Organ-specific immune profiling of Leishmania donovani-infected hamsters.

Visceral leishmaniasis (VL) is a neglected disease with a broad spectrum of clinical manifestations and involvement of visceral organs. Organ-specific immune response against the Leishmania donovani (Ld) complex is not yet understood due to the unavailability of an appropriate experimental model. In reference to our recent work on comparing the hamster model with VL patients, it is now possible to understand immune profiling in different visceral organs. This may offer an answer to varying parasite loads in different visceral organs in the same host. Herein, we analysed a panel of immune markers (Th-2/Th-1) in visceral organs of Ld-infected hamsters and quantified parasitic load in the same tissues using qPCR assay. In spleen, liver, bone marrow and lymph node (mesenteric) from Ld-infected hamsters, the parasite burden was quantified along with mRNA expression of a panel of Th-2 and Th-1 type immune markers, namely IL-10, IL-4, Arginase-I, GATA-3, SOCS-3, IL-12, IFN-γ, iNOS, T-bet and SOCS-5. A clear dichotomy was absent between Th-2 and Th-1 type immune markers and the major players of this immune response were IFN-γ, IL-10, T-bet, GATA-3, SOCS-5 and SOCS-3.

Leishmania donovani secretory protein nucleoside diphosphate kinase b localizes in its nucleus and prevents ATP mediated cytolysis of macrophages.

Leishmania donovani pathogenicity is closely linked to its ability to live and replicate in the hostile environment of macrophages. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. For some pathogens, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor mediated, ATP-induced death of infected macrophages. Here, Leishmanaia donovani nucleoside diphosphate kinase (LdNDKb) was cloned, expressed and purified by Ni2+-NTA affinity chromatography to elucidate its biological significance. The presence of secreted form of LdNDKb in the medium was confirmed by Western blot analysis. Interestingly, cellular localization by confocal microscopy showed that this protein was localized in the nucleus, inner leaflet of membrane and on the flagella of this parasite which indicates its multiple role in the life cycle of Leishmania donovani. Its possibility to bind with DNA was confirmed by gel retardation assay and electrophoretic mobility shift assay (EMSA) which show the binding with linear and supercoiled is not sequence specific. Further, treatment of J774 macrophages with recombinant LdNdKb and periodate oxidized ATP - a P2X7 receptor antagonist, inhibited ATP-induced cytolysis in vitro, as determined by lactate dehydrogenise release from J774 macrophages. Thus, LdNDKb prevents ATP-mediated host-cell plasma membrane permeabilization by hydrolyzing extracellular ATP, thereby, preserving the integrity of the host cells for the benefit of the parasite. This study indicates that LdNDKb could be explored for its potentiality as a drug/vaccine target against visceral leishmaniasis.

Status of IL-4 and IL-10 driven markers in experimental models of Visceral Leishmaniasis.

AIM: Leishmania donovani, the causative agent for visceral leishmaniasis (VL), modulates host monocytes/macrophages to ensure its survival. However, knowledge regarding the host-parasite interactions underpinning the disease remains limited. As disease progression is associated with polarization of monocytes/macrophages towards M2, which is regulated by cytokines IL-4/IL-13 and IL-10, this study evaluated the status of key IL-4- and IL-10 driven markers in experimental models of VL, as also evaluated their correlation, if any, with parasite load. METHODS: In liver and splenic tissues from L donovani-infected hamsters and BALB/c mice, the parasite burden was determined along with mRNA expression of IL-4-driven markers, that is CD206, Arginase-I, CCL17, CCL22, PPAR-γ, STAT6, KLF4, FIZZ1 and YM1 along with IL-10-driven markers, CXCL13, IL-10, TGF-ß, VDR, CCR2 and CYP27A1. RESULTS: The mRNA expression of IL-4- and IL-10-driven markers was enhanced in both models, but only in the hamster model, the splenic tissues demonstrated a positive correlation between all the IL-10-driven markers and parasite load. CONCLUSIONS: Contrary to human VL, both models demonstrated an increased expression of IL-4- and IL-10-driven markers.

Purified Splenic amastigotes of Leishmania donovani-Immunoproteomic approach for exploring Th1 stimulatory polyproteins.

Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylactic vaccines against disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub-fractionation of purified splenic amastigotes revealed four sub-fractions, belonging to 97 to 68 kDa and 68 to 43 kDa ranges, which showed long-lasting protection with remarkable Th1-type cellular responses in hamsters vaccinated with these sub-fractions (LTT, NO, QRT-PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub-fractions, identified a total of 47 proteins including twenty-five hypothetical proteins/unknowns. Amastigote stage has potential Th1-stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.

Parasitic load determination by differential expressions of 5-lipoxygenase and PGE2 synthases in visceral leishmaniasis.

Infection with L. donovani affects mainly visceral organs. Importantly, the parasitic load differs in different visceral organs; therefore there is a need to understand the organ specific immune regulation, particularly in the spleen and liver. Comparative studies between these organs in Leishmania infected hamster (Mesocricetus auratus) are lacking. Our study highlights the importance of eicosanoids in the organ specific pathology of visceral leishmaniasis. Among other immune cells, macrophages (mφ) which harbor Leishmania parasite are major producers of eicosanoids. In this study, we intend to explore linkage between organ specific immune response and eicosanoids. We suggest that eicosanoids (early immune modulators) and their organ specific expressions, possibly tune the outcome of mφ differently at different sites. We have observed that liver showed better containment of parasitic load than spleen, where we have found higher expression of 5-lipoxygenase (5-LO) enzyme along with IL-12 and iNOS. However, in spleen, enzymes of the PGE2 pathway i.e. PGE2 synthases (cytosolic and microsomal) along with IL-10 were predominantly higher. To further corroborate our findings, in vitro assays were carried out using purified eicosanoids (LTB4 and PGE2) and the inhibitors of these pathways. Findings establish that the 5-lipoxygenase pathway (i.e. LTB4) is anti-parasitic and its inhibition increases the parasitic load (qPCR based kDNA detection). On the contrary, PGES pathway (i.e. PGE2) supports establishment of infection in mφ. Taken together, 5-LO pathway plays a protective role in liver during L. donovani infection. However, the PGES pathway favors the parasite growth, particularly in the spleen at a later stage.

Fabrication of 3-O-sn-Phosphatidyl-L-serine Anchored PLGA Nanoparticle Bearing Amphotericin B for Macrophage Targeting.

PURPOSE: To fabricate, characterize and evaluate 3-O-sn-Phosphatidyl-L-serine (PhoS) anchored PLGA nanoparticles for macrophage targeted therapeutic intervention of VL. MATERIALS AND METHODS: PLGA-AmpB NPs were prepared by well-established nanoprecipitation method and decorated with Phos by thin film hydration method. Physico-chemical characterization of the formulation was done by Zetasizer nano ZS and atomic force microscopy. RESULTS: The optimized formulation (particle size, 157.3 ± 4.64 nm; zeta potential, - 42.51 ± 2.11 mV; encapsulation efficiency, ∼98%) showed initial rapid release up to 8 h followed by sustained release until 72 h. PhoS generated 'eat-me' signal driven augmented macrophage uptake, significant increase in in-vitro (with ∼82% parasite inhibition) and in-vivo antileishmanial activity with preferential accumulation in macrophage rich organs liver and spleen were found. Excellent hemo-compatibility justified safety profile of developed formulation in comparison to commercial formulations. CONCLUSION: The developed PhoS-PLGA-AmpB NPs have improved efficacy, and necessary stability which promisingly put itself as a better alternative to available commercial formulations for optimized treatment of VL.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Bioinspired Calcium Phosphate Nanoparticles Featuring as Efficient Carrier and Prompter for Macrophage Intervention in Experimental Leishmaniasis.

PURPOSE: To develop a biocompatible and bioresorbable calcium phosphate (CaP) nanoparticles (NPs) bearing Amphotericin B (AmB) with an aim to provide macrophage specific targeting in visceral leishmaniasis (VL). MATERIALS & METHODS: CaP-AmB-NPs were architectured through emulsion precipitation method. The developed formulation was extensively characterized for various parameters including in-vitro and in-vivo antileishmanial activity. Moreover, plasma pharmacokinetics, tissue biodistribution and toxicity profile were also assessed. RESULTS: Optimized CaP-AmB-NPs exhibited higher entrapment (71.1 ± 6.68%) of AmB. No trend related to higher hemolysis was apparent in the developed formulation as evidenced in commercially available colloidal and liposomal formulations. Cellular uptake of the developed CaP-AmB-NPs was quantified through flow cytometry in J774A.1 cell line, and a 23.90 fold rise in uptake was observed. Fluorescent microscopy also confirmed the time dependent rise in uptake. In-vivo multiple dose toxicity study demonstrated no toxicity upto 5 mg/kg dose of AmB. Plasma kinetics and tissue distribution studies established significantly higher concentration of AmB in group treated with CaP-AmB-NPs in liver and spleen as compared to CAmB, LAmB and AmB suspension group. In-vivo animal experimental results revealed that the CaP-AmB-NPs showed higher splenic parasite inhibition compared to CAmB and LAmB in leishmania parasite infected hamsters. CONCLUSIONS: The investigated CaP-AmB-NPs are effective in provoking macrophage mediated uptake and collectively features lower toxicity and offers a suitable replacement for available AmB-formulations for the obliteration of intra-macrophage VL parasite.

Antigen presenting cells targeting and stimulation potential of lipoteichoic acid functionalized lipo-polymerosome: a chemo-immunotherapeutic approach against intracellular infectious disease.

Antigen presenting cells (APC) are well-recognized therapeutic targets for intracellular infectious diseases, including visceral leishmaniasis. These targets have raised concerns regarding their potential for drug delivery due to overexpression of a variety of receptors for pathogen associated molecular pathways after infection. Since, lipoteichoic acid (LTA), a surface glycolipid of Gram-positive bacteria responsible for recognition of bacteria by APC receptors that also regulate their activation for pro-inflammatory cytokine secretion, provides additive and significant protection against parasite. Here, we report the nanoarchitechture of APC focused LTA functionalized amphotericin B encapsulated lipo-polymerosome (LTA-AmB-L-Psome) delivery system mediated by self-assembly of synthesized glycol chitosan-stearic acid copolymer (GC-SA) and cholesterol lipid, which can activate and target the chemotherapeutic agents to Leishmania parasite resident APC. Greater J774A and RAW264.7 macrophage internalization of FITC tagged LTA-AmB-L-Psome compared to core AmB-L-Psome was observed by FACSCalibur cytometer assessment. This was further confirmed by higher accumulation in macrophage rich liver, lung and spleen during biodistribution study. The LTA-AmB-L-Psome overcame encapsulated drug toxicity and significantly increased parasite growth inhibition beyond commercial AmB treatment in both in vitro (macrophage-amastigote system; IC50, 0.082 ± 0.009 µg/mL) and in vivo (Leishmania donovani infected hamsters; 89.25 ± 6.44% parasite inhibition) models. Moreover, LTA-AmB-L-Psome stimulated the production of protective cytokines like interferon-γ (IFN-γ), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase and nitric oxide with down-regulation of disease susceptible cytokines, like transforming growth factor-ß (TGF-ß), IL-10, and IL-4. These data demonstrate the potential use of LTA-functionalized lipo-polymerosome as a biocompatible lucrative nanotherapeutic platform for overcoming toxicity and improving drug efficacy along with induction of robust APC immune responses for effective therapeutics of intracellular diseases.

Overexpressed Macrophage Mannose Receptor Targeted Nanocapsules- Mediated Cargo Delivery Approach for Eradication of Resident Parasite: In Vitro and In Vivo Studies.

PURPOSE: Since, Leishmania protozoans are obligate intracellular parasites of macrophages, an immunopotentiating macrophage-specific Amphotericin B (AB) delivery system would be ideally appropriate to increase its superiority for leishmaniasis treatment and to eliminate undesirable toxicity. Herein, we report AB entrapped mannose grafted chitosan nanocapsules (MnosCNc-AB) that results in effective treatment of visceral leishmaniasis, while also enhancing L. donovani specific T-cell immune responses in infected host. METHODS: MnosCNc-AB were prepared via synthesized mannosylated chitosan deposition on interface of oil/water nanoemulsion intermediate and were characterized. J774A.1 macrophage uptake potential, antileishmanial activity and immunomodulatory profile were evaluated in hamster. Tissue localization, biodistribution and toxicity profile were also investigated. RESULTS: MnosCNc-AB had nanometric size (197.8 ± 8.84 nm), unimodal distribution (0.115 ± 0.04), positive zeta potential (+31.7 ± 1.03 mV) and 97.5 ± 1.13% cargo encapsulation efficiency. Superior macrophage internalization of mannosylated chitosan nanocapsules compared to unmodified chitosan nanocapsules was observed by fluorescence-based assessment, further confirmed by rapid blood clearance and, greater localization and higher accumulation in macrophage rich liver and spleen. While, MnosCNc-AB mediated cargo distribution to kidney decreased. Augmented in vitro antileishmanial activity and in vivo pro-inflammatory mediator's expression were observed with MnosCNc-AB, led to significant reduction (∼90%) in splenic parasite burden. CONCLUSIONS: Results demonstrated that mannose ligand grafted chitosan nanocapsules could improve selective delivery of AB into macrophages via interactions with overexpressed mannose receptors thus reduce undesirable toxicity. Study provides evidence for MnosCNc-AB potential to leishmaniasis therapeutics and presents valuable therapeutic strategies for combating chronic macrophage-resident microbial infections.

Self assembled ionically sodium alginate cross-linked amphotericin B encapsulated glycol chitosan stearate nanoparticles: applicability in better chemotherapy and non-toxic delivery in visceral leishmaniasis.

OBJECTIVES: To investigate the applicability, localization, biodistribution and toxicity of self assembled ionically sodium alginate cross-linked AmB loaded glycol chitosan stearate nanoparticles for effective management of visceral leishmaniasis. METHODS: Here, we fabricated Amphotericin B (AmB) encapsulated sodium alginate-glycol chitosan stearate nanoparticles (AmB-SA-GCS-NP) using strong electrostatic interaction between oppositely charged polymer and copolymer by ionotropic complexation method. The tagged FAmB-SA-GCS-NP was compared with tagged FAmB for in vitro macrophagic uptake in J774A macrophages and in vivo localization in liver, spleen, lung and kidney tissues. The AmB-SA-GCS-NP and plain AmB were compared for in vitro and in vivo antileishmanial activity, pharmacokinetics, organ distribution and toxicity profiling. RESULTS: The morphology of SA-GCS-NP revealed as nanocrystal (size, 196.3 ± 17.2 nm; PDI, 0.216 ± 0.078; zeta potential, (-) 32.4 ± 5.1 mV) by field emission scanning electron microscopy and high resolution transmission electron microscopy. The macrophage uptake and in vivo tissue localization studies shows tagged FAmB-SA-GCS-NP has significantly higher (~1.7) uptake compared to tagged FAmB. The biodistribution study of AmB-SA-GCS-NP showed more localized distribution towards Leishmania infected organs i.e. spleen and liver while lesser towards kidney. The in vitro (IC50, 0.128 ± 0.024 µg AmB/ml) and in vivo (parasite inhibition, 70.21 ± 3.46%) results of AmB-SA-GCS-NP illustrated significantly higher (P < 0.05) efficacy over plain AmB. The monomeric form of AmB within SA-GCS-NP, observed by UV-visible spectroscopy, favored very less in vitro and in vivo toxicities compared to plain AmB. CONCLUSION: The molecular organization, toxicity studies, desired localization and biodistribution of cost effective AmB-SA-GCS-NP was found to be highly effective and can be proved as practical delivery platform for better management of leishmaniasis.

Immunological consequences of stress-related proteins--cytosolic tryparedoxin peroxidase and chaperonin TCP20--identified in splenic amastigotes of Leishmania donovani as Th1 stimulatory, in experimental visceral leishmaniasis.

In earlier studies, proteomic characterization of splenic amastigote fractions from clinical isolates of Leishmania donovani, exhibiting significant cellular responses in cured Leishmania subjects, led to the identification of cytosolic tryparedoxin peroxidase (LdcTryP) and chaperonin-TCP20 (LdTCP20) as Th1-stimulatory proteins. Both the proteins, particularly LdTCP20 for the first time, were successfully cloned, overexpressed, purified and were found to be localized in the cytosol of purified splenic amastigotes. When evaluated against lymphocytes of cured Leishmania-infected hamsters, the purified recombinant proteins (rLdcTryP and rLdTCP20) induced their proliferations as well as nitric oxide production. Similarly, these proteins also generated Th1-type cytokines (IFN-γ/IL-12) from stimulated PBMCs of cured/endemic Leishmania patients. Further, vaccination with rLdcTryP elicited noticeable delayed-type hypersensitivity response and offered considerably good prophylactic efficacy (~78% inhibition) against L. donovani challenge in hamsters, which was well supported by the increased mRNA expression of Th1 and Th2 cytokines. However, animals vaccinated with rLdTCP20 exhibited comparatively lesser prophylactic efficacy (~55%) with inferior immunological response. The results indicate the potentiality of rLdcTryP protein, between the two, as a suitable anti-leishmanial vaccine. Since, rLdTCP20 is also an important target, for optimization, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up.

Characterization of the proliferating cell nuclear antigen of Leishmania donovani clinical isolates and its association with antimony resistance.

Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥ 3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ~5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb(V)) and trivalent antimonial (Sb(III)) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of Sb(V) (from 41.2 ± 0.6 µg/ml to 66.5 ± 3.9 µg/ml) and Sb(III) (from 24.0 ± 0.3 µg/ml to 43.4 ± 1.8 µg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb(III) treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.

Exploitation of lectinized lipo-polymerosome encapsulated Amphotericin B to target macrophages for effective chemotherapy of visceral leishmaniasis.

We have designed lectin functionalized Lipo-polymerosome bearing Amphotericin B (Lec-AmB-L-Psome) for specific internalization via lectin receptors overexpressed on infected macrophages of mononuclear phagocytic system (MPS) for the effective management of intramacrophage diseases such as visceral leishmaniasis. The lipo-polymerosome composed of glycol chitosan-stearic acid copolymer (GC-SA25%) and model lipid cholesterol was surface-functionalized with lectin by the EDC/NHS carbodiimide coupling method. Our designed Lec-AmB-L-Psome showed >2-fold enhanced uptake and significantly higher internalization in macrophages as compared to AmB-L-Psome. Importantly, pharmacokinetic and organ distribution studies illustrate significantly higher accumulation of Lec-AmB-L-Psome in MPS especially in liver, spleen, and lung as compared to AmB-L-Psome, Ambisome, and Fungizone. The IC50 value demonstrated that Lec-AmB-L-Psome has 1.63, 2.23, and 3.43 times higher activity than AmB-L-Psome (p < 0.01), Ambisome (p < 0.05), and Fungizone (p < 0.05), respectively. Additionally, the Lec-AmB-L-Psome showed significantly higher splenic parasite inhibition (78.66 ± 3.08%) compared to Fungizone and Ambisome that caused only 56.54 ± 3.91% (p < 0.05) and 66.46 ± 2.08% (p < 0.05) parasite inhibition, respectively, in Leishmania-infected hamsters. The toxicity profile revealed that Lec-AmB-L-Psome is a safe delivery system with diminished nephrotoxicity which is a limiting factor of Fungizone application. Taken together, these studies suggest that this surface functionalized self-assembled Lec-AmB-L-Psome can introduce a new platform to specifically target macrophages for effective management of intramacrophage diseases.

Covalent functionalized self-assembled lipo-polymerosome bearing amphotericin B for better management of leishmaniasis and its toxicity evaluation.

Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosan-stearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 ± 17.9 nm, PDI of 0.168 ± 0.08 and zeta potential of (+) 27.15 ± 0.46 mV with 25.59 ± 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An in vitro study showed comparatively sustained AmB release (66.082 ± 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. In vitro and in vivo toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV-visible spectroscopy. Experimental results of in vitro (macrophage amastigote system) and in vivo (Leishmania donovani infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-α, IL-12 and IFN-γ) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-ß, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RT-PCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system.

Efficacy of Leishmania donovani trypanothione reductase, identified as a potent Th1 stimulatory protein, for its immunogenicity and prophylactic potential against experimental visceral leishmaniasis.

In visceral leishmaniasis (VL), Th1-type of immune responses play an important role which correlates with recovery from and resistance to disease resulting in lifelong immunity. Based on this rationale, the soluble leishmanial antigens that elicit cellular responses in peripheral blood mononuclear cells (PBMCs) from cured Leishmania patients were characterized through immunoproteomic approach which led to the identification of trypanothione reductase (TPR) (a cytosolic enzyme explored as a drug target), as one of the potent Th1 stimulatory protein. In this study, the immunogenicity of recombinant Leishmania donovani TPR (rLdTPR) was assessed in PBMCs of cured Leishmania-infected patients/hamsters and further evaluated its prophylactic efficacy against L. donovani challenges in hamsters. Substantial proliferative responses to rLdTPR, as compared to soluble L. donovani antigen, were observed in Leishmania-infected cured patients as well as in hamsters. Moreover, rLdTPR reasonably stimulated PBMCs of cured Leishmania patients to produce IFNγ, IL-12, and TNF-α but not IL-4 or IL-10. On the other hand, the protein downregulated LPS-induced IL-10 as well as soluble L. donovani antigen-induced IL-4 production in PBMCs of Leishmania patients. In case of cured hamsters, rLdTPR generates mixed Th1 and Th2 immune response. Vaccination with rLdTPR along with Bacillus Calmette-Guerin (BCG) was able to provide considerably good prophylactic efficacy (~60%) against L. donovani challenge in hamsters. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFNγ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Since rLdTPR protein is an important target, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up to optimize its prophylactic efficacy.

Immunoadjuvant chemotherapy of visceral leishmaniasis in hamsters using amphotericin B-encapsulated nanoemulsion template-based chitosan nanocapsules.

The accessible treatment options for life-threatening neglected visceral leishmaniasis (VL) disease have problems with efficacy, stability, adverse effects, and cost, making treatment a complex issue. Here we formulated nanometric amphotericin B (AmB)-encapsulated chitosan nanocapsules (CNC-AmB) using a polymer deposition technique mediated by nanoemulsion template fabrication. CNC-AmB exhibited good steric stability in vitro, where the chitosan content was found to be efficient at preventing destabilization in the presence of protein and Ca(2+). A toxicity study on the model cell line J774A and erythrocytes revealed that CNC-AmB was less toxic than commercialized AmB formulations such as Fungizone and AmBisome. The results of in vitro (macrophage-amastigote system; 50% inhibitory concentration [IC(50)], 0.19 ± 0.04 µg AmB/ml) and in vivo (Leishmania donovani-infected hamsters; 86.1% ± 2.08% parasite inhibition) experiments in conjunction with effective internalization by macrophages illustrated the efficacy of CNC-AmB at augmenting antileishmanial properties. Quantitative mRNA analysis by real-time PCR (RT-PCR) showed that the improved effect was synergized with the upregulation of tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and inducible nitric oxide synthase and with the downregulation of transforming growth factor ß (TGF-ß), IL-10, and IL-4. These research findings suggest that a cost-effective CNC-AmB immunoadjuvant chemotherapeutic delivery system could be a viable alternative to the current high-cost commercial lipid-based formulations.

Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge.

In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12- and IFN-γ-mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

Unresponsiveness of Mycobacterium w vaccine in managing acute and chronic Leishmania donovani infections in mouse and hamster.

The role of Mycobacterium w (Mw) vaccine as an immunomodulator and immunoprophylactant in the treatment of mycobacterial diseases (leprosy and pulmonary tuberculosis) is well established. The fact that it shares common antigens with leishmanial parasites prompted its assessment as an immunostimulant and as an adjunct to known anti-leishmanials that may help in stimulating the suppressed immune status of Leishmania donovani-infected individuals. The efficacy of Mw vaccine was assessed as an immunomodulator, prophylactically either alone or in combination with anti-leishmanial vaccine, as well as therapeutically as an adjunct to anti-leishmanial treatment in L. donovani-infected hamsters, representing a chronic human Visceral Leishmaniasis (VL) model. Similarly, its efficacy was also evaluated in L. donovani-infected BALB/c mice, representing an acute VL model. The preliminary studies revealed that Mw was ineffective as an immunostimulant and/or immunoprophylactant in hamsters infected with L. donovani, as estimated by T-cell immunological responses. However, in the BALB/c mice-VL model it appeared as an effective immunostimulant but a futile prophylactic agent. It is therefore inferred that, contrary to its role in managing tuberculosis and leprosy infections, Mw vaccine has not been successful in controlling VL infection, emphasizing the need to find detailed explanations for the failure of this vaccine against the disease.

Amplified fragment length polymorphism: an adept technique for genome mapping, genetic differentiation, and intraspecific variation in protozoan parasites.

With the advent of polymerase chain reaction (PCR), genetic markers are now accessible for all organisms, including parasites. Amplified fragment length polymorphism (AFLP) is a PCR-based marker for the rapid screening of genetic diversity and intraspecific variation. It is a potent fingerprinting technique for genomic DNAs of any origin or complexity and rapidly generates a number of highly replicable markers that allow high-resolution genotyping. AFLPs are convenient and reliable in comparison to other markers like random amplified polymorphic DNA, restriction fragment length polymorphism, and simple sequence repeat in terms of time and cost efficiency, reproducibility, and resolution as it does not require template DNA sequencing. In addition, AFLP essentially probes the entire genome at random, without prior sequence knowledge. So, AFLP markers have emerged as an advance type of genetic marker with broad application in genomic mapping, population genetics, and DNA fingerprinting and are ideally suited as screening tool for molecular markers linked with biological and clinical traits. This review describes the AFLP procedure and its applications and overview in the fingerprinting of a genome, which has been currently used in parasite genome research. We outline the AFLP procedure adapted for Leishmania genome study and discuss the benefits of AFLPs for assessing genetic variation and genome mapping over other existing molecular techniques. We highlight the possible use of AFLPs as genetic markers with its broad application in parasitological research because it allows random screening of the entire genome for linkage with genetic and clinical properties of the parasite. In this review, we have taken a pragmatic approach on the study of AFLP for genome mapping and polymorphism in protozoan parasites and conclude that AFLP is a very useful tool.