Leishmania major

Due to its low toxicity and high absorbance in the range of 650 to 900 nm, indocyanine green (ICG) has garnered significant attention for its applications in photodynamic therapy (PDT) and photothermal therapy (PTT). However, its tendency to aggregate in aqueous environments limits its efficacy in both in vitro and in vivo applications. Encapsulating ICG in a biocompatible nanomicelle can improve its aqueous stability and photophysical properties. The present study investigated the synergistic effect of ICG-loaded nanomicelles upon irradiation by an 808-nm laser on Leishmania major (L. major) parasites. 

Initially, a nanomicelle comprised ICG was synthesized and characterized. Then, the temperature increase during irradiation and promastigote viability were evaluated in vitro. Subsequently, the prepared samples’ in vitro dark toxicity and phototoxicity were assessed via the MTS assay. Finally, the in vivo antileishmanial efficacy of the ICG-loaded nanomicelles formulation was investigated in BALB/c mice. 

The absorbance of ICG-loaded nanomicelles at 808 nm was more than 2 times greater than Free-ICG. Also, the prepared formulation exhibited a mean diameter of ~25 nm and a zeta potential of -2.3 ± 1 mV. The combination of ICG-loaded nanomicelles and 808 nm laser with a power density of 2.5 W cm−2 led to a significant reduction in the survival rate of promastigotes and lesion size of infected mice compared to control groups. 

The PDT/PTT mediated by ICG-loaded nanomicelles can be considered a promising and efficient therapeutic method for L. major, as it is inexpensive, safe, and easy to implement.

Background and purpose:

Cutaneous leishmaniasis poses significant health and socioeconomic challenges, making vaccine development a top priority, especially in endemic regions. Cysteine proteases, KMP-11, and HASPB proteins are promising candidates for leishmaniasis vaccine development owing to their immunogenic properties and capacity to provoke robust immune responses, as evidenced by different investigations. This study aimed to design a recombinant chimeric protein (MEV-Fc) vaccine using multi-epitopes from these Leishmania major proteins.

Experimental approach:

The antigens were subjected to immunoinformatic prediction and screening of HTL, CTL, and B-cell epitopes. The multi-epitope protein was designed with significantly high-scoring epitopes and suitable linkers. Natural adjuvants were then added to enhance immunogenicity. Vaccine potency was innovatively improved by covalently fusing human IgG1 Fc with multi-epitope protein. To investigate how the MEV-Fc vaccine interacts with Toll-like receptors, molecular docking, multi-scale normal mode analysis simulation, and computational immune simulation were employed to study humoral and cellular immune responses.

Findings/Results:

The results demonstrated the vaccine's antigenicity, stability, and nontoxicity. The structural validation confirmed the accuracy of the 3D models, indicating robust interactions with TLR2 and TLR4, with binding free energies of -1269.9 and -1128.7 (kcal/mol), respectively. Immune simulation results showed significant increases in IgM and IgG antibody levels following three vaccinations, along with enhanced activation of B cells, helper T cells, and cytotoxic T lymphocytes.

Conclusion and implications:

These findings provide novel insights for developing effective candidates for cutaneous leishmaniasis vaccines. However, laboratory experiments are necessary to evaluate its protective effects.

The promastigotes of Leishmania major (MRHO/IR/75/ER) were grown as previously described [1]. J774A.1 which is a murine macrophage Cell Line was purchased from Pasteur Institute of Iran and cultured using Roswell Park Memorial Institute medium (RPMI-1640) (Biowest, France) enriched with 10% heat-inactivated Fetal Bovine Serum (FBS) (Gibco, USA) with 2 mM L.glutamine (Sigma, Australia) and 1% penicillin/streptomycin (Gibco, USA). 7 × 106 macrophage cells were seeded in a T75 cell culture flask (SPL life sciences, South Korea). After 24 hours, 7 × 107 stationary phase promastigotes of L. major were added to the T75 flask containing J774A.1 macrophage cells. The video was recorded immediately and 3 hours after the infection of macrophages with L. major parasites under an inverted microscope (OPTIKA, Italy) with Samsung A13 mobile phone camera (Samsung, USA).

Current leishmaniasis treatment shows limitations such as side effects, low efficacy, and resistance. Thymus species are well-known as medicinal plants. This study evaluated antileishmanial efficacy and cytotoxicity of Thymus kotschyanus hydroalcoholic extract in in vitro experiments. Aerial parts of T. kotschyanus were extracted by maceration method with 70% ethanol. The hydroalcoholic extract was evaluated against Leishmania major promastigote and amastigote stages by directly counting the number of parasites in a macrophage model. Cytotoxicity of the extract was evaluated by MTT assay. The infectivity rate and amastigote numbers in macrophages were determined compared to control and glucantime (as a reference drug) groups. The crude extract inhibited the growth of promastigotes and amastigotes with the highest effect observed at 48 h. The IC50 values of T. kotschyanus on uninfected macrophages were 381.8 and 392.9 μg/mL at 48 and 72 hours, respectively. The extract of 300 and 350 μg/mL showed a significant reduction in the percentage of infected macrophages (p=0.042, p=0.001, respectively) and intra-macrophage amastigote numbers (p<0.05 and p=0.001, respectively) compared to control groups. The concentrations of 300 and 350 µg/mL were significantly more effective on the mean number of amastigote forms than 5 µg/mL glucantime for each point time (p<0.05, p<0.01, respectively); whereas these values had equal effects with 10 and 15 μg/mL glucantime. Thymus kotschyanus extract might be considered an effective and safe herbal material against L. major. Nevertheless, supplementary investigations will be necessary to recognize the plant as a new herbal therapy.

This research aimed to investigate the impact of ozone on Leishmania major. Leishmania major promastigotes were exposed to ozone flow at varying concentrations (5, 10, 20, 30, and 40 ppm) for different durations (5, 10, 20, 30, and 40 minutes). The DNA of the treated promastigotes was extracted to evaluate DNA fragmentation. Apoptosis induction in the treated L. major promastigotes was analyzed using a specific kit. Additionally, scanning electron microscopy was employed to observe ultrastructural changes in the treated promastigotes. The highest fatality rate (100%) occurred after 30 minutes of exposure to 40 ppm ozone gas, while the lowest fatality rate was 16.14% after 5 minutes of exposure to 5 ppm ozone gas. The DNA of the treated parasites was degraded, and significant morphological changes were observed in the body and flagella of the promastigotes. Induction of apoptosis was also noted. Ozone exhibits anti-Leishmania activity and induces apoptosis in Leishmania major. Furthermore, clear ultrastructural changes were observed in Leishmania major following ozone exposure. These findings suggest that ozone warrants further investigation as an anti-leishmanial agent.

Curcumin is an extract of rhizome turmeric (diferuloylmethane), with antioxidant, anti-inflammatory, antimicrobial, and anti-parasitic properties, which making it a potential candidate for the treatment of leishmaniasis. The aim of the presented study was to evaluate curcumin as possible candidate for treatment of cutaneous leishmaniasis. 

We investigated the physicochemical properties and anti-leishmanial effects of nanoliposomal curcumin (40, 80, and 120 μM) in Leishmania major (MRHO/IR/75/ER) infected BALB/c mice at the faculty of Veterinary Medicinem University of Tehran, Iran. For this aim, L. major promastigotes (MHROM/IR/75/ER) at stationary phase (2×106) were inoculated sub-cutaneously into the upper area of the tail in BALB/c mice (six groups, n= 10 per group). For evaluation of nanoliposomal curcumin, the zeta potential, particle size and stability of nanoliposomal curcumin was determined. Furthermore, the anti-leishmanial effects of nanoliposomal curcumin formulation on the lesion sizes was determined and the parasite burden in the leishmania induced lesion was performed using semi quantitative PCR.

Treatment of L. major infected BALB/c mice with nanoliposomal curcumin led to a reduction in the kinetic of the skin lesion size development. The semi quantitative PCR analysis of DNA extracted from the lesions showed reduction of parasite burden. The most effective treatment could be found in 80 μM nanoliposomal curcumin. Treatment with Glucantime, as a positive control, also showed a nearly similar effect compared to the effect of 80 μM nanoliposomal curcumin.

Nanoliposomal curcumin could be considered as a potential drug against cutaneous leishmaniasis caused by L. major in susceptible animal models.

Leishmaniasis is a vector-borne parasitic disease caused by an obligate intracellular protozoan. Despite the significant frequency of cutaneous leishmaniasis, there is still no appropriate prevention, control and treatment. In recent years, a few studies have been performed on the effects of garlic extract for the treatment of cutaneous leishmaniasis. Current drugs, such as glucantime, have several side effects and lead to direct cellular damage. If an herbal-based drug reduces Leishmania by augmenting the immune system, it has the potential to possess a wider margin of safety. This study aimed to investigate the effects of in vitro systematic review of the effects of garlic (Allium sativum) and its compounds on Leishmania major.

Five English databases (PubMed, Google Scholar, Embase, Scopus, and Web of Science) were searched until the end of December 2022. The syntax and specific tags have been used for each database. The studies with poor methodology, inadequate information, inappropriate analysis, and confusing presentation were excluded from the current study. The quality of articles was assessed by eleven questions developed by the authors and the data were extracted from the selected articles and discussed.

In this study, 198 articles were selected in the search step, of which five eligible articles were included for examination. The most commonly used solvent for the preparation of garlic extract was distilled water (60%), followed by methanol (40%). In these studies, the bulb of garlic is mostly used (80%). Cell type of J774 was used in only one study (20%), and murine peritoneal macrophages were used in the other studies (80%). The effect of garlic on Leishmania major was strong (80%). Studies have shown that garlic extract or its active compounds can increase cellular immune responses, which play an important role in inhibiting the Leishmania parasite. This is associated with the activation of macrophages and increased IFN-γ levels and NO production.

According to the studies, the effect of different combinations of garlic on the Leishmania parasite has been shown. However, the exact mechanism of the anti-Leishmanial effect of garlic has not been determined. Thus, this issue needs further investigation.

Leishmania spp. protozoa cause leishmaniasis by infecting macrophages. Long non-coding RNAs (lncRNAs), such as H19, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT), HOX Antisense Intergenic RNA (HOTAIR), and TNF and HNRNPL Related Immuno-regulatory lncRNA (THRIL), play a role in macrophage polarization and gene regulation. Additionally, leukocytes can synthesize and respond to melatonin, yet the regulatory role of melatonin in Leishmania major-infected macrophages is not well understood.

This study aimed to assess the impact of melatonin on the expression of lncRNAs like H19, MALAT, HOTAIR, and THRIL, as well as on nitric oxide synthase (NOS) activity in L. major-infected macrophages.

Leishmania major promastigotes and U937 cell lines were cultured. Macrophages were infected with the promastigotes and subsequently treated with melatonin at concentrations of 3, 10, 30, and 100 nM for durations of 4, 24, and 48 hours. The expression levels of the lncRNAs and NOS activity were measured using quantitative Polymerase Chain Reaction (q-PCR) and spectrophotometry, respectively.

Melatonin treatment (100 nM) significantly increased the expression of H19 compared to the control after 48 hours (P = 0.002). There was also a significant upregulation of MALAT and HOTAIR in macrophages treated with 3 nM melatonin compared to controls after 48 hours (P = 0.02 and P = 0.003, respectively). Additionally, THRIL expression significantly increased in the melatonin group (10 nM) compared to the control after 4 hours of treatment (P = 0.003). An increase in NOS activity was observed in the melatonin group (100 nM) compared to the control at 4 hours, 24 hours, and 48 hours (P = 0.034, P = 0.011, and P = 0.014, respectively).

The findings suggest that melatonin may enhance the expression of H19, THRIL, MALAT, and HOTAIR, as well as NOS activity in macrophages infected with L. major. The upregulation of these lncRNAs by melatonin could potentially improve the macrophages' ability to combat L. major infection.

Current medications especially the pentavalent antimonial compounds have been used as the first line treatment of cutaneous leishmaniasis (CL), but they have limitations due to serious side effects such as drug resistance, cardio and nephrotoxicity, and high costs. Hence, the demand to find more usable drugs is evident. Synthesis and devel-opment of natural, effective, biocompatible, and harmless compounds against Leishmania major is the principal priority of this study.

By electrospinning method, a new type of nanofiber were synthesized from royal jelly and propolis with dif-ferent ratios. Nanofibers were characterized by Scanning Electron Microscope (SEM), Transmission Electron Micros-copy (TEM), Thermogravimetric Analysis (TGA), Contact angle, and Fourier-transform infrared spectroscopy (FTIR). The Half-maximal inhibitory concentration (IC50), Half-maximal effective concentration (EC50) and the 50% cytotoxic concentration (CC50) for different concentrations of nanofibers were determined using quantitative calorimetric meth-ods. Inductively coupled plasma-optical emission spectrometry (ICP-OES) and flow cytometry were performed as com-plementary tests.

The results showed that the proposed formulas provide a new achievement that, despite the significant killing activity on L. major, has negligible cytotoxicity on the host cells. Royal jelly nanofibers have significantly shown the best 72 hours results (IC50= 35 μg/ml and EC50=16.4 μg/ml) and the least cytotoxicity.

This study presents a great challenge to introduce a new low-cost treatment method for CL, accelerate wound healing, and reduce scarring with minimal side effects and biocompatible materials. Royal jelly and propolis nanofibers significantly inhibit the growth of L. major in-vitro.

 Cutaneous leishmaniasis (CL) is an ulcerative skin disease caused by some species of the genus Leishmania. Evidence shows that Perovskia abrotanoides is an important herbal medicine against Leishmania. This study was conducted to investigate the killing effect of terpenoid-rich fractions on promastigotes of L. major (MRHO/IR/75/ER).

 The eluates of reverse phased medium pressure liquid chromatography (RP-MPLC) of the extract were subjected to thin-layer chromatography (TLC) and categorized into six final fractions. Primary proton nuclear magnetic resonance (H-NMR) spectroscopy confirmed fractions' nature. Fractions 4, 5, and 6 (F4, F5, F6) were identified as terpenoid-rich content. Two concentrations of 50 and 100 μg/ml were prepared to test leishmanicidal activity. Followed by treating promastigotes of L. major by the fractions in incubation times of 12, 24, and 48 hours, their viability was determined using a cell proliferation MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay.

 F4, F5, and F6 showed significant killing activity on promastigotes of L. major in a concentration-dependent manner. The viability of promastigotes was significantly reduced at a concentration of 100 μg/ml compared to 50 μg/ml (P-value <0.05). Also, over time a significant decreasing trend in the viability of promastigotes confirmed the time-dependent manner of the fractions (P-value <0.01). Furthermore, F5 had the highest leishmanicidal activity at the first incubation time compared with other fractions.

 Terpenoid-rich fractions of the P. abrotanoides have a leishmanicidal activity that depends on time and concentration. Among them, F5 has the highest potency that may contain potent terpenoid constituents.

 Many studies in the past have evaluated the role of immune system boosters in the treatment of leishmania major infection. Protein A (PA) is one of the structural components in peptidoglycan cell wall of gram-negative bacteria such as staphylococcus aurous which functions as a stimulator in the cellular immune system. The present study aims to evaluate the anti-inflammatory effect of PA on the recovery of leishmania major infection.

 This study was conducted on 24 female Balb/c-infected mice. The experimental group received PA at a dose of 60 mg/kg for four weeks. There was no intervention for the negative control group; the third group received the solvent of PA and sterile H2O; and the positive control group received Amphotericin B at a dose of 1 mg/kg body weight. At the end of the treatment period, a real-time polymerase chain reaction (PCR) assay was performed to determine parasitic burden, and the size of the lesions was measured by caliper with an accuracy of 0.01 mm.

 Results showed that PA did slightly decrease the wound spread and growth but not to an extent that can be considered statistically significant. Also, differences in cycle threshold (Ct) values between the treated group and the untreated group was not impressive.

 Although findings showed that PA isn't such a good candidate for leishmania treatment, it may still be suitable for therapies that use multiple drugs in combination to speed up the healing of leishmaniosis, an issue that merits evaluation in future studies.

Pathogen recognition receptors (PRRs), which play a crucial role in responding to pathogens, affect the function of mesenchymal stem cells (MSCs). One important group of PRRs is the toll-like receptors (TLRs). When PRRs are activated, they can alter the expression of specific surface markers, the ability of MSCs to differentiate, and the types of substances they secrete. These modifications in MSC function may have unexpected consequences for patients. In this study, we examined how Leishmania major (L. major) promastigotes affect the properties of MSCs. MSCs were isolated from adipose tissue and categorized into two groups: one group left untreated and the other group exposed to L. major. Giemsa staining was employed to accurately quantify the number of parasites that entered the cells. After 72 hours, real-time polymerase chain reaction was utilized to assess the expression of TLRs. Additionally, the flow cytometry technique was used to evaluate the expression of surface markers on the MSCs. Our results showed that MSCs can engulf parasites and increase the expression of TLR4 and TLR6. The pro-inflammatory cytokine increased, and the transforming growth factor-β decreased significantly. The parasite exposure increased reactive oxygen species production. Additionally, the percentage of cluster differentiation (CD) 73 decreased, and the mean fluorescent index of CD29 and CD73 was down-regulated by L. major. Exposure to parasites diminishes the immunomodulatory capacity of MSCs. This discovery holds significance for the application of MSCs in addressing parasite infections and underscores the need for additional research to enhance their therapeutic effectiveness.

Recent studies have shown an increasing number of patients with cutaneous leishmaniasis (CL) who do not respond to pentavalent antimonials as the first line of treatment for CL. Nanocarriers such as extracellular vesicles (EVs) are efficient vehicles that might be used as drug delivery systems for the treatment of diseases. Therefore, we aimed to isolate and characterize the EVs of Leishmania major, load them with Amphotericin B (AmB), and investigate the toxicity and efficacy of the prepared drug form.

The EVs of L. major were isolated, characterized, and loaded with amphotericin B (AmB), and the EVs-Amphotericin B (EVs-AmB) form was synthesized. Relevant in vitro and in vivo methods were performed to evaluate the toxicity and efficacy of EVs-AmB compared to the control.

The anti-leishmanial activity of the EVs-AmB showed a higher percentage inhibition (PI%) (P = 0.023) compared to the AmB at different concentrations and time points. Obtained data showed a significant increase in the lesion size and parasite load in the lesion, PBS, and EVs mice groups in comparison with EVs-AmB, AmB, and Glucantime groups (P < 0.05), EVs-AmB had a significant decrease in lesion sizes in comparison with AmB (P < 0.05). Results showed that EVs-AmB decreased its toxicity to the kidneys and liver (P < 0.05).

EVs-AmB improved the efficacy of AmB in mouse skin lesions and reduced hepatorenal toxicity. Furthermore, EVs could be a promising nanoplatform for the delivery of AmB in CL caused by L. major.

We aimed to assess the in vitro effects of the green synthesized silver nanoparticles (Ag NPs) via Thymus vulgaris (thyme) against Leishmania major infection.

We have prepared T. vulgalis silver nanoparticles (TSNPs) by adding thyme extract to the silver nitrate aqueous solution (0.2 mM), and evaluated their antileishmanial activity. The viability of L. major promastigotes was assessed in the presence of various concentrations of TSNPs by direct counting after 24 h. The MTT assay was used to identify the viability of promastigotes. The same procedures were assessed in uninfected macrophage cells. The apoptotic effects of nanoparticles on L. major promastigotes were determined by flow cytometry assay using annexin staining. To evaluate anti-amastigotes activity of TSNPs, light microscopic observation was used to determine the number of parasites within the macrophages in each well.

The effect of TSNPs on promastigotes and amastigotes of L. major was effective and had a reverse relationship with its concentration. TSNPs, inhibited the growth rate of L. major amastigotes and, the IC50 value of these nanoparticles was estimated 3.02 μg/mL (28 μM) after 72h. The results of flow cytometry showed that the toxic effects of TSNPs on promastigotes after 24 hours were statistically significant (P<0.05) and showed 69.51% of apoptosis.

TSNPs had an inhibitor effect on promastigote and amastigote forms of L. major in vitro. It might be considered as a candidate for the treatment of this infection.

The most commonly available drugs for leishmaniasis are pentavalent antimony compounds; whereas the recent studies showed various complications and limitations of these drugs. We aimed to green synthesized silver nanoparticles (AgNPs) and study the promising antileishmanial and synergic effects of green synthesized silver nanoparticles alone and combined with glucantime.

The precipitation technique was used to drop silver ions via an extract of Astragalus spinosus to AgNPs at Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University, Saudi Arabia in 2022. Then, its anti-amastigotes, caspase-3-like activity, triggering the nitric oxide (NO) as well as its cytotoxicity effects on macrophage cells as well as effects on leishmaniasis in BALB/c mice infected by L. major were measured.

The size of the AgNPs were ranging from 30-40 nm. The IC50 value for AgNPs, AgNPs+ meglumine antimoniate (MA), and MA was 59.3, 18.6, and 51.2 μg/mL, respectively. The determined FIC value for AgNPs and MA was found to be 0.31 and 0.36, respectively; demonstrating the synergistic potency of AgNPs when combined with MA. The diameter of CL lesions treated with various doses of AgNPs and AgNPs+MA notably (p<0.001) decreased. AgNPs, particularly at the concentrations of ½ IC50 and IC50, considerably triggered the caspase-3 activation. The calculated CC50 of AgNPs and MA was 612.5 and 789.8 μg/mL, respectively. Green synthesized AgNPs, especially in combination with MA had synergic antileishmanial effects and displayed a promising drug candidate for treating L. major CL.

We found satisfactory findings in the parasite reduction in both in vitro and animal models. Still, more studies are expected to explain the precise action mechanisms of AgNPs and their efficacy in humans.

The Leishmaniasis is a zoonosis disease with a global spread that occurs in two, cutaneous and visceral forms. In Iran, three species of Leishmania tropica, Leishmania major, and Leishmania infantum has been reported from different regions of the country. Nowadays, molecular methods are usually used for the diagnosis and identification of parasite species. Amplification of the ITS1 gene can also be used to differentiate symptomatic and asymptomatic visceral leishmaniasis as well as types of cutaneous leishmaniasis.

The main aim of our research was to diagnose and identify the common Leishmania species in Iran through ITS1 gene amplification and using the PCR-RFLP method.

First, L. major, L. tropica, and L. infantum parasites were proliferated in an RPMI culture medium, and DNA was extracted from these species separately then ITS1 gene of the parasite was amplified using the PCR-RFLP method.

The result of PCR-RFLP after enzymatic cutting indicated, L. tropica produced 4 fragments of 139, 76, 56, 20 bp bands; L. major showed two fragments of 165, 139 bp bands and L. infantum produced three fragments of 141, 91, 54 bp bands.

The results of the present study showed that the use of ITS1 gene and HaeIII enzyme in the PCR-RFLP method is efficient for identifying L. tropica, L. major, and L. infantum.

Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-β and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-β overexpression.

The miRNAs that target TGF-β -3´UTR were predicted and scored by bioinformatic tools. After cloning of TGF-β-3'UTR in psi-CHECK TM- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-β, as well as Nitric Oxide concentration was evaluated.

miR-27a received the highest score for targeting TGF-β in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-β-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-β expression and Nitric Oxide concentration were declined in L. major infected macrophages.

Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-β. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.

Current therapies for Leishmaniasis are associated with several side effects as well as drug resistance. Sensitivity and resistance of Leishmania major to Glutamine are referred to as those isolates which are responsive or non-responsive to one or two full courses of treatment by Glucantime systematically and/or intra-lesionally, respectively. In this study, We assessed a new approach to the investigation of the therapeutic efficiency of novel Carum copticum Nanoparticles against Leishmania major Promastigotes. First, the Carum copticum Nanoparticles were synthesized and liposomal Carum copticum was applied as a new therapeutic approach substituted for current therapy. In this experimental study, liposomal Carum copticum was prepared using the thin film hydration method and characterized based on encapsulation efficiency, size, and zeta potential. Carum copticum was successfully loaded into the liposome. The surface charge of the nanoparticle was neutral and the size of the nanoparticle was 176.5 nm. Liposomal Carum copticum beared spherical shape without any agglomeration. Results revealed that liposomal Carum copticum carried a significant effect, compared to the control sample, on parasite growth in both logarithmic and stationary phases. The result of this study signifies that the Carum copticum Nanoparticles induces a better and more tangible effect on the survival of Leishmania major promastigotes.

Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8+ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8+ T lymphocytes.

Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8+ T cell priming. Cytotoxic activity of the primed CD8+ T cell against L. major infected macrophages was measured using apoptosis assay.

α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8+ T cells augmented CD8+ T cells priming and proliferation more than in other study groups. These primed CD8+ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8+ T cells.

α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8+ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.

 The available drugs for the treatment of leishmaniasis are highly toxic and extremely expensive, with low efficiency; therefore, the development of effective therapeutic compounds is essential.

 The present study aimed to explore the antileishmanial effects of ethyl acetate extract, methanol extract, and fractions 1-4 (F1-F4) of Ferula tabasensis, alone or in combination with shark cartilage extract (ShCE), on L. major in vitro.

 In this study, ethyl acetate, methanol, and n-hexane extracts were extracted from the aerial roots of F. tabasensis by the maceration method. The silica gel column chromatography was used to separate n-hexane extracts at varying polarities (F1-F4 fractions). Subsequently, the effects of extracts and fractions against promastigotes were assessed by the parasite counting method microscopic inhibition test and MTT assay. Besides, their effects on the infected macrophage cells and the number of amastigotes were investigated. Cytotoxicity was evaluated in non-infected J774A.1 macrophage cells. Finally, apoptosis induction of promastigotes, including infected and non-infected macrophages, was evaluated.

 The results indicated the highly potent activity of F. tabasensis extracts and F1-F4 fractions, alone or in combination with ShCE, against L. major promastigotes and amastigotes in a dose-dependent manner (P < 0.05). The F1 fraction and methanol extract showed markedly higher toxicity compared to the other extracts and fractions, with 50% inhibitory concentrations (IC50/72h) of 2.4 ± 0.29 and 2.9 ± 0.55 µg/mL against promastigotes and 1.79 ± 0.27 µg/mL and 1.39 ± 0.27 µg/mL against amastigotes (P < 0.001). Moreover, they had a high selectivity index (SI) due to the low toxicity of macrophages (P < 0.0001). The results of flow cytometry indicated that the percentages of apoptotic promastigote cells in contact with IC50 concentrations of F1 and methanol extract alone after 72 h were 43.83 and 43.93%, as well as 78.4%, and 65.45% for their combination with ShCE, respectively.
Also, apoptosis of infected macrophages induced by F1 and methanol extracts was estimated at 68.5% and 83.7%, respectively.

 In this study, the F1 fraction and methanol extract of F. tabasensis showed potent efficacy against L. major, associated with low toxicity and apoptosis induction. Therefore, they can be promising therapeutic candidates in future animal and even human studies.