In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum.

OBJECTIVE: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infantum proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. METHODS: L. infantum promastigotes were treated with 50 and 100 µg/mL AEPa for 72 h and then antipromastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. RESULTS: Treatment with AEPa reduced the numbers of L. infantum promastigotes (50% inhibitory concentration (IC50) = 65.9 µg/mL; selectivity index (SI) = 22.1) and amastigotes (IC50 = 37.9 µg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2 µg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 µg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration >1000 µg/mL). CONCLUSION: AEPa presented antileishmanial activity against the promastigotes and amastigotes of L. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.

Exploring Leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using immunoinformatics approach.

Visceral leishmaniasis (VL) is a fatal form of leishmaniasis which affects 70 countries, worldwide. Increasing drug resistance, HIV co-infection, and poor health system require operative vaccination strategy to control the VL transmission dynamics. Therefore, a holistic approach is needed to generate T and B memory cells to mediate long-term immunity against VL infection. Consequently, immunoinformatics approach was applied to design Leishmania secretory protein based multi-epitope subunit vaccine construct consisting of B and T cell epitopes. Further, the physiochemical characterization was performed to check the aliphatic index, theoretical PI, molecular weight, and thermostable nature of vaccine construct. The allergenicity and antigenicity were also predicted to ensure the safety and immunogenic behavior of final vaccine construct. Moreover, homology modeling, followed by molecular docking and molecular dynamics simulation study was also performed to evaluate the binding affinity and stability of receptor (TLR-4) and ligand (vaccine protein) complex. This study warrants the experimental validation to ensure the immunogenicity and safety profile of presented vaccine construct which may be further helpful to control VL infection.

The role of vitamin D in the control of Leishmania infection.

Vitamin D has been described as an essential element for maintaining the homeostasis of mineral content in the body and bone architecture. However, our view of the physiological functions of this micronutrient has radically changed, owing to the vast number of properties, not calcium-related, mediated by its nuclear receptor. This receptor has been found in a variety of cells, including the immune cells, where many of the functions performed by vitamin D are related to inflammation. Although the effect of vitamin D has been widely studied in many diseases caused by viruses or bacteria, very little is known about its role in parasitic diseases, such as leishmaniasis, which is a vector-borne disease caused by different species of the intracellular parasite Leishmania spp. This disease occurs as a spectrum of different clinical syndromes, all of them characterized by a large amount of tissue damage, sometimes leading to necrosis. Owing to the involvement of vitamin D in inflammation and wound healing, its role in leishmaniasis must be relevant, and could be used as an adjuvant for the control of this parasitic disease, opening a possibility for a therapeutic application.

Investigations into an alternate approach to target mannose receptors on macrophages using 4-sulfated N-acetyl galactosamine more efficiently in comparison with mannose-decorated liposomes: an application in drug delivery.

In this study the potential of 2 different ligands, i.e., palmitoyl mannose (Man-Lip) and 4-SO(4)GalNAc (Sulf-Lip) to target resident macrophages was investigated after surface decoration of Amphotericin B (AmB) loaded liposomes. In the case of Sulf-Lip, the 4-SO(4)GalNAc was adsorbed through electrostatic interaction on cationic liposomes, which was confirmed by change in zeta potential from +48.2 ± 3.7 mV for Lip to +12.2 ± 1.3 mV for Sulf-Lip. The mean particle size of Sulf-Lip and Man-Lip was found to be 139.4 ± 7.4 nm and 147.4 ± 8.6 nm, respectively. Flow cytometric data reveal enhanced uptake of Sulf-Lip in both J774 and RAW cell lines in comparison with the uptake of Man-Lip. Intracellular localization studies indicate that the fluorescence intensity of Sulf-Lip was much higher in comparison with that of Man-Lip and Lip formulations. Sulf-Lip and Man-Lip showed significantly higher localization of AmB at all time points in comparison with Lip (P < 0.05) after intravenous (IV) administration. The studies provide evidence that 4-SO(4)GalNAc possesses a promising feature for targeting resident macrophages and its application in the conditions of leishmaniasis is in the offing. FROM THE CLINICAL EDITOR: This in vivo study compares two different ligands to deliver Amphotericin B l(AmB) loaded liposomes to resident macrophages. Targeted approaches showed significantly higher localization of AmB at all time points in comparison to non-targeted liposomes, and future applications in leishmaniasis are already under preparation.

Cationic solid lipid nanoparticles loaded by cysteine proteinase genes as a novel anti-leishmaniasis DNA vaccine delivery system: characterization and in vitro evaluations.

PURPOSE: Leishmaniasis is a major health problem in many tropical and sub-tropical countries and development of a safe and easily-available vaccine has high priority. Although several antigens potentially capable of inducing protective immunity have been studied, in the absence of pharmaceutical industry interest they have remained as fine publications only. Amongst them, Cathepsin L-like cysteine proteinases (CPs) have received considerable attention and type I and II CPs have been used in a heterologous prime-boost vaccination regime for experimental visceral leishmaniasis in dogs. Due to the promising results of the mentioned vaccination regime, we aimed to evaluate cationic solid lipid nanoparticles (cSLNs) for in vitro delivery of cpa, cpb and cpb(CTE) intended to be used as a cocktail DNA vaccine in our forthcoming studies. METHODS: cSLNs were formulated of cetyl palmitate, cholesterol, DOTAP and Tween 80 via melt emulsification method followed by high shear homogenization. Different formulations were prepared by anchoring pDNAs on the surface of cSLNs via charge interaction. The formulations were characterized according to their size and zeta potential as well as pDNA integrity and stability against DNase I treatment. Lipoplexes' cytotoxicity was investigated on COS-7 cells by MTT test. The effect of the DOTAP:pDNA ratio on protection ability and cytotoxicity was also studied. In vitro transfection efficiency was qualified by fluorescent microscopy and quantified using flow cytometry technique. RESULTS: cSLN-pDNA complexes were formulated with suitable size and zeta potential. Efficiency/cytotoxicity ratio of cSLN-pDNAs formulations was comparable to linear PEI-25KD-pDNAs polyplexes while exhibiting significantly lower cytotoxicity. CONCLUSION: Tested formulations were able to deliver immunogenic CP genes efficiently. This data proves the ability of this system as a promising DNA vaccine carrier for leishmaniasis to cover the main drawback of naked pDNA delivery that is rapid elimination from the circulation.

Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis.

The balance between T helper (Th) 1 and Th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized Th1 or Th2 responses are not sufficient to account for healing or nonhealing. Here we show that high arginase activity, a hallmark of nonhealing disease, is primarily expressed locally at the site of pathology. The high arginase activity causes local depletion of L-arginine, which impairs the capacity of T cells in the lesion to proliferate and to produce interferon-gamma, while T cells in the local draining lymph nodes respond normally. Healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. Moreover, competitive inhibition of arginase as well as supplementation with L-arginine restored T cell effector functions and reduced pathology and parasite growth at the site of lesions. These results demonstrate that in nonhealing leishmaniasis, arginase-induced L-arginine depletion results in impaired T cell responses. Our results identify a novel mechanism in leishmaniasis that contributes to the failure to heal persistent lesions and suggest new approaches to therapy.

6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: exploitation of GSK-3 for treating leishmaniasis.

Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3'-oxime (6-BIO), 6-Br-indirubin-3'acetoxime and 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) were the most potent inhibitors of Leishmania donovani promastigote and amastigote growth (half maximal inhibitory concentration (IC(50)) values < or =1.2 microM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3beta, for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), whilst 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death. Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s. This finding strongly supports that LdGSK-3s is: (i) the intracellular target of 5-Me-6-BIO, and (ii) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3 and apoptosis-like death. These effects were partially reversed in parasites over-expressing LdGSK-3s suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human GSK-3beta and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals.

The influence of dietary fish-oil supplementation on cutaneous Leishmania amazonensis infection in mice.

Dietary fish-oil (FO) supplementation has been shown to inhibit inflammation in various clinical disease states and to be beneficial in experimental models of inflammation and bacterial and plasmodial infection. In mice, FO increases macrophage production of tumour necrosis factor alpha (TNF). Production of TNF has been reported to be important in the resistance of mice against various Leishmania spp. We investigated whether dietary supplementation with FO protects susceptible Balb/c mice against infection with Leishmania amazonensis. No influence of the FO diet on the course of infection was observed, as evaluated by the increase in thickness of infected footpads over forty days. Lipopolysaccharide (LPS)-induced TNF production of peritoneal cells was however significantly increased in FO fed mice (P<0.01). When L. amazonensis was used as a stimulus, the in-vitro production of TNF by isolated peritoneal cells was minimal and did not differ between the various treatment groups. Addition of interferon gamma did not restore the effect of FO on TNF production capacity. We conclude that dietary supplementation with FO is of no benefit in Leishmaniasis in susceptible Balb/c mice, and that L. amazonensis is an insufficient trigger for TNF production in this model.

Leishmania tropica major: effect of paromomycin and pentamidine on polyamine levels in the skin of normal and infected mice.

The polyamine content of the skin of BALB/c and C3H mice was determined at intervals, after injecting Leishmania tropica major. In BALB/c mice, putrescine and spermidine levels increased three- to seven-fold; in C3H mice, spontaneous recovery occurred after 3 weeks, accompanied by a reduction in putrescine and spermidine levels. Ornithine decarboxylase activity was negligible in normal, uninfected skin of both BALB/c and C3H mice, but increased steadily during infection. Treatment with drugs that inhibit the growth of leishmanial amastigotes in the skin of mice also reduced polyamine levels and ornithine decarboxylase activity of previously infected skin. There was a close correlation between the therapeutic activity of the drugs and their effect on polyamine content and synthesis. The aminoglycoside paromomycin, which was chemotherapeutically more effective than pentamidine, also had a greater effect on polyamine levels. S-adenosyl-L-Methionine decarboxylase activity in the skin of BALB/c and C3H mice was only slightly affected by the parasites. Polyamine levels and ornithine decarboxylase activity could possibly serve as means for measuring the growth of leishmanial parasites in skin and other tissues and as a measure of the efficacy of anti-leishmanial chemotherapeutics.