Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

A mushroom derived 'carbohydrate-fraction' reinstates host-immunity and protects from Leishmania donovani infection.

The anti-leishmanial effect of the 'carbohydrate-fraction', isolated from an edible mushroom Astraeus hygrometricus, was evaluated against Leishmania donovani infection both in vitro and in vivo. Ahf-Car induced the expression of inducible nitric oxide synthase 2 (iNOS2) and pro-inflammatory cytokines like TNF-α and IL-12, with subsequent downregulation of the anti-inflammatory cytokines as TGF-ß and IL-10, in vitro and in vivo along with a remarkable increase in the expressions of IL-6, IL-1ß, IFN-γ and IRFs, IRF-7 and IRF-8 in vivo. Ahf-Car also reduced the parasite burden in the spleen and liver dose-dependently with a simultaneous proliferation of Ly6C+ cells in the bone marrow of Leishmania-infected experimental animals. It also increased the monocyte population dose-dependently and the expression of the myeloid transcription factor PU.1, in vivo, which presumably signifies the expansion of protective macrophages. Thus, Ahf-Car might be a potent anti-leishmanial lead with unique and effective adjuvant capacity.

Cytokine profile and nitric oxide levels in peritoneal macrophages of BALB/c mice exposed to the fucose-mannose ligand of Leishmania infantum combined with glycyrrhizin.

BACKGROUND: The fucose-mannose ligand (FML) of Leishmania infantum is a complex glycoprotein which does not elicit adequate immunogenicity in humans. In recent years, adjuvant compounds derived from plants have been used for improving the immunogenicity of vaccines. Glycyrrhizin (GL) is a natural triterpenoid saponin that has known immunomodulatory activities. In the present study, we investigated the effects of co-treatment with FML and GL on the production of cytokines and nitric oxide (NO) by macrophages, in vitro. METHODS: Lipopolysaccharide (LPS) stimulated murine peritoneal macrophages were treated with FML (5 µg/ml) of L. infantum and various concentrations of GL (1 µg/ml, 10 µg/ml and 20 µg/ml). After 48 h of treatment, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 were measured by sandwich ELISA and NO concentration by Griess reaction. RESULTS: Our results indicate that the treatment of activated macrophages with FML plus GL leads to enhanced production of NO, TNF-α and IL-12p70, and reduction of IL-10 levels in comparison with FML treatment alone. CONCLUSIONS: Therefore, we concluded that GL can improve the immunostimulatory effect of FML on macrophages and leads to their polarization towards an M1-like phenotype.

Leishmania infantum pyridoxal kinase evaluated in a recombinant protein and DNA vaccine to protects against visceral leishmaniasis.

Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.

Preventive as well as therapeutic significances of linoleic acid in the containment of Leishmania donovani infection.

People suffering from malnutrition show compromised levels of ω-6 fatty acid and malnutrition is frequently observed among visceral leishmaniasis (VL) patients as disease inflicts primarily the socioeconomic destitute communities. Dietary linoleic acid (LA, 18:2; ω-6 fatty acid) is the principal source of essential fatty acid and its derivatives i.e. eicosanoids possess immune-modulatory activities. However, its role in VL is not yet established. LA was measured in VL human subjects (serum) as well as in Leishmania(L.)donovani infected hamsters (serum and visceral organs). Organ-specific mRNA expressions of various enzymes of the LA metabolic pathway were measured in visceral organs of infected hamsters. Our findings showed a decrease in the concentrations of LA in the serum samples of VL patients, suggesting malnutrition among these patients. However, in L. donovani infected hamsters, its level was not altered in the early infection (15 days) and then increased at late infection (60 days). Importantly, the supplementation of LA restored the Th-1 type of immune response and significantly reduced the parasite load within infected macrophages in vitro. This protective response of LA was mediated through 5-lipoxygenase pathway not via the cyclooxygenase pathway. Preventive usage of LA to mϕ followed by L. donovani infection also showed the strengthening of Th-1 immune response and significantly fewer parasite loads. Our findings demonstrate the protective role of LA in the containment of the parasite load. Incorporating LA rich oils in daily food habits across highly inflicted regions may be a significant advancement towards the eradication of the disease.

Evaluation of in vitro antileishmanial efficacy of cyclosporin A and its non-immunosuppressive derivative, dihydrocyclosporin A.

BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Vitamins (A&D) and Isoprenoid (Chenodeoxycholic acid) molecules are accompanied by Th1 immunostimulatory response and therapeutic cure in vivo: possible antileishmanial drugs.

Investigation of immune modulatory anti-leishmanial molecules is now being strongly encouraged to overcome the immunosuppression manifested during visceral leishmaniasis (VL), resistance, toxicity and high cost associated with conventional therapeutics. In the present study, we explored the protective efficacy of vitamin D3, retinoic acid and isoprenoid chenodeoxycholic acid (CDCA) combinations against L. donovani infected BALB/c mice. We also probed the immune modulatory response (Th1 & Th2 cytokines) and infection dynamics following experimental infections with drug treated animals. Our results indicate that Vit.D3/RA and CDCA/RA combination treatment led to significant inhibition of parasite load on days 21 and 28 post treatment. Furthermore, there was a marked inhibition of Th2 type immune responses in IL-4, IL-5 and polarization of Th1 biased immunity along with upregulation of IL-1, IFN-γ, and TNF-α levels on day 28 post treatment. In addition, mice treated with Vit.D3/RA and CDCA/RA demonstrates here that splenic histological recovery against the virulent challenge of L. donovani by day 28 was comparable to control group. The conclusions derived from this study suggests that a combination of vitamin A, D3 and isoprenoids may have a potential immunomodulatory therapeutic role against leishmaniasis.

Cinnamomum cassia exhibits antileishmanial activity against Leishmania donovani infection in vitro and in vivo.

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.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis.

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

3M-052 as an adjuvant for a PLGA microparticle-based Leishmania donovani recombinant protein vaccine.

It is believed that an effective vaccine against leishmaniasis will require a T helper type 1 (TH 1) immune response. In this study, we investigated the adjuvanticity of the Toll-like receptor (TLR) 7/8 agonist 3M-052 in combination with the Leishmania donovani 36-kDa nucleoside hydrolase recombinant protein antigen (NH36). NH36 and 3M-052 were encapsulated in separate batches of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). The loading efficiency for NH36 was 83% and for 3M-052 was above 95%. In vitro stimulation of bone marrow-derived dendritic cells, measured by IL-12 secretion, demonstrated that 3M-052 (free or MP-formulated) had a concentration-dependent immunostimulatory effect with an optimum concentration of 2 µg/mL. In immunogenicity studies in BALB/c mice, MP-formulated NH36 and 3M-052 elicited the highest serum titers of TH 1-associated IgG2a and IgG2b antibodies and the highest frequency of IFNγ-producing splenocytes. No dose dependency was observed among MP/NH36/3M-052 groups over a dose range of 4-60 µg 3M-052 per injection. The ability of MP-formulated NH36 and 3M-052 to elicit a TH 1-biased immune response indicates the potential for PLGA MP-formulated 3M-052 to be used as an adjuvant for leishmaniasis vaccines. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1587-1594, 2018.

Homeopathic medicines cause Th1 predominance and induce spleen and megakaryocytes changes in BALB/c mice infected with Leishmania infantum.

The prevalence of Th1/Th2 response, spleen changes and megakaryocytes were investigated in BALB/c mice (n=138) infected with Leishmania infantum, and treated with Leishmania infantum 30× (10-30) biotherapy - BioLi30×. We performed controlled experiments using 8-to-12-week-old mice, infected with 5×107L. infantum promastigotes, divided into eight groups: G1 (healthy), G2 (infected with L. infantum), G3 (BioLi30× pre-treated), G4 (BioLi30× pre/post-treated), G5 (BioLi30× post-treated), G6 (Water 30× post-treated), G7 (Antimonium crudum 30× post-treated) and G8 (Glucantime® post-treated). G3-G7 groups were orally treated with their respective drugs diluted in filtered water (1:10), and G8 received Glucantime® (0.6mg/100µl of PBS), intraperitoneally. Spleen fragments were submitted to double blind histopathological evaluation and the number of megakaryocytes was counted. Besides, animals' serum was measured after 49days of infection, and cytokines (IFN-γ, IL-4, IL-10, IL-12), as well as the Th1/Th2 correlation (IFN-γ/IL-4 and IFN-γ/IL-10), were analyzed. Spleen histological parameters were classified as: healthy appearance (G1); discreet (G3-G7), moderate (G2) and moderate to severe (G8) white pulp hyperplasia; proliferation of megakaryocytes (G2-G8), and intense disruption (G2-G8). All groups, except for G7, showed higher percentages of megakaryocytes per field ranging from 87% to 15%, when compared to healthy animals (G1). Th1 predominance in IFN-γ/IL-4 ratio (comparing to G2) was detected in G4, G5, G6 and G7. Finally, pre/post (BioLi30x) and post-treatment (Antimonium crudum 30x) presented reduction of megakaryocytes/spleen changes due to immunomodulation animal process, controlling the infection process, probably by the Th1 cytokine predominance.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis.

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±16 nm, polydispersity index of 0.2±0.03, zeta potential of -27.4±5.7 mV, 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±1.4 µg/mL and 5.1±0.9 µg/mL, respectively. Following treatment in a murine model of VL, NLA demonstrated superior efficacy compared to artemisinin with a percentage inhibition of 82.4%±3.8% in the liver and 77.6%±5.5% in spleen at the highest dose of 20 mg/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.

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.

Nanotized Curcumin and Miltefosine, a Potential Combination for Treatment of Experimental Visceral Leishmaniasis.

Leishmaniasis chemotherapy remains very challenging due to high cost of the drug and its associated toxicity and drug resistance, which develops over a period of time. Combination therapies (CT) are now in use to treat many diseases, such as cancer and malaria, since it is more effective and affordable than monotherapy. CT are believed to represent a new explorable strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania In the present study, we investigated the effect of a combination of a traditional Indian medicine (ayurveda), a natural product curcumin and miltefosine, the only oral drug for visceral leishmaniasis (VL) using a Leishmania donovani-hamster model. We developed an oral nanoparticle-based formulation of curcumin. Nanoformulation of curcumin alone exhibited significant leishmanicidal activity both in vitro and in vivo In combination with miltefosine, it exhibited a synergistic effect on both promastigotes and amastigotes under in vitro conditions. The combination of these two agents also demonstrated increased in vivo leishmanicidal activity accompanied by increased production of toxic reactive oxygen/nitrogen metabolites and enhanced phagocytic activity. The combination also exhibited increased lymphocyte proliferation. The present study thus establishes the possible use of nanocurcumin as an adjunct to antileishmanial chemotherapy.

Immune Response and Protective Efficacy of a Heterologous DNA-Protein Immunization with Leishmania Superoxide Dismutase B1.

Growing evidence shows that antioxidant proteins of Leishmania could be used as vaccine candidates. In this study, we report the efficacy of Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-α or IL-2 by antigen-stimulated T cells. Eight weeks after L. major infection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γ production by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high dose L. major infection.

The leishmanicidal activity of oleuropein is selectively regulated through inflammation- and oxidative stress-related genes.

BACKGROUND: Much research effort has been focused on investigating new compounds derived from low-cost sources, such as natural products, for treating leishmaniasis. Oleuropein derived from numerous plants, particularly from the olive tree, Olea europaea L. (Oleaceae), is a biophenol with many biological activities. Our previous findings showed that oleuropein exhibits leishmanicidal effects against three Leishmania spp. in vitro, and minimizes the parasite burden in L. donovani-infected BALB/c mice. The aim of the present study is to investigate the possible mechanism(s) that mediate this leishmanicidal activity. METHODS: We determined the efficacy of oleuropein in elevating ROS and NO production in L. donovani-infected J774A.1 macrophages and in explanted splenocytes and hepatocytes obtained from L. donovani-infected BALB/c mice. We also assessed the expression of genes that are related to inflammation, T-cell polarization and antioxidant defense, in splenocytes. Finally, we determined the ratios of specific IgG2a/IgG1 antibodies and DTH reactions in L. donovani-infected BALB/c mice treated with oleuropein. RESULTS: Oleuropein was able to elevate ROS production in both in vitro and in vivo models of visceral leishmaniasis and raised NO production in ex vivo cultures of splenocytes and hepatocytes. The extensive oxidative stress found in oleuropein-treated mice was obviated by the upregulation of the host's antioxidant enzyme (mGCLC) and the simultaneous downregulation of the corresponding enzyme of the parasite (LdGCLC). Moreover, oleuropein was able to mount a significant Th1 polarization characterized by the expression of immune genes (IL-12ß, IL-10, TGF-ß1, IFN-γ) and transcription factors (Tbx21 and GATA3). Moreover, this immunomodulatory effect was also correlated with an inhibitory effect on IL-1ß gene expression, rather than with the expression of IL-1α, IL-1rn and TNF-α. Furthermore, oleuropein-treated BALB/c mice mounted a delayed-type hypersensitivity (DTH) response and an elevated Leishmania-specific IgG2a/IgG1 ratio that clearly demonstrated an in vivo protective mechanism. CONCLUSION: The ability of Oleuropein to promote a Th1 type immune response in L. donovani-infected BALB/c mice points towards the candidacy of this bioactive compound as an immunomodulatory agent that may complement therapeutic approaches to leishmaniasis.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis.

The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the BALB/c mice proved to be similar when comparing the F2 and F4 fractions with a known Th1 adjuvant (saponin), though animals vaccinated with saponin did present a slight to moderate inflammatory edema on their hind footpads. In conclusion, the F2 and F4 fractions appear to induce a Th1-type immune response and, in this context, they could be evaluated in association with other protective antigens against Leishmania, as well as in other disease models.

Protective effect of Croton caudatus Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo.

In the present state of overwhelming emergence of drug-unresponsive phenotypes of Leishmania donovani and persistent severe toxicity in conventional anti-leishmanial therapy, in search for novel leads, the aim of this study has been fixed to identify the active extract(s) of Croton caudatus Geisel. var. tomentosus Hook effective against the parasitic protozoans in vitro and in vivo. C. caudatus Geisel. is often used by Chakma and Hmar community, the local tribes of north-east India for medicinal and veterinary purposes. Among the five semi-purified extracts tested, C. caudatus leaves, extracted in hexane and subsequently semi-purified in a column packed with silica gel (70-130 µM; mesh size 60 A°) using ethyl acetate-hexane solvent (9:1), was found to be the most effective growth inhibitor (JDHex) against the Leishmania promastigotes and amastigotes. JDHex significantly altered the biochemical parameters (protein, lipid and carbohydrates) in promastigotes followed by the morphological changes, DNA condensation and subsequent apoptosis in L. donovani. In consequent steps, it has been also proved that JDHex reduced the replication of intracellular amastigotes with concomitant release of nitric oxide and pro-inflammatory cytokines, IL-12 and TNF-α in vitro. Significantly, the 50% inhibitory concentration of JDHex was estimated much lower against the intracellular amastigotes (2.5 µg/mL) in comparison to promastigotes (10 µg/mL). JDHex was also found efficient in reducing parasite burden in spleen and liver when treated in vivo and increased the intracellular IFN-γ and decreased the IL-10 in CD4+ T cells in splenocytes of orally treated animals. The results of this study support the importance in exploration of novel anti-leishmanial leads from C. caudatus Geisel. var. tomentosus Hook. against the L. donovani (MHOM/IN/83/AG83) infection. Partial chemical characterization of JDHex revealed the presence of terpenoids. However, the further chemical investigation of JDHex is warranted.