Lupeol stimulates iNOS, TNF-α, and IL-10 expression in the U937 cell line infected with old-world Leishmania donovani.

OBJECTIVE: Visceral leishmaniasis is a neglected tropical disease that can be lethal if not treated. The available medicines have severe side effects, such as toxicity and drug resistance. Various investigations are looking into new anti-leishmanial compounds from natural products that have little impact on host cells. Lupeol, a triterpenoid present in the flora of many edible plants, has been shown to have antimicrobial properties. The present study investigated the immunomodulatory effects of lupeol on U937 macrophages infected with Leishmania donovani, focusing on the expression of key cytokines and enzymes involved in the immune response. METHODS: U937 macrophages were infected with Leishmania donovani amastigotes and treated with varying concentrations of lupeol throughout three days. The expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured using real-time polymerase chain reaction (RT-PCR). A positive simulation of gene expression was estimated using ΔΔCT to assess relative expression. RESULTS: The results demonstrated that lupeol significantly upregulated iNOS and TNF-α expression, especially at higher concentrations, indicating enhanced pro-inflammatory and anti-leishmanial activity. Interestingly, IL-10 expression also increased, suggesting a complex immunomodulatory role of lupeol that involves both pro-inflammatory and anti-inflammatory pathways. Pearson correlation analysis revealed a strong association between iNOS and TNF-α (0.97692), as well as a moderate correlation between iNOS and IL-10 (0.51603). CONCLUSION: These findings suggest that lupeol may promote a balanced immune response, enhancing the body's ability to combat L. donovani while potentially mitigating excessive inflammation. Lupeol can potentially serve as a novel therapeutic agent against visceral leishmaniasis.

Detection of artemisinin effect on macrophage inducible nitric oxide gene expression in macrophage infected with Leishmania donovani.

Leishmaniosis is a parasitic infection spreads to humans by sand flies. Over 20 different species of Leishmania are responsible for the disease, which infect over 14 million people around the world. Chemotherapy is one of the most effective treatments for leishmaniosis, however it is restricted by the high cost and/or toxicity. In this study, the possible effect of artemisinin (ART) was detected on intracellular amastigotes of Iraqi strain of Leishmania donovani in ex vivo condition in U937 macrophage cell line. Gene expression of inducible nitric oxide synthase (iNOS) in U937 macrophage was investigated, before and after treatment with artemisinin. Kinetic result by real-time PCR demonstrated that the iNOS expression folding reached the maximum at concentration of 500 µM after 24 hours, at 750 µM after 48 hours and at 1000 µM after 72 hours, which was 56, 11, and 6, respectively. The copy number of iNOS gene expression was also significantly higher in treated infected U937 cells compared to both non-treated-infected cells and intact macrophages, under different concentration of ART along the three times of follow-up. Moreover, stained macrophages with fluorescent DAPI proved that the percentage of intracellular infective amastigotes was decreased to the minimum in treated U937 cells, in comparison to non-treated cells. The minimal amastigote-invasion percentage was recorded at 1000 µM, which was 26%, 27%, 21% compared to 61%, 87%, 75% in untreated cells after 24, 48, 72 hours respectively. These findings demonstrated ART positive efficacy against iNOS expression and this compound can be further studied as novel therapeutic rather than toxic available chemotherapies.

Artemisinin efficacy against old world Leishmania donovani: in vitro and ex vivo study.

Visceral leishmaniosis is one of the most fatal old-world neglected disease with estimated 90 thousand worldwide cases emerge each year. In Iraq, the cutaneous and visceral form are endemic but available chemotherapies are either toxic with diverse side effects, expensive available drugs or parasite resistant is arising. Artemisinin (ART) is a semi-synthetic compound which proved its effectiveness against protozoan parasites, such as malaria and Leishmania. In this study, the efficacy of different concentrations of pure artemisinin was screened in vitro against promastigotes and axenic amastigotes by MTT assay after 24, 48 and 27 hours follow up. In addition, the infectivity ability and number was investigated of intra-cellular Leishman bodies in treated murine peritoneal macrophages after 24 and 48 hours ART treatment. The results verified ART efficacy against the promastigotes and axenic amastigotes viability with IC50 measured after 24, 48- and 72-hours treatment. Infectivity percentage of murine macrophages and parasite burden were significantly reduced in treated cells. These findings indicate the leishmanicidal activity of ART against the Iraqi isolate of L. donovani and further in vivo study is recommended for assigning ART as a natural anti visceral leishmaniosis compound.

Endocytosis and Sphingolipid Scavenging in Leishmania mexicana Amastigotes.

Leishmania species are the causative agents of the leishmaniases, a spectrum of neglected tropical diseases. Amastigote stage parasites exist within macrophages and scavenge host factors for survival, for example, Leishmania species utilise host sphingolipid for synthesis of complex sphingolipid. In this study L. mexicana endocytosis was shown to be significantly upregulated in amastigotes, indicating that sphingolipid scavenging may be enhanced. However, inhibition of host sphingolipid biosynthesis had no significant effect on amastigote proliferation within a macrophage cell line. In addition, infection itself did not directly influence host biosynthesis. Notably, in contrast to L. major, L. mexicana amastigotes are indicated to possess a complete biosynthetic pathway suggesting that scavenged sphingolipids may be nonessential for proliferation. This suggested that Old and New World species differ in their interactions with the macrophage host. This will need to be considered when targeting the Leishmania sphingolipid biosynthetic pathway with novel therapeutics.

Studies on the antileishmanial properties of the antimicrobial peptides temporin A, B and 1Sa.

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector-borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite.

Exploring Leishmania major inositol phosphorylceramide synthase (LmjIPCS): insights into the ceramide binding domain.

The synthesis of set of ceramide analogues exploring hydrophobicity in the acyl chains and the degree and nature of hydroxylation is described. These have been assayed against the parasitic protozoan enzyme LmjIPCS. These studies showed that whilst the C-3 hydroxyl group was not essential for turnover it provided enhanced affinity. Reflecting the membrane bound nature of the enzyme a long (C(13)) hydrocarbon ceramide tail was necessary for both high affinity and turnover. Whilst the N-acyl chain also contributed to affinity, analogues lacking the amide linkage functioned as competitive inhibitors in both enzyme and cell-based assays. A model that accounts for this observation is proposed.

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase.

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V(max)=2.31 pmol min(-1)U(-1). Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.