Coccinia grandis (L.) Voigt Leaf Extract Exhibits Antileishmanial Effect Through Pro-inflammatory Response: An In Vitro Study.

The conventional drugs used for the treatment of human visceral leishmaniasis have concerns about the toxicity and most importantly parasite resistance. To overcome these troubles, more efforts are made for the development of innovative therapeutic agents having effective antileishmanial activity and simultaneously stimulate adaptive immune system of host cells. Hence, search for new leishmanicidal from the natural origin like plants has shown its effectiveness for the treatment of this tropical disease. The aim of this study is to investigate and characterize the antileishmanial efficacy of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) with its immunomodulatory property against Leishmania donovani in an in vitro experimental model. Cg-Ex significantly reduces the intracellular L. donovani parasite load with IC50 value 193 ± 0.78 µg/ml, but it has lower cytotoxicity on the murine RAW 264.7 macrophage cell line. Interestingly, Cg-Ex induces the generation of potent antimicrobials like reactive oxygen species and nitric oxide dose dependently in infected murine macrophages. Moreover, the increased production of Th1 cytokines (IL-12, TNF-α) with a concurrent decrease of Th2 cytokines (IL-10, TGF-ß) was also observed in Cg-Ex-treated infected host macrophages. Our results thus confirm that serine protease inhibitor(s)-rich Cg-Ex exhibits antileishmanial activity in vitro, and this was mediated through the modulation of pro-inflammatory cytokines. On the whole, the present findings first demonstrate the antileishmanial property of Cg-Ex targeting the Leishmania serine protease resulting protection of host cells with Th1 cytokine expression. Thus, these data indicate that C. grandis leaf extract (Cg-Ex) might be considered as a new lead for designing alternative and novel natural therapeutic against visceral leishmaniasis.

Protective inflammatory response against visceral leishmaniasis with potato tuber extract: A new approach of successful therapy.

The increasing number of drug resistance issue of Leishmania donovani strain to common drugs compels to develop new therapeutics against leishmaniasis with minimal toxicity. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The important roles of Leishmania proteases in the virulence of Leishmania parasite make them very hopeful targets for the improvement of current remedial of leishmaniasis. As part of a hunt for new drugs, we have evaluated in vivo anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx), isolated by sodium bisulfite extraction from potato tuber. The amastigote load of 25mg/kg body weight/day treated BALB/c mice showed 86.9% decrease in liver and 88.7% in case of spleen. This anti-leishmanial effect was also supported by PTEx induced immunomodulatory activity like acute formation of ROS and prolonged NO generation. The Th1/Th2 cytokine balance in splenocytes of PTEx treated animals was estimated and evaluated by ELISA assay as well as by mRNA expression using RT-PCR. Furthermore, significant survival rate (80%) was observed in PTEx treated hamsters. Thus, from the present observations we could accentuate the potential of PTEx to be employed as a new therapeutics from natural source against L. donovani. This might also provide a novel perception of natural serine protease inhibitor from potato tuber as an alternate approach for the treatment of visceral leishmaniasis.

Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection.

Infection with antimony-resistant Leishmania donovani (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.

Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.

TLR mediated GSK3ß activation suppresses CREB mediated IL-10 production to induce a protective immune response against murine visceral leishmaniasis.

During Leishmania donovani (LD) infection Interleukin (IL)-10 favors parasite replication and plays a central role as a target for immune-based therapy. Glycogen synthase kinase 3 (GSK3)ß differentially regulates TLR-mediated cytokine production. CREB, an important transcription factor that induces IL-10 production is negatively regulated by GSK3ß. However, down regulation of IL-10 via CREB suppression has not been well explored in controlling LD infection. Here we demonstrate that, the TLR4 agonist 29 KDa ß 1,4-galactose terminal glycoprotein (GP29) of LD activated GSK3ß through TLR4 to induce IL-12-mediated Nitric oxide (NO) production that resulted in effective parasite clearance from macrophages. GSK3ß activation abrogated both CREB phosphorylation and IL-10 production. Two subcutaneous injections of GP29 at fortnightly intervals in a 4-week infected mouse model of LD resulted in a dominant IL-12-mediated NO production and 100% animals were protected against a subsequent challenge with virulent LD parasites. Complete absence of GP29 mediated protection with down regulated NO and IL-12 production and dominant IL-10 production in presence of the GSK3ß inhibitor, Lithium chloride reiterated the role of GSK3ß in disease resolution in the murine model of visceral leishmaniasis.

Class II MHC/peptide interaction in Leishmania donovani infection: implications in vaccine design.

We show that Leishmania donovani-infected macrophages (MΦs) are capable of stimulating MHC class II (MHC-II)-restricted T cells at 6 h of infection. At 48 h, infected MΦs (I-MΦs) failed to stimulate MHC-II-restricted T cells but not MHC class I-restricted ones, in contrast to normal MΦs. Such I-MΦs could stimulate T cells at a higher Ag concentration, indicating that general Ag processing and trafficking of peptide-MHC-II complexes are not defective. Analysis of the kinetic parameters, like "kon" and "koff," showed that peptide-MHC-II complex formation is compromised in I-MΦs compared with normal MΦs. This indicates interference in loading of the cognate peptide to MHC-II, which may be due to the presence of a noncognate molecule. This notion received support from the finding that exposure of I-MΦs to low pH or treatment with 2-(1-adamantyl)-ethanol, a molecule that favors peptide exchange, led to T cell activation. When treated with 2-(1-adamantyl)-ethanol, splenocytes from 8 wk-infected BALB/c mice showed significantly higher antileishmanial T cell expansion in vitro compared with untreated controls. Hence, it is tempting to speculate that high, but not low, concentrations of cognate peptide may favor peptide exchange in I-MΦs, leading to expansion of the antileishmanial T cell repertoire. The results suggest that a high Ag dose may overcome compromised T cell responses in visceral leishmaniasis, and this has an important implication in therapeutic vaccine design.

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa ß 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-κB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-κB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88-IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication.

Heterologous priming-boosting with DNA and vaccinia virus expressing kinetoplastid membrane protein-11 induces potent cellular immune response and confers protection against infection with antimony resistant and sensitive strains of Leishmania (Leishmania) donovani.

BACKGROUND: Emergence of resistance against commonly available drugs poses a major threat in the treatment of visceral leishmaniasis (VL), particularly in the Indian subcontinent. Absence of any licensed vaccine against VL emphasizes the urgent need to develop an effective alternative vaccination strategy. METHODOLOGY: We developed a novel heterologous prime boost immunization strategy using kinetoplastid membrane protein-11 (KMP-11) DNA priming followed by boosting with recombinant vaccinia virus (rVV) expressing the same antigen. The efficacy of this vaccination regimen in a murine and hamster model of visceral leishmaniasis caused by both antimony resistant (Sb-R) and sensitive (Sb-S) Leishmania (L.) donovani is examined. RESULT: Heterologous prime-boost (KMP-11 DNA/rVV) vaccination was able to protect mice and hamsters from experimental VL induced by both Sb-S and Sb-R-L. (L.) donovani isolates. Parasite burden is kept significantly low in the vaccinated groups even after 60 days post-infection in hamsters, which are extremely susceptible to VL. Protection in mice is correlated with strong cellular and humoral immune responses. Generation of polyfunctional CD8(+) T cell was observed in vaccinated groups, which is one of the most important prerequisite for successful vaccination against VL. Protection was accompanied with generation of antigen specific CD4(+) and CD8(+) cells that produced effector cytokines such as IFN-γ, IL-2 and TNF-α. KMP-11-DNA/rVV vaccination also developed strong cytotoxic response and reversed T-cell impairment to induce antigen specific T cell proliferation. CONCLUSION: KMP-11 is a unique antigen with high epitope density. Heterologous prime boost vaccination activates CD4(+) and CD8(+) T-cell mediated immunity to confer resistance to VL. This immunization method also produces high quality T-cells secreting multiple effector cytokines thus enhancing durability of the immune response. Thus the vaccination regime as described in the present study could provide a potent strategy for future anti-leishmanial vaccine development.

Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1.

The molecular mechanism of antimony-resistant Leishmania donovani (Sb(R)LD)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Ms) has been investigated. This study showed that both promastigote and amastigote forms of Sb(R)LD, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in Sb(R)LD (KD Sb(R)LD), compromises the ability to induce the above effects. Infection of Ms with KD Sb(R)LD enhanced the sensitivity toward antimonials compared with infection with Sb(R)LD, and infection of BALB/c mice with KD Sb(R)LD caused significantly less organ parasite burden compared with infection induced by Sb(R)LD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by Sb(R)LD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and Sb(R)LD up-regulate MDR1 in M with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Ms derived from IL-10(-/-) mice are unable to show MDR1 up-regulation on infection with Sb(R)LD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs.

Hyperlipidemia offers protection against Leishmania donovani infection: role of membrane cholesterol.

Leishmania donovani (LD), the causative agent of visceral leishmaniasis (VL), extracts membrane cholesterol from macrophages and disrupts lipid rafts, leading to their inability to stimulate T cells. Restoration of membrane cholesterol by liposomal delivery corrects the above defects and offers protection in infected hamsters. To reinforce further the protective role of cholesterol in VL, mice were either provided a high-cholesterol (atherogenic) diet or underwent statin treatment. Subsequent LD infection showed that an atherogenic diet is associated with protection, whereas hypocholesterolemia due to statin treatment confers susceptibility to the infection. This observation was validated in apolipoprotein E knockout mice (AE) mice that displayed intrinsic hypercholesterolemia with hepatic granuloma, production of host-protective cytokines, and expansion of antileishmanial CD8(+)IFN- γ (+) and CD8(+)IFN- γ (+)TNF- α (+) T cells in contrast to the wild-type C57BL/6 (BL/6) mice when infected with LD. Normal macrophages from AE mice (N-AE-MΦ) showed 3-fold higher membrane cholesterol coupled with increased fluorescence anisotropy (FA) compared with wild-type macrophage (N-BL/6-MΦ). Characterization of in vitro LD-infected AE macrophage (LD-AE-MΦ) revealed intact raft architecture and ability to stimulate T cells, which were compromised in LD-BL/6-MΦ. This study clearly indicates that hypercholesterolemia, induced intrinsically or extrinsically, can control the pathogenesis of VL by modulating immune repertoire in favor of the host.

Asiaticoside induces tumour-necrosis-factor-α-mediated nitric oxide production to cure experimental visceral leishmaniasis caused by antimony-susceptible and -resistant Leishmania donovani strains.

OBJECTIVES: The aim of this study was to investigate and characterize the efficacy of asiaticoside in an experimental model of visceral leishmaniasis caused by antimony-susceptible (AG83) and -resistant (GE1F8R and K39) Leishmania donovani. METHODS: The effect of asiaticoside was evaluated by microscopic counting of intracellular amastigotes in cultured macrophages stained with Giemsa. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by estimation of splenic and liver parasite burdens in Leishman Donovan units. Cytokines were measured by real-time PCR and ELISA. Intracellular tumour necrosis factor-α (TNF-α) was measured by fluorescence-activated cell sorting. Nitric oxide was measured by the Griess reaction. RESULTS: Besides effectively inhibiting in vitro replication of the parasite within macrophages, asiaticoside treatment resulted in almost complete clearance of the liver and splenic parasite burden when administered at a dose of 5 mg/kg × 10 starting on day +30 of challenge with antimony-susceptible (AG83) and -resistant (GE1F8R and K39) L. donovani. Asiaticoside treatment was associated with a switch in the host from a Th2- to a Th1-type immune response accompanied by the induction of TNF-α-mediated nitric oxide production, all of which are important elements for macrophage function in antileishmanial defence mechanisms. CONCLUSIONS: These results suggest that oral therapy with asiaticoside shows promising antileishmanial efficacy in animals infected by antimony-susceptible (AG83) and -resistant (GE1F8R and K39) L. donovani.

Imipramine is an orally active drug against both antimony sensitive and resistant Leishmania donovani clinical isolates in experimental infection.

BACKGROUND: In an endeavor to find an orally active and affordable antileishmanial drug, we tested the efficacy of a cationic amphiphilic drug, imipramine, commonly used for the treatment of depression in humans. The only available orally active antileishmanial drug is miltefosine with long half life and teratogenic potential limits patient compliance. Thus there is a genuine need for an orally active antileishmanial drug. Previously it was shown that imipramine, a tricyclic antidepressant alters the protonmotive force in promastigotes, but its in vivo efficacy was not reported. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the drug is highly active against antimony sensitive and resistant Leishmania donovani in both promastigotes and intracellular amastigotes and in LD infected hamster model. The drug was found to decrease the mitochondrial transmembrane potential of Leishmania donovani (LD) promastigotes and purified amastigotes after 8 h of treatment, whereas miltefosine effected only a marginal change even after 24 h. The drug restores defective antigen presenting ability of the parasitized macrophages. The status of the host protective factors TNF α, IFN γ and iNOS activity increased with the concomitant decrease in IL 10 and TGF ß level in imipramine treated infected hamsters and evolution of matured sterile hepatic granuloma. The 10-day therapeutic window as a monotherapy, showing about 90% clearance of organ parasites in infected hamsters regardless of their SSG sensitivity. CONCLUSIONS: This study showed that imipramine possibly qualifies for a new use of an old drug and can be used as an effective orally active drug for the treatment of Kala-azar.

Characterisation of antimony-resistant Leishmania donovani isolates: biochemical and biophysical studies and interaction with host cells.

Recent clinical isolates of Leishmania donovani from the hyperendemic zone of Bihar were characterised in vitro in terms of their sensitivity towards sodium stibogluconate in a macrophage culture system. The resulting half maximal effective concentration (EC(50)) values were compared with those of known sensitive isolates. Fifteen of the isolates showed decreased sensitivity towards SSG with an average EC(50) of 25.7 ± 4.5 µg/ml pentavalent antimony (defined as antimony resistant), whereas nine showed considerable sensitivity with an average EC(50) of 4.6 ± 1.7 µg/ml (defined as antimony sensitive). Out of those nine, seven were recent clinical isolates and the remaining two were known sensitive isolates. Compared with the antimony sensitive, resistant isolates showed enhanced expression of thiol metabolising enzymes in varying degrees coupled with increased intracellular non-protein thiol content, decreased fluorescence anisotropy (inversely proportional with membrane fluidity) and over-expression of the terminal glycoconjugates (N-acetyl-d-galactosaminyl residue). Macrophages infected with resistant but not with sensitive showed up-regulation of the ATP Binding Cassette transporter multidrug resistance protein 1 and permeability glycoprotein, while the supernatant contained abundant IL-10. The above results reinforce the notion that antimony resistant parasites have undergone a number of biochemical and biophysical changes as part of their adaptation to ensure their survival in the host.

Complete protection against experimental visceral leishmaniasis with complete soluble antigen from attenuated Leishmania donovani promastigotes involves Th1-immunity and down-regulation of IL-10.

Compared with cutaneous leishmaniasis, vaccination against visceral leishmaniasis has received limited attention. Most available drugs are toxic, and relapse after cure remains a chronic problem. Growing limitations in available chemotherapeutic strategies due to emerging resistant strains and lack of an effective vaccine strategy against visceral leishmaniasis deepens the crisis. Complete soluble antigen (CSA), from a beta1-4 galactosyltransferase expressing attenuated Leishmania donovani parasite, induced protection against subsequent challenge and during active infections. CSA immunization was effective against both pentavalent antimony sensitive and resistant strains of L. donovani. Majority ( approximately 85%) of the immunized animals showed sterile protection. Resolution of the disease required the presence of T cells, and the recovered animals remained immune to re-challenge. Control of the parasites was dependent on type 1 CD4(+) helper cells, which evolved in the presence of IL-12 and activated macrophages through the production of IFN-gamma. Immunity was adoptively transferable and was dependent on both CD4(+) and CD8(+) cells. CSA immunization led to enhanced IFN-gamma production, while suppressing the IL-10 production. However, CSA immunization did not abrogate IL-4 production. Our results accentuate the need to establish a favorable cellular immunity while intervening with the development of Th2 cells during leishmania infection.

Sub-optimal dose of Sodium Antimony Gluconate (SAG)-diperoxovanadate combination clears organ parasites from BALB/c mice infected with antimony resistant Leishmania donovani by expanding antileishmanial T-cell repertoire and increasing IFN-gamma to IL-10 ratio.

We demonstrate that the combination of sub-optimal doses of Sodium Antimony Gluconate (SAG) and the diperoxovanadate compound K[VO(O2)2(H2O)], also designated as PV6, is highly effective in combating experimental infection of BALB/c mice with antimony resistant (Sb(R)) Leishmania donovani (LD) as evident from the significant reduction in organ parasite burden where SAG is essentially ineffective. Interestingly, such treatment also allowed clonal expansion of antileishmanial T-cells coupled with robust surge of IFN-c and concomitant decrease in IL-10 production. The splenocytes from the treated animals generated significantly higher amounts of IFN-c inducible parasiticidal effector molecules like superoxide and nitric oxide as compared to the infected group. Our study indicates that the combination of sub-optimal doses of SAG and PV6 may be beneficial for the treatment of SAG resistant visceral leishmaniasis patients.

Designing therapies against experimental visceral leishmaniasis by modulating the membrane fluidity of antigen-presenting cells.

The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-gamma signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.

KMP-11 DNA immunization significantly protects against L. donovani infection but requires exogenous IL-12 as an adjuvant for comparable protection against L. major.

As vaccine potential of cross-species protection by a candidate antigen is less explored, in this study we compared cross-specific protective efficacy of Kinetoplastid Membrane Protein-11 (KMP-11) as a DNA vaccine alone and in conjunction with exogenous IL-12 administration in experimental BALB/c model against two most widely prevalent forms of clinical diseases caused by Leishmania major (LM) and Leishmania donovani (LD). Whereas, KMP-11 DNA vaccination alone showed significant potential in terms of resolution of splenic and hepatic parasite burden against virulent LD challenge, it showed considerably less efficacy (<70% reduction) against virulent LM challenge in terms of presence of parasite in lymph node. Remarkably exogenous IL-12 administration in the form of IL-12 p35/p40 expression vectors or recombinant protein along with KMP-11 DNA had exactly opposing effect on protection against LM and LD. Exogenous IL-12 administration significantly increased residual LD-burden but enhanced the protective efficacy of KMP-11 DNA vaccine against LM compared to KMP-11 immunization alone. Elucidation of effector mechanism showed KMP-11 DNA induced protection against LD was associated with the generation of mixed Th1/Th2 response, while KMP-11/IL-12-induced comparable protection against LM was associated with high IgG2a titre indicative of a polarized Th1 response. Exogenous IL-12 administration resulted in robust gamma interferon (IFN-gamma) production and suppression of IL-4 from CD4+ T cell against both LM and LD. Nevertheless protective immune response was only compromised against LD infection where frequency of anti-KMP-11 CTL response was significantly reduced after exogenous IL-12 administration. Our study provides a comparative evaluation of effector mechanisms in the assessment of cross-specific protection by KMP-11 and KMP-11/IL-12 immunization against these two prevalent forms of leishmaniasis.

Hybrid cell vaccination resolves Leishmania donovani infection by eliciting a strong CD8+ cytotoxic T-lymphocyte response with concomitant suppression of interleukin-10 (IL-10) but not IL-4 or IL-13.

There is an acute dearth of therapeutic interventions against visceral leishmaniasis that is required to restore an established defective cell-mediated immune response. Hence, formulation of effective immunotherapy requires the use of dominant antigen(s) targeted to elicit a specific antiparasitic cellular immune response. We implemented hybrid cell vaccination therapy in Leishmania donovani-infected BALB/c mice by electrofusing dominant Leishmania antigen kinetoplastid membrane protein 11 (KMP-11)-transfected bone marrow-derived macrophages from BALB/c mice with allogeneic bone marrow-derived dendritic cells from C57BL/6 mice. Hybrid cell vaccine (HCV) cleared the splenic and hepatic parasite burden, eliciting KMP-11-specific major histocompatibility complex class I-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses. Moreover, splenic lymphocytes of HCV-treated mice not only showed the enhancement of gamma interferon but also marked an elevated expression of the Th2 cytokines interleukin-4 (IL-4) and IL-13 at both transcriptional and translational levels. On the other hand, IL-10 production from splenic T cells was markedly suppressed as a result of HCV therapy. CD8+ T-cell depletion completely abrogated HCV-mediated immunity and the anti-KMP-11 CTL response. Interestingly, CD8+ T-cell depletion completely abrogated HCV-induced immunity, resulting in a marked increase of IL-10 but not of IL-4 and IL-13. The present study reports the first implementation of HCV immunotherapy in an infectious disease model, establishing strong antigen-specific CTL generation as a correlate of HCV-mediated antileishmanial immunity that is reversed by in vivo CD8+ T-cell depletion of HCV-treated mice. Our findings might be extended to drug-nonresponsive visceral leishmaniasis patients, as well as against multiple infectious diseases with pathogen-specific immunodominant antigens.

Kinetoplastid membrane protein-11 DNA vaccination induces complete protection against both pentavalent antimonial-sensitive and -resistant strains of Leishmania donovani that correlates with inducible nitric oxide synthase activity and IL-4 generation: evidence for mixed Th1- and Th2-like responses in visceral leishmaniasis.

The emergence of an increasing number of Leishmania donovani strains resistant to pentavalent antimonials (SbV), the first line of treatment for visceral leishmaniasis worldwide, accounts for decreasing efficacy of chemotherapeutic interventions. A kinetoplastid membrane protein-11 (KMP-11)-encoding construct protected extremely susceptible golden hamsters from both pentavalent antimony responsive (AG83) and antimony resistant (GE1F8R) virulent L. donovani challenge. All the KMP-11 DNA vaccinated hamsters continued to survive beyond 8 mo postinfection, with the majority showing sterile protection. Vaccinated hamsters showed reversal of T cell anergy with functional IL-2 generation along with vigorous specific anti-KMP-11 CTL-like response. Cytokines known to influence Th1- and Th2-like immune responses hinted toward a complex immune modulation in the presence of a mixed Th1/Th2 response in conferring protection against visceral leishmaniasis. KMP-11 DNA vaccinated hamsters were protected by a surge in IFN-gamma, TNF-alpha, and IL-12 levels along with extreme down-regulation of IL-10. Surprisingly the prototype candidature of IL-4, known as a disease exacerbating cytokine, was found to have a positive correlation to protection. Contrary to some previous reports, inducible NO synthase was actively synthesized by macrophages of the protected hamsters with concomitant high levels of NO production. This is the first report of a vaccine conferring protection to both antimony responsive and resistant Leishmania strains reflecting several aspects of clinical visceral leishmaniasis.