration of the dampened immune system coupled with antileishmanial effect would be a rational approach in the quest for antileishmanial drugs. Plant derived secondary metabolites have been recommended for the containment of antiparasitic disease including leishmaniasis that synergistically aid in lifting the immune suppression. We previously reported in vitro antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) that was mediated by apoptosis. In this study, we found significant reduction in liver and spleen parasite burden of Leishmania donovani infected BALB/c mice upon oral administration of AAL and AAS with concomitant immunostimulation and induction of immunological memory. The immunotherapeutic potentiation by AAL and AAS with no adverse toxic effects validates their use for treatment of this debilitating disease.

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.

Sojourners visiting high-altitude (HA) (>2500 m) are susceptible to HA disorders; on the contrary, HA natives are well adapted to the extreme hypoxic environment. High aldosterone levels are believed to be involved in HA disorders, we, therefore, envisaged role of CYP11B2 gene variants in HA adaptation and therefore investigated the -344T/C, intron-2 conversion (Iw/Ic), K173R, and A5160C polymorphisms. In addition, polymorphisms in AGT, AT1R, ATP1A1, ADRB2, and GSTP1 genes were also investigated. The study comprised of 662 subjects, comprising of 426 Himalayan highlanders (HLs) and 236 lowlanders (LLs). The -344T/C and K173R polymorphisms were found to be in complete linkage disequilibrium. The wild-type allele -344T and combination of wild-type homozygous genotypes between -344T/C, Iw/Ic, and A5160C polymorphisms, containing all the six wild-type alleles were over-represented in the HLs (p < 0.0001, and p = 0.008, respectively). The wild-type haplotypes -344T-Iw, -344T-5160A, and -344T-Iw-5160A also showed over-representation in the HLs (p < 0.0001). Furthermore, greater the number of wild-type alleles, lower was the ARR (p < 0.05). The genotype distribution in remaining genes did not differ. To conclude, the over-representation of wild-type -344T allele, genotype combinations and haplotypes of CYP11B2, and their correlation with lower aldosterone levels associate with HA adaptation in the HLs. Such an allelic presentation in sojourners may help them cope with adverse HA environment.

Podophyllum hexandrum , a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25–200 μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20 Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100 μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.

High-altitude natives are adapted to hypobaric hypoxia, suggestive of genetic basis of adaptation. Since endothelin-1 (ET-1) is of prime importance in high-altitude disorders in sojourners, we envisaged the role of allelic variants of ET-1 in high-altitude adaptation. Four ET-1 polymorphisms, viz., (CT)(n)-(CA)(n) repeat, -3A/-4A, G2288T, and Lys198Asn, were investigated in 426 highlanders (HLs) and 236 lowlanders (LLs). The plasma ET-1 levels, SBP and BMI were significantly lower in the HLs than those in LLs (p

Hippophae rhamnoides or seabuckthorn is used extensively in Indian and Tibetan traditional medicine for the treatment of circulatory disorders, ischemic heart disease, hepatic injury, and neoplasia. In the present study, we have evaluated the radioprotective potential of REC-1001, a fraction isolated from the berries of H. rhamnoides. Chemical analysis of the extract indicated that REC-1001 was approximately 68% by weight polyphenols, and contained kaempferol, isorhamnetin, and quercetin. The effect of REC-1001 on modulating radiation-induced DNA damage was determined in murine thymocytes by measuring nonspecific nuclear DNA damage at the whole genome level using the alkaline halo assay and by measuring sequence/gene-specific DNA damage both in nuclear DNA (beta-globin gene) and in mitochondrial DNA using a quantitative polymerase chain reaction. Treatment with 10 Gy resulted in a significant amount of DNA damage in the halo assay and reductions in the amplification of both the beta-globin gene and mitochondrial DNA. REC-1001 dose-dependently reduced the amount of damage detected in each assay, with the maximum protective effects observed at the highest REC-1001 dose evaluated (250 micro g/ml). Studies measuring the nicking of naked plasmid DNA further established the radioprotective effect of REC-1001. To elucidate possible mechanisms of action, the antioxidant properties and the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependently scavenged radiation-induced hydroxyl radicals, chemically-generated superoxide anions, stabilized DPPH radicals, and reduced Fe(3+) to Fe(2+). The results of the study indicate that the REC-1001 extract of H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage. The polyphenols/flavonoids present in the extract might be responsible for the free radical scavenging and DNA protection afforded by REC-1001.

BACKGROUND High salt intake is the main determinant of hypertension. The alleles, which once had adaptive value in the salt-poor environment, by promoting salt retention, now induce hypertension. It would be interesting to determine whether the variant alleles of the aldosterone synthase gene (CYP11B2), if related to exaggerated expression/altered activity, are associated with hypertension when combined with a salt-rich diet. OBJECTIVE To investigate the -344T/C, K173R and intron-2 conversion polymorphisms of CYP11B2 for an association with hypertension in highlanders accustomed to a high salt intake. DESIGN AND METHODS Three CYP11B2 polymorphisms were compared with respect to frequencies and clinical characteristics in 190 normotensive highlanders (NHLs) and 100 hypertensive highlanders (HHLs). One-way ANOVA, chi2 test and logistic regression analysis were carried out to investigate the association of these polymorphisms with hypertension. RESULTS The HHLs had significantly higher systolic blood pressure (SBP), diastolic blood pressure (DBP) (P < 0.0001), body mass index (BMI) (P = 0.0002), plasma aldosterone levels (P = 0.03) and aldosterone to plasma renin ratio (ARR) (P < 0.0001) and lower plasma renin activity (PRA) (P = 0.007). The -344T/C and K173R polymorphisms were in complete linkage disequilibrium with each other and the intron-2 conversion allele was in absolute association with the T allele. The TC/CC genotypes correlated with higher BMI when compared with TT genotype in the NHLs and the HHLs (P = 0.002 and 0.004, respectively). The intron-2 conversion heterozygotes/homozygotes correlated with higher SBP in the HHLs (P = 0.03) and significantly higher ARR when compared to IwIw (P = 0.02). Genotype combinations between the -344T/C and intron-2 conversion polymorphisms revealed that combinations with TC or CC genotypes inclined towards higher BMI in both the groups (P < 0.05). CONCLUSIONS Our findings showed a correlation of C allele with high BMI, suggesting that -344T/C polymorphism is in linkage disequilibrium with a functional polymorphism on the adjacent 11-beta hydroxylase gene. The correlation of the intron-2 conversion allele with high SBP and ARR associates it with hypertension. The intron-2 conversion could be a functional variant, since it has been suggested to lead to overexpression of the gene; however, the presence of another functional variant in linkage disequilibrium within the gene cannot be ruled out.

Leishmania donovanipromastigote membrane antigens (LAg) encapsulated in positively charged liposomes have been found to induce very significant levels of protection against experimental visceral leishmaniasis. The protectively immunized animals exhibited profound delayed-type hypersensitivity and antibody responses. The extent of protection induced by the same antigens, however, varied depending on the charge of the vesicles, with maximum induction by positively charged liposomes, followed by neutral liposomes and last negatively charged liposomes. Characterization of LAg and LAg entrapped in liposomes of different charges by Western blot analysis revealed the immunodominance of gp63 in all three vaccine preparations. The strong reactivity of antigens in a restricted antigen profile that included, in addition to gp63, 72-, 52-, 48-, 45-, 39-, and 20-kDa components in neutral and positively charged liposomes contrasted with the reactivity of a greater number of LAg components in negatively charged liposomes. Resistance to visceral leishmaniasis appears to depend on the immunity induced by gp63 and a few select antigens in association with the right liposomes. A striking similarity between the immunogenic profile of partially purified soluble antigens and that of LAg in neutral and positively charged liposomes suggests the potentiality of these antigens in future vaccine studies ofL. donovani.

Leishmaniasis is a pestilent affliction that importunately needs better therapeutics necessitated by absence of effective vaccine, emergence as HIV co-infection and the dread of debilitating chemotherapy. The Leishmania parasites incapacitate host macrophages by preventing the formation of phagolysosomes, impeding antigen presentation to T cells, leading to suppression of cell-mediated immunity. An ideal approach to cure leishmaniasis includes administration of antileishmanial compounds that can concomitantly establish an effective Th1 response via restoration of requisite signaling between macrophages and T cells, for subsequent activation of macrophages to eliminate intracellular amastigotes. Plants have provided an opulent treasure of biomolecules that have fuelled the discovery of antileishmanial drugs. Modulation of immune functions using medicinal plants and their products has emerged as an effective therapeutic strategy. Herein, we review the plant extracts and natural products that have resulted in therapeutic polarization of host immunity to cure leishmaniasis. These immunostimulatory phytochemicals as source of potential antileishmanials may provide new strategies to combat leishmaniasis, alone or as adjunct modality.

Background Fumaria parviflora Lam. (Fumaraceae) is widely used in traditional as well as folkloric system of medicine from ancient. It is commonly known as ‘Pitpapra’ or ‘Shahtrah’ in Indian traditional system of medicine and used for treating numerous ailments like diarrhea, fever, influenza, blood purifier and other complications. The object of the present study was to evaluate the Antileishmanial, antibacterial, antifungal and cytotoxic potential of isolated compound. Methods Methanolic extract of whole plant of Fumaria parviflora was dried under reduced pressure to obtain a dark brown residue which was adsorbed on silica gel column grade (60–120 mesh) to obtain a slurry and chromatographed over silica gel loaded column in petroleum ether – chloroform (3:1, 1:1 and 1:3 v/v). The in vitro antileishmanial evaluation of isolated compound against Leishmania donovani promastigotes was investigated by growth kinetics assay, reversibility assay, analysis of cellular morphology, adverse toxicity and determination of 50% growth inhibitory concentration (GI50). Disc diffusion and broth micro dilution methods were used to study the antibacterial (Gram + Staphylococcus epidermidis and Bacillus subtilis; Gram - Escherichia coli and Salmonella typhimurium) and antifungal (Candida albicans and Aspergillus niger) potential in vitro. Results Structure elucidation by spectral data analysis revealed a novel compound, n-octacosan-7β-ol (OC), yield (0.471%), having significant antimicrobial activity against Leishmania donovani promastigotes, Staphylococcus epidermidis, Escherichia coli, Candida albicans and Aspergillus niger in vitro with GI50 = 5.35, MIC 250, MIC 250 and MFC 500 and MIC 250 μg ml-1 respectively. The isolated compound did not show adverse effect against mammalian macrophages. Conclusions The available evidence of compound suggested that it may be used as antimicrobial agent in future and may provide new platform for drug discovery programmes for leishmaniasis.

Human malaria is a major parasitic infection, and the situation has worsened mainly due to the emergence of resistant malaria parasites to several anti-malarial drugs. Thus, an urgent need to find suitable drug targets has led to the development of newer classes of anti-malarial drugs. Helicases have been targeted to develop therapeutics for viral, bacterial, and other microorganism infections. Recently, Plasmodium falciparum RuvB ATPases/helicases have been characterized and proposed as a suitable antimalarial drug target. In the present study, the screening of various compounds was done and the results suggest that PfRuvB1 ATPase activity is inhibited considerably by the novobiocin and partially by cisplatin and ciprofloxacin. Helicase assay of PfRuvB1 in the presence of various compounds suggest novobiocin, actinomycin, and ethidium bromide as potent inhibitors. Novobiocin inhibits the helicase activity of PfRuvB1 possibly by blocking the ATPase activity of PfRuvB1. This study is unique in respect to the identification of novobiocin as inhibitor of PfRuvB1, partially by competing with ATP binding at its active site and provides evidence for PfRuvB1 as target of novobiocin after DNA gyrase-B and HSP90. These studies will certainly help the pharmacologist to design and develop some novel inhibitor specific to PfRuvB1, which may serve as suitable chemotherapeutics to target malaria.

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain "A" mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4+cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4(+) naïve cells with concomitant increase in activated CD4+ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4(+)CD25(+)FoxP3(+)). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.

Pathogenesis in kala-azar is associated with depressed cellular immunity and significant elevation of antileishmanial antibodies. Since these antibodies are present even after cure, analysis of the parasite-specific isotypes and immunoglobulin G (IgG) subclasses in kala-azar patients may shed new light on the immune responses during progression and resolution of infection. Using leishmanial membrane antigenic extracts, we investigated the relative levels of specific IgG, IgM, IgA, IgE, and IgG subclasses in Indian kala-azar patient sera during disease, drug resistance, and cure. Acute-phase sera showed strong stimulation of IgG, followed by IgE and IgM and lastly by IgA antibodies. IgG subclass analysis revealed expression of all of the subclasses, with a predominance of IgG1 during disease. Following sodium stibogluconate (SAG) resistance, the levels of IgG, IgM, IgE, and IgG4 remained constant, while there was a decrease in the titers of IgG2 and IgG3. In contrast, a significant (2.2-fold) increase in IgG1 was observed in these individuals. Cure, in both SAG-responsive and unresponsive patients, correlated with a decline in the levels of IgG, IgM, IgE, and all of the IgG subclasses. The stimulation of IgG1 and the persistence, most importantly, of IgE and IgG4 following drug resistance, along with a decline in IgE, IgG4, and IgG1 with cure, demonstrate the potential of these isotypes as possible markers for monitoring effective treatment in kala-azar.

Leishmaniasis consists of a complex spectrum of infectious diseases with worldwide distribution of which visceral leishmaniasis or kala-azar caused by Leishmania donovani is the most devastating. In the absence of vaccines, chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice are expensive and associated with multiple adverse side effects. Because of these limitations, the development of new antileishmanial compounds is imperative and plants offer prospects in this regard. The present work was conducted to study the antileishmanial potential of oil from Syzygium aromaticum flower buds (clove). The S. aromaticum oil was characterized by gas chromatography and GC-MS and eugenol as well as eugenyl acetate were found to be the most abundant compounds, composing 59.75 % and 29.24 %, respectively of the oil. Our findings have shown that eugenol-rich essential oil from S. aromaticum (EROSA) possesses significant activity against L. donovani, with 50 % inhibitory concentration of 21±0.16 µg ml−1 and 15.24±0.14 µg ml−1, respectively, against promastigotes and intracellular amastigotes. Alterations in cellular morphology and growth reversibility assay substantiated the leishmanicidal activity of EROSA. The leishmanicidal effect was mediated via apoptosis as confirmed by externalization of phosphatidylserine, DNA nicking by TdT-mediated dUTP nick-end labelling (TUNEL) assay, dyskinetoplastidy, cell cycle arrest at sub-G0–G1 phase, loss of mitochondrial membrane potential and reactive oxygen species generation. EROSA presented no adverse cytotoxic effects against murine macrophages even at 200 µg ml−1. Our studies authenticate the promising antileishmanial activity of EROSA, which is mediated by programmed cell death, and, accordingly, EROSA may be a source of novel agents for the treatment of leishmaniasis.

Recently discovered R2TP complex is an important multiprotein complex involved in multiple cellular process like snoRNP biogenesis, PIKK signaling, RNA polymerase II assembly and apoptosis. Within R2TP complex, Pih1 tightly interacts with Rvb1/Rvb2 and with Tah1 to form R2TP macromolecular complex. R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex, which has been found critical in many cellular process. The genome wide screening of Leishmania donovani and Plasmodium falciparum led to the identification of RuvB like1, RuvB like 2, Pih1, and Tah1. Therefore, we speculate that this complex is also important for these parasites as in the yeast. The detailed analysis of crucial components of R2TP complex, Ld-RuvB like 1, and Ld-RuvB like 2, revealed the presence of characteristic motifs like DNA binding motif and ATPase motifs. Hsp90 is also reported from Leishmania donovani and Plasmodium falciparum suggesting that the R2TP complex further interacts with Hsp90 to form R2TP-Hsp90 complex. Recently it has been discovered that RuvB like proteins are overexpressed in many cancers and their ATPase activity is crucial for cancer cell proliferation and the human RuvBs have been proposed as suitable drug target for cancer. Similarly one of the Plasmodium falciparum RuvB like protein (PfRuvB3) has been found to be specific to the stage where nuclear division led multiplication of parasite take place. Considering all these it seems that the R2TP complex may be playing some critical role both in the cancer cell proliferation in human and rapid multiplication of the parasites Leishmania donovani and Plasmodium falciparum.

SDF-1/CXCR4 axis plays a principle role in the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs), a process that defines cells ability to reach and seed recipient bone marrow niche following their intravenous infusion. However, the proper functioning of CXCR4 downstream signaling depends upon consistent optimal expression of both SDF-1 ligand and its receptor CXCR4, which in turn is variable and regulated by several factors. The constitutive active mutants of CXCR4 (N119A and N119S) being able to induce autonomous downstream signaling, overcome the limitation of ligand-receptor interaction for induction of CXCR4 signaling. Therefore, we intended to explore their potential in chemotaxis; a key cellular process which crucially regulates cells homing to bone marrow. In present study, Tet-on inducible gene expression vector system was used for doxycycline inducible regulated transgene expression of CXCR4 active mutants in hematopoietic stem progenitor cell line K-562. Both of these mutants revealed significantly enhanced chemotaxis to SDF-1 gradient as compared to wild type. Furthermore, gene expression profiling of these genetically engineered cells as assessed by microarray analysis revealed the up-regulation of group of genes that are known to play a crucial role in CXCR4 mediated cells homing and engraftment. Hence, this study suggest the potential prospects of CXCR4 active mutants in research and development aimed to improve the efficiency of cells in the mechanism of homing and engraftment process.