In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Preparation and characterization of artemether-loaded niosomes in Leishmania major-induced cutaneous leishmaniasis.

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Role of Macrophage PIST Protein in Regulating Leishmania major Infection.

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently emerged as a key player in regulating viral pathogenesis. Nevertheless, the involvement of PIST in parasitic infections remains unexplored. Leishmania parasites infiltrate their host macrophage cells through phagocytosis, where they subsequently multiply within the parasitophorous vacuole (PV). Host cell autophagy has been found to be important in regulating this parasite infection. Since PIST plays a pivotal role in triggering autophagy through the Beclin 1-PI3KC3 pathway, it becomes interesting to identify the status of PIST during Leishmania infection. We found that while macrophage cells are infected with Leishmania major (L. major), the expression of PIST protein remains unaltered; however, it traffics from the Golgi compartment to PV. Further, we identified that in L. major-infected macrophage cells, PIST associates with the autophagy regulatory protein Beclin 1 within the PVs; however, PIST does not interact with LC3. Reduction in PIST protein through siRNA silencing significantly increased parasite burden, whereas overexpression of PIST in macrophages restricted L. major infectivity. Together, our study reports that the macrophage PIST protein is essential in regulating L. major infectivity.

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Comparison of Intralesional Sodium Stibogluconate versus Intralesional Meglumine Antimoniate for the Treatment of Leishmania major Cutaneous Leishmaniasis.

Israel is endemic for Old-World cutaneous leishmaniasis. The most common species is Leishmania major. However, the available treatment options are limited. This study's objective was to compare the authors' experience with different antimony intralesional treatments of Leishmania major cutaneous leishmaniasis. A retrospective evaluation was undertaken for cases of Leishmania major cutaneous leishmaniasis treated by pentavalent antimony in a university-affiliated medical centre in Israel. The previous treatment of intralesional sodium stibogluconate (Pentostam®) was compared with the current treatment of meglumine antimoniate (Glucantime®). One hundred cases of cutaneous leishmaniasis were treated during the study period, of whom 33 were treated with intralesional sodium stibogluconate and 67 were treated with intralesional meglumine antimoniate. The patients were 78 males and 22 females, mean age 24 (range 10-67) and there was a total of 354 skin lesions. Within 3 months from treatment, 91% (30/33) of the intralesional sodium stibogluconate group and 88% (59/67) of the intralesional meglumine antimoniate group had complete healing of the cutaneous lesions after an average of 3 treatment cycles (non-statistically significant). In conclusion, the 2 different medications have the same efficacy and safety for treating cutaneous leishmaniasis. Pentavalent antimoniate intralesional infiltration treatment is safe, effective, and well tolerated with minimal side effects for Old-World cutaneous leishmaniasis.

Phylogenetic analysis and antimony resistance of Leishmania major isolated from humans and rodents.

Cutaneous Leishmaniasis (CL) is one of the world's neglected diseases which is caused by Leishmania spp. The aim of this study was to assess molecular profile and antimony resistance of Leishmania isolated from human and rodent hosts. Samples were collected from suspected CL patients referred to health centres and wild rodent's traps in Gonbad-e-Qabus region, north-eastern Iran. Smears were subjected to PCR-RFLP to identify Leishmania species. In addition, ITS1-PCR products were sequenced for phylogenetic analysis. Clinical isolates and rodent samples were subjected to MTT assay to determine IC50 values and in vitro susceptibilities. Expression levels of antimony resistance-related genes were determined in CL isolates. Out of 1,949 suspected patients with CL and 148 rodents, 1,704 (87.4%) and 6 (4.05%) were positive with direct smear, respectively. Digestion patterns of BusRI (HaeIII) endonuclease enzyme were similar to what expected for Leishmania major. Phylogenetic analysis revealed that the highest interspecies similarity was found between current L. major sequences with L. major obtained from Russia and Uzbekistan. Out of 20 L. major samples tested, 13 (65%) were resistant to meglumine antimoniate (MA) treatment, with an activity index (AI) exceeding 4. The remaining 7 samples (35%) responded to MA treatment and were classified as sensitive isolates, with a confirmed sensitive phenotype based on their AI values. The comparison expression analysis of three major antimony resistance-associated genes in unresponsive clinical isolates demonstrated significant fold changes for TDR1 (4.78-fold), AQP1 (1.3-fold), and γ-GCS (1.17-fold) genes (P < 0.05). Herein, we demonstrate genetic diversity and antimony resistance of L. major isolated from human and reservoir hosts in north-eastern Iran, which could be the basis for planning future control strategies.

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics.

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Genetic Diversity of Leishmania major Isolated from Different Dermal Lesions Using ITS2 Region.

BACKGROUND AND OBJECTIVE: Cutaneous leishmaniasis (CL) is still considered to be an uncontrolled endemic disease that spreads in many countries. The current study aimed to determine intra-species relationships of L. major using ITS2 sequencing. METHODS: The study was conducted from the beginning of March to the end of November 2022. All medical information regarding CL was collected from patients of Thi-Qar province who attended the Dermatology Department of Al-Hussein Teaching Hospital in Nasiriyah city. Seventy-three samples were selected for the molecular identification after confirming microscopy with Giemsa stain. In this study, the primers were designed using NCBI GenBank sequence database and Primer 3 plus primer design online software. RESULTS: The results recorded 21 (28.77%) positive samples of L. major using the internal transcribed spacer 2 region (ITS2) in ribosomal RNA gene. The local L. major IQN.1-IQN.10 were submitted to NCBI GenBank database with accession numbers OM069357.1-OM069366.1, respectively. The phylogenetic analysis revealed that local isolates of L. major showed a close relationship with NCBI-BLAST L. major Iran isolate (KU680848.1). CONCLUSION: ITS2-PCR is suitable for identifying Leishmania spp. and determining genetic diversity. A phylogenetic data analysis may provide an idea on the genetic homogeneity of local isolates and knowing the genetic origin of the dermal lesion. However, the local isolates showed genetic proximity to the KU680848.1 isolate. This signifies the possibility of infection prevalence from Iranian areas. In general, genetic variation of L. major isolates may give several clinical manifestations of the cutaneous lesion. Therefore, determination of the heterogeneity is important for detecting the infection origin, epidemiology, therapy, and control strategies.

A Geomedical Survey: Is There an Association Between Climatic Conditions and Leishmania Species Distribution in Iran During the Years 1999-2021?

PURPOSE: Iran is among the high-risk leishmaniasis regions in the world. WHO recommends the use of GIS as an ideal tool for healthcare authorities to predict the evolution of a disease, delimit the risk of outbreaks and identify critical areas. The aim of this research is to find the association between the main species of Leishmania (L. major, L. tropica, L. infantum) dispersion and climatic variables in Iran. METHODS: All molecular-based reports of leishmaniasis from Iran between 1999 and 2021 were gathered from reliable medical sources. Meteorological data (air and soil temperatures, annual rainfall and humidity) of the country along the study period were obtained from the Iranian Climatological Research Centre. The data concerning species distribution and climatic conditions during this period were moved to a base-map through raster layers using ArcGIS 10.4.1 software. The relationship between parasitological and climatic models was examined using ANOVA. RESULTS: High risk area maps, based on the cut-off thresholds, were generated for Leishmania major, L. tropica and L. infantum. According to the molecular-based reports, the L. major distribution was significantly related to all climatic variables, while L. tropica was merely related to rainfall and humidity, and the L. infantum distribution was significantly associated with rainfall, soil and air temperatures. CONCLUSION: The association between climatic conditions and Leishmania species distribution in Iran has been confirmed. Consequently, both, the relationship between climatic conditions and the geographical distribution of Leishmania species, and the use of GIS to better understand the spatial epidemiology of leishmaniasis, have been reaffirmed.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes.

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Adipose-Derived Stem Cells' Secretome Attenuates Lesion Size and Parasite Loading in Leishmaniasis Caused by Leishmania Major in Mice.

Background: Stem cell-derived secretome (SE) released into the extracellular space contributes to tissue repair. The present study aimed to investigate the impact of isolated secretome (SE) from adipose-derived mesenchymal stem cells (ASCs) on Leishmania major (L. major) lesions in BALB/c mice. Methods: This experimental study was conducted at Ahvaz University of Medical Sciences (Ahvaz, Iran) in 2021. Forty female BALB/c mice were infected with stationary phase promastigotes through intradermal injection in the bottom of their tail and randomly divided into four groups (n=10 per group). The mice were given SE (20 mg/mL), either alone or in combination with Glucantime (GC, 20 mg/mL/Kg), meglumine antimoniate (20 mg/mL/Kg) for the GC group, and phosphate-buffered saline (PBS) for the control group. After eight weeks, the lesion size, histopathology, the levels of Interleukin 10 (IL-10), and Interleukin 12 (IL-12) were assessed. For the comparison of values between groups, the parametric one-way ANOVA was used to assess statistical significance. Results: At the end of the experiment, the mice that received SE had smaller lesions (4.56±0.83 mm versus 3.62±0.59 mm, P=0.092), lower levels of IL-10 (66.5±9.7 pg/mL versus 285.4±25.2 pg/mL, P<0.001), and higher levels of IL-12 (152.2±14.2 pg/mL versus 24.2±4.4 pg/mL, P<0.001) than the control. Histopathology findings revealed that mice treated with SE had a lower parasite burden in lesions and spleen than the control group. Conclusion: The current study demonstrated that ADSC-derived SE could protect mice infected with L. major against leishmaniasis.

Geographical and Molecular Analysis of Haplotype Variations in Leishmania major Among Infected Iranian Phlebotomus papatasi.

PURPOSE: Leishmania major is main causative agent and Phlebotomus papatasi is only proven vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in Iran. Human leishmaniasis is mostly susceptible to climatic conditions and molecular variations of Leishmania parasites within sandflies. METHODS: L. major was analyzed based on geographical, environmental, climatic changes and haplotype variations within P. papatasi. Molecular tools and different geographical aspects were employed using Arc-GIS software for mapping the geographic distribution of samples and other statistics tests. Fragments of ITS-rDNA, k-DNA, and microsatellite genes of Leishmania were used for PCR, RFLP, sequencing, and phylogenetic analyses. RESULTS: Totally 81 out of 1083 female P. papatasi were detected with Leishmania parasites: 70 and five were L. major and L. turanica, respectively. Golestan and Fars provinces had the highest (13.64%) and lowest (4.55%) infection rates, respectively. The infection rate among female P. papatasi collected from gerbil burrows was significantly higher (15.15%) than animal shelters, yards, and inside houses (4.48%) (P < 0.0%). Microsatellite was more sensitive (22.72%) than k-DNA (18.8%) and ITS-rDNA (7.48%). More molecular variations of L. major were found in Isfahan province. CONCLUSIONS: Arc-GIS software and other statistics tests were employed to find Leishmania positive and haplotype variations among sand flies. Geographical situations, altitude, climate, precipitation, humidity, temperature, urbanization, migrations, regional divergences, deforestation, global warming, genome instability, ecology, and biology of the sand flies intrinsically, and the reservoir hosts and neighboring infected locations could be reasons for increasing or decreasing the rate of Leishmania infection and haplotype variations.

In vitro and In vivo Effects of Ethanolic Extract of Fumaria parviflora Lam. Embedded in Chitosan Nanoparticles Against Leishmania major.

INTRODUCTION: Fumaria has been traditionally used to treat skin damages due to anti-inflammatory properties. In the present study, we evaluated the effect of the ethanolic extract of Fumaria parviflora Lam. (F. parviflora) against Leishmania major (L. major) using chitosan biopolymer drug delivery system both In vitro and In vivo models. MATERIALS AND METHODS: The ethanolic extract of F. parviflora was analyzed by HPLC to determine its active ingredients content. The extract was then loaded on chitosan nanoparticles (CNPs). The parasite was treated with various concentrations of the ethanolic extract, CNPs and CNPs loaded with F. parviflora extract (CNPs@ F. parviflora). The size of lesions of treated mice were measured on a weekly basis. The parasite burden was evaluated 8 weeks after treatment. RESULTS: The HPLC analysis showed the presence of Fumaric acid at a high concentration. The percentage of the drug released from CNPs@ F. parviflora within 24 and 72 h were 65% and 90% respectively. The results showed that F. parviflora extract and CNPs@ F. parviflora caused 84% and 96% growth inhibition of L. major promastigotes as revealed by Neubauer chamber counting and MTT test respectively. The IC50 values of F. parviflora extract and CNPs@ F. parviflora were 450 and 68.4 µg/ml respectively. In amastigote assay, the best results showed in CNPs@ F. parviflora that only 2% of macrophages were infected with amastigotes. In vivo experiments for mice treated with F. parviflora and CNPs @ F. parviflora in comparison to control group showed a significant reduction (P < 0.05) in the mean diameter of the lesions (2.3 and 1.72 mm and 9.91 mm respectively). CONCLUSION: The ethanolic extract of F. parviflora both as standalone and loaded in CNPs showed promising inhibitory effects against L. major both upon In vitro and In vivo experimentation as well as therapeutic effects for wound healing in infected mice.

Investigating the Effect of Satureja khuzestanica Essential oil on MDR1 Gene Expression in Leishmania major.

BACKGROUND: Cutaneous leishmaniasis is among the neglected diseases in the world. Pentavalent antimonial compounds are considered the first-line treatment for this disease. However, using alternative natural products has received great attention due to the side effects of chemical drugs and drug resistance of the Leishmania parasite. The present study aims to investigate the effect of Satureja khuzestanica essential oil (SKEO) on MDR1 gene expression. METHODS: In this study, standard strains of Leishmania major promastigotes were exposed to 5, 10, 15, and 20 µg/ml of SKEO. MDR1 gene expression of parasites exposed to essential oil was evaluated using real-time PCR. GAPDH was employed as the housekeeping gene for internal control. RESULTS: Despite the increase, no statistically significant difference was observed in the relative expression of the MDR1 gene between the control group and the groups containing 5, 10, and 20 µg/ml of SKEO (P > 0.05). The relative expression of the MDR1 gene significantly increased in the group containing 15 µg/ml of essential oil compared to the control one (P < 0.05). CONCLUSION: This study showed that the use of essential oil of Satureja khuzestanica plant can have an increasing effect on the expression of MDR1 gene of Leishmania promastigotes, which is the best case if Satureja khuzestanica essential oil reduces the expression of MDR1 gene. So it seems that the use of essential oil of Satoria plant is effective in controlling Leishmania parasite, but its concentrations induce drug resistance. As a result, concentrations of essential oil should be used that have a controlling effect on the growth and proliferation of Leishmania parasite and also have the least effect on the induction of MDR1 gene expression.

Comparison of biosynthetic zinc oxide nanoparticle and glucantime cytotoxic effects on Leishmania major (MRHO/IR/75/ER).

Currently, zinc oxide (ZnO) particles are used in nanotechnology to destroy a wide range of microorganisms. Although pentavalent antimony compounds are used as antileishmanial drugs, they are associated with several limitations and side effects. Therefore, it is always desirable to try to find new and effective treatments. The aim of this research is to determine the antileishmanial effect of ZnO particles in comparison to the Antimoan Meglumine compound on promastigotes and amastigotes of Leishmania major (MRHO/IR/75/ER). After the extraction and purification of macrophages from the peritoneal cavity of C57BL/6 mice, L. major parasites were cultured in Roswell Park Memorial Institute-1640 culture medium containing fetal bovine serum (FBS) 10% and antibiotic. In this experimental study, the effect of different concentrations of nanoparticles was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method, in comparison to the glucantime on promastigotes, amastigotes and healthy macrophages in the culture medium. The amount of light absorption of the obtained color from the regeneration of tetrazolium salt to the product color of formazan by the parasite was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the IC50 value was calculated. IC50 after 24 h of incubation was calculated as IC50 = 358.6 µg/mL. The results showed, that the efficacy of ZnO nanoparticles was favorable and dose-dependent. The concentration of 500 µg/mL of ZnO nanoparticles induced 84.67% apoptosis after 72. Also, the toxicity of nanoparticles was less than the drug. Nanoparticles exert their cytotoxic effects by inducing apoptosis. They can be suitable candidates in the pharmaceutical industry in the future.

Designing of a new multi-epitope vaccine against Leishmania major using Leish-F1 epitopes: An In-silico study.

Cutaneous leishmaniasis (CL) is the most common form of the disease which can cause malignant lesions on the skin. Vaccination for the prevention and treatment of leishmaniasis can be the most effective way to combat this disease. In this study, we designed a novel multi-epitope vaccine against Leishmania major (L. major) using immunoinformatics tools to assess its efficacy in silico. Sequences of Leish-F1 protein (TSA, Leif, and LMSTI1) of L. major were taken from GenBank. The helper T (Th) and cytotoxic T (Tc) epitopes of the protein were predicted. The final multi-epitope consisted of 18 CTL epitopes joined by AAY linker. There were also nine HTL epitopes in the structure of the vaccine construct, joined by GPGPG linker. The profilin adjuvant (the toll-like receptor 11 agonist) was also added into the construct by AAY Linker. There were 613 residues in the structure of the vaccine construct. The multi-epitope vaccine candidate was stable and non-allergic. The data obtained from the binding of final multi-epitope vaccine-TLR11 residues (band lengths and weighted scores) unveiled the ligand and the receptor high score of binding affinity. Moreover, in silico assessment of the vaccine construct cloning achieved its suitable expression in E. coli host. Based on these results, the current multi-epitope vaccine prevents L. major infection in silico, while further confirmatory assessments are required.