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.

A comparative genomics approach reveals a local genetic signature of Leishmania tropica in Morocco.

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is an important health problem. Despite the high incidence of CL in the country, the genomic heterogeneity of these parasites is still incompletely understood. In this study, we sequenced the genomes of 14 Moroccan isolates of L. tropica collected from confirmed cases of CL to investigate their genomic heterogeneity. Comparative genomics analyses were conducted by applying the recently established Genome Instability Pipeline (GIP), which allowed us to conduct phylogenomic and principal components analyses (PCA), and to assess genomic variations at the levels of the karyotype, gene copy number, single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) variants. Read-depth analyses revealed a mostly disomic karyotype, with the exception of the stable tetrasomy of chromosome 31. In contrast, we identified important gene copy number variations across all isolates, which affect known virulence genes and thus were probably selected in the field. SNP-based cluster analysis of the 14 isolates revealed a core group of 12 strains that formed a tight cluster and shared 45.1 % (87 751) of SNPs, as well as two strains (M3015, Ltr_16) that clustered separately from each other and the core group, suggesting the circulation of genetically highly diverse strains in Morocco. Phylogenetic analysis, which compared our 14 L. tropica isolates against 40 published genomes of L. tropica from a diverse array of locations, confirmed the genetic difference of our Moroccan isolates from all other isolates examined. In conclusion, our results indicate potential regional variations in SNP profiles that may differentiate Moroccan L. tropica from other L. tropica strains circulating in endemic countries in the Middle East. Our report paves the way for future research with a larger number of strains that will allow correlation of diverse phenotypes (resistance to treatments, virulence) and origins (geography, host species, year of isolation) to defined genomic signals such as gene copy number variations or SNP profiles that may represent interesting biomarker candidates.

[Investigation of Cytotoxic and Antileishmanial Activity of Hybrid Silver Nanoparticle Complexes: Potential Drug Candidates against Leishmania Species]. / Hibrit Gümüs Nanoparçacik Komplekslerinin Sitotoksik ve Antilaysmanyal Aktivitesinin Arastirilmasi: Leishmania Türlerine Karsi Potansiyel Ilaç Adaylari.

In recent years, isolation of resistant Leishmania species to drugs in use has made it necessary to search alternative molecules that may be drug candidates. In this study, it was aimed to investigate the cytotoxic and in vitro antileishmanial activity of hybrid silver nanoparticle (AgNP) complexes. In this study, three types of nanoparticles (NPs), oxidized amylose-silver (OA-Ag) NPs, oxidized amylose-curcumin (OA-Cur) NPs and oxidized amylose-curcumin-silver (OA-CurAgNP) nanoparticles were synthesized. The cytotoxic activity of the synthesized nanoparticles was determined against L929 mouse fibroblasts and the in vitro antileishmanial activity was determined against Leishmania tropica, Leishmania infantum and Leishmania donovani isolates by the broth microdilution method. It was observed that the hybrid OA-CurAgNP complex obtained by combining curcumin and silver nanoparticles showed cytotoxic effects against L929 mouse fibroblasts at concentrations of 1074 µg/mL and above. IC50 values expressing the antileishmanial activity of the hybrid OA-CurAgNP complex against L.tropica, L.infantum and L.donovani isolates, were found to vary between 95-121 µg/mL, 202-330 µg/mL and 210-254 µg/mL, respectively. Resistance development has emerged as a major challenge in the treatment of leishmaniasis in recent times. Metallic nanoparticles are considered excellent candidates for medical applications due to their chemical and physical properties, as well as their prolonged circulation in the body. The current drugs used for leishmaniasis treatment are highly toxic, while nanoparticles offer advantages such as low toxicity and easy cellular uptake due to their nanoscale dimensions. The identification of strong efficacy in these particles may contribute scientific evidence for their potential use in leishmaniasis treatment. Therefore, the therapeutical value of OA-CurAgNP complex alone in combination with existing drugs should be examined.

ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Cocrystals of a coumarin derivative: an efficient approach towards anti-leishmanial cocrystals against MIL-resistant Leishmania tropica.

Leishmaniasis is a neglected parasitic tropical disease with numerous clinical manifestations. One of the causative agents of cutaneous leishmaniasis (CL) is Leishmania tropica (L. tropica) known for causing ulcerative lesions on the skin. The adverse effects of the recommended available drugs, such as amphotericin B and pentavalent antimonial, and the emergence of drug resistance in parasites, mean the search for new safe and effective anti-leishmanial agents is crucial. Miltefosine (MIL) was the first recommended oral medication, but its use is now limited because of the rapid emergence of resistance. Pharmaceutical cocrystallization is an effective method to improve the physicochemical and biological properties of active pharmaceutical ingredients (APIs). Herein, we describe the cocrystallization of coumarin-3-carboxylic acid (CU, 1a; 2-oxobenzopyrane-3-carboxylic acid, C10H6O4) with five coformers [2-amino-3-bromopyridine (1b), 2-amino-5-(trifluoromethyl)-pyridine (1c), 2-amino-6-methylpyridine (1d), p-aminobenzoic acid (1e) and amitrole (1f)] in a 1:1 stoichiometric ratio via the neat grinding method. The cocrystals 2-6 obtained were characterized via single-crystal X-ray diffraction, powder X-ray diffraction, differential scanning calorimetry and thermogravimetric analysis, as well as Fourier transform infrared spectroscopy. Non-covalent interactions, such as van der Waals, hydrogen bonding, C-H...π and π...π interactions contribute significantly towards the packing of a crystal structure and alter the physicochemical and biological activity of CU. In this research, newly synthesized cocrystals were evaluated for their anti-leishmanial activity against the MIL-resistant L. tropica and cytotoxicity against the 3T3 (normal fibroblast) cell line. Among the non-cytotoxic cocrystals synthesized (2-6), CU:1b (2, IC50 = 61.83 ± 0.59 µM), CU:1c (3, 125.7 ± 1.15 µM) and CU:1d (4, 48.71 ± 0.75 µM) appeared to be potent anti-leishmanial agents and showed several-fold more anti-leishmanial potential than the tested standard drug (MIL, IC50 = 169.55 ± 0.078 µM). The results indicate that cocrystals 2-4 are promising anti-leishmanial agents which require further exploration.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Metallic nanoparticles and treatment of cutaneous leishmaniasis: A systematic review.

BACKGROUND: Cutaneous leishmaniasis (LC) is an infectious vector-borne disease caused by parasites belonging to the genus Leishmania. Metallic nanoparticles (MNPs) have been investigated as alternatives for the treatment of LC owing to their small size and high surface area. Here, we aimed to evaluate the effect of MNPs in the treatment of LC through experimental, in vitro and in vivo investigations. METHODS: The databases used were MEDLINE/ PubMed, Scopus, Web of Science, Embase, and Science Direct. Manual searches of the reference lists of the included studies and grey literature were also performed. English language and experimental in vitro and in vivo studies using different Leishmania species, both related to MNP treatment, were included. This study was registered in PROSPERO (CRD42021248245). RESULTS: A total of 93 articles were included. Silver nanoparticles are the most studied MNPs, and L. tropica is the most studied species. Among the mechanisms of action of MNPs in vitro, we highlight the production of reactive oxygen species, direct contact of MNPs with the biomolecules of the parasite, and release of metal ions. CONCLUSION: MNPs may be considered a promising alternative for the treatment of LC, but further studies are needed to define their efficacy and safety.

In vitro evaluation of antileishmanial activity of Boswellia serrata essential oil nanoliposome.

BACKGROUND: Leishmaniasis poses a significant health risk. OBJECTIVES: This study aimed to evaluate the effects of Boswellia serrata (B. serrata) essential oil nanoliposomes on Leishmania tropica (L. tropica) in vitro. METHODS: A mixture of B. serrata essential oil, phosphatidylcholine and Tween 80 were used to prepare B. serrata essential oil nanoliposomes, followed by drying, hydration and size characterisation. The promastigotes of L. tropica were cultured in Roswell Park Memorial Institute medium (RPMI-1640) containing streptomycin, penicillin and fetal bovine serum. Different concentrations of B. serrata essential and nanoliposomes were tested for their antileishmanial properties by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide tests (MTT). RESULTS: Results of Dynamic Light Scattering (DLS) for B. serrata nanoliposomes indicate that they are successful at producing nanoliposomes with dimensions of 74.8 nm. At 1 µg/mL dose, B. serrata essence caused 17 ± 1.73% mortality, while B. serrata nanoliposomes induced 26 ± 1.15% mortality. B. serrata essence achieved a mortality of 55 ± 2.88% at 10 µg/mL, whereas B. serrata nanoliposomes demonstrated a mortality of 63.66±0.88% at 10 µg/mL. Furthermore, there was a significant difference between similar concentrations of B. serrata and B. serrata nanoliposomes. The LC50 of B. serrata essential oil is 7.26 µg/mL in the 95% confidence interval (12.13-5.25). The LC90 value of B. serrata essential oil is 129.37 µg/mL in the 95% confidence interval (50.07-852.58). The LC50 value of B. serrata nanoliposome is 4.20 µg/mL in the 95% confidence interval (6.13-3.10). LC90 value for B. serrata nanoliposome is calculated as 91.89 µg/mL in the 95% confidence interval (37.09-583.29). CONCLUSIONS: In vitro experiments have shown that B. serrata oil and the nanoliposome suppress the proliferation of L. tropica promastigotes, which suggests it may be a promising option for treating leishmaniasis.

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.

Epidemiological survey, molecular profiling and phylogenetic analysis of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cutaneous leishmaniasis (CL), an emerging vector-borne ailment in Khyber Pakhtunkhwa (KPK), Pakistan, exhibits diverse spread patterns and outbreaks. METHODS: To comprehend its epidemiology and identify parasite species, we conducted an active survey on suspected CL cases (n=8845) in KPK. RESULTS: Microscopy and internal transcribed spacer-1 PCR-restriction fragment length polymorphism (RFLP) molecular techniques detected Leishmania spp. in blood samples. Phylogenetic analysis gauged genetic affinities with other areas. District Bannu displayed the highest CL impact (14.58%), while Swat had the lowest impact (4.33%) among cases. Annual blood examination rate, parasite incidence and slide positivity rate were 4.96 per 1000 people, 0.0233 and 0.047%, respectively. CL infections were prevalent in 1- to 20-y-olds, with males (57.17%) more vulnerable than females (42.82%). Single lesions occurred in 43.73% of patients, while 31.2% people had two lesions, 17.31% had three lesions and 7.74% had more than three lesions. Most had sand-fly exposure but lacked preventive measures like repellents and bed nets. Leishmania tropica was confirmed via RFLP analysis in amplified samples. Phylogenetic analysis unveiled genetic parallels between L. tropica of KPK and isolates from China, Iran, Afghanistan, India, Syria and Morocco. CONCLUSIONS: Urgent comprehensive control measures are imperative. Early detection, targeted interventions and raising awareness of CL and sand-fly vectors are vital for reducing the disease's impact. International collaboration and monitoring are crucial to tackle Leishmania spp.'s genetic diversity and curtail its cross-border spread.

Epithelial homelessness: an atypical form of anoikis triggered by Leishmania interaction with epithelial cells.

Aim: Leishmaniasis is characterized by a spectrum of diseases with two main clinical forms, cutaneous and visceral, caused by Leishmania tropica and Leishmania donovani, respectively. Studying Leishmania's interaction with the epithelial barrier at the initial site of a bite is crucial to understanding the establishment of the disease. Materials & methods: To discern parasite-host epithelial interaction, we developed in vitro cellular models involving co-cultures of Leishmania and MDCK epithelial cells. Results: Both L. donovani-MDCK and L. tropica-MDCK co-culture models demonstrated a phenomenon known as atypical anoikis apoptosis, typically identified by distinctive 'flipping in' of cell membranes and disordered cytoskeletal frameworks. Conclusion: This study bridges the gap in the fundamental understanding of the intricate latticework involving vector-Leishmania-host and may inform drug development strategies.

Small parasites called Leishmania are passed to humans through the bites of sandflies. These parasites cause three deadly forms of disease: one that affects the organs, one that causes skin lesions and one that affects organ linings. This study looked at how Leishmania parasites behave when they enter through the skin. We found that when the parasites were in contact with cells, the cells changed their shape and lost contact with neighboring cells. This led to a type of cell death known as anoikis, a Greek term meaning 'homelessness'.

Entomological, parasitological and molecular investigations in a new focus of cutaneous leishmaniasis in Youssoufia region, Morocco.

BACKGROUND AND AIMS: Leishmaniasis is a neglected tropical infection caused by Leishmania parasite that affect human and animal. In Morocco, the cutaneous leishmaniasis has spread substantially to the new areas. The surveillance limited to active foci may underestimate the occurrence of cutaneous leishmaniasis (CL). This study aims to investigate the local transmission of CL in rural districts of Youssoufia province, central Morocco, as a potential focus of CL. METHODS: For this purpose, parasitological, molecular and entomological investigations were carried out in this area. Data collection concerns potential vectors and human cases. Thus, 402 patients were examined for suspected leishmaniasis lesions in three localities of the province of Youssoufia. In these same localities, 983 sand flies were collected by CDC light traps and sticky paper during one-night per month during 6 months. These sand flies were all identified morphologically using the Moroccan identification key. RESULTS: The results showed that among the 25 skin lesions detected in a population of 402 individuals, 18 were confirmed by kDNA nested PCR as CL positive patients, of which only 25% were positive by direct examination. Leishmania tropica and Leishmania major were identified as causative agents of CL in the study area. Direct parasitological examination showed a low sensitivity (27.78%), especially for L. major, although its specificity was evaluated at 100%. Regarding entomological results, both genera of the Moroccan sand fly were collected in the study area: Genus/Phlebotomus (75.28%) and Sergentomyia (24.72%). Phlebotomus (P) papatasi, the proven vector of L. major, was the most abundant species (33.98%), followed by Paralongicollum sergenti (22.58%), the confirmed vector of L. tropica; while Sergentomyia (S) minuta, P. longicuspis, S. fallax and P. kazeruni were collected with, respectively, 17.60%, 16.99%, 7.12% and 1.73%. CONCLUSION: This study constitutes the first report of CL in the study areas, as well as the coexistence of L. tropica and L. major in these rural localities. Local transmission of CL is highly probable, as indicated by the prevalence of the two proven vectors of L. major and L. tropica. To control the spread of this disease, our results suggest the use of highly sensitive molecular methods to detect CL cases in potential leishmaniasis foci, which will improve surveillance.

Molecular identification of Leishmania tropica in mammals occurring in human-inhabited areas of a cutaneous leishmaniasis endemic focus in North-West Pakistan.

Cutaneous Leishmaniasis is endemic in the tribal district of Khyber near the Pak-Afghan border and is caused by Leishmania tropica. In Pakistan, cutaneous leishmaniasis caused by L. tropica is considered anthroponotic and is thought to be maintained by a human-sand fly-human transmission cycle. Along with humans, other mammals may also be acting as reservoir hosts of leishmaniasis in the study area. To investigate the role of non-human mammals in the transmission of leishmaniasis, blood samples were collected from 245 animals from the CL endemic district of Khyber, Pakistan. Leishmania parasite in these samples was detected by amplifying the species-specific sequences in minicircle kinetoplast DNA, using PCR. L. tropica DNA was detected in 18 (7.35%) samples, comprising 11 cows (Bos taurus), 6 goats (Capra hircus), and 1 dog (Canus lupus familiaris). Only a single cow and dog had a leishmaniasis-like lesion, and the remaining positive samples were asymptomatic. None of the tested sheep (Ovis aries) and rat (Rattus rattus, Rattus norvegicus) was positive. The present study reports the first instance of molecular detection of L. tropica in domestic animals. Our study indicates that along with humans' cows, goats and dogs may also be playing an important role in the transmission of anthroponotic cutaneous leishmaniasis in district Khyber in particular and Pakistan in general.

Vaccinomics-based next-generation multi-epitope chimeric vaccine models prediction against Leishmania tropica - a hierarchical subtractive proteomics and immunoinformatics approach.

Leishmania tropica is a vector-borne parasitic protozoa that is the leading cause of leishmaniasis throughout the global tropics and subtropics. L. tropica is a multidrug-resistant parasite with a diverse set of serological, biochemical, and genomic features. There are currently no particular vaccines available to combat leishmaniasis. The present study prioritized potential vaccine candidate proteins of L. tropica using subtractive proteomics and vaccinomics approaches. These vaccine candidate proteins were downstream analyzed to predict B- and T-cell epitopes based on high antigenicity, non-allergenic, and non-toxic characteristics. The top-ranked overlapping MHC-I, MHC-II, and linear B-cell epitopes were prioritized for model vaccine designing. The lead epitopes were linked together by suitable linker sequences to design multi-epitope constructs. Immunogenic adjuvant sequences were incorporated at the N-terminus of the model vaccine constructs to enhance their immunological potential. Among different combinations of constructs, four vaccine designs were selected based on their physicochemical and immunological features. The tertiary structure models of the designed vaccine constructs were predicted and verified. The molecular docking and molecular dynamic (MD) simulation analyses indicated that the vaccine design V1 demonstrated robust and stable molecular interactions with toll-like receptor 4 (TLR4). The top-ranked vaccine construct model-IV demonstrated significant expressive capability in the E. coli expression system during in-silico restriction cloning analysis. The results of the present study are intriguing; nevertheless, experimental bioassays are required to validate the efficacy of the predicted model chimeric vaccine.

Comparative analysis of the severity and progression of cutaneous leishmaniasis caused by Leishmania tropica in untreated and glucantime-treated patients.

Millions of people worldwide are affected by cutaneous leishmaniasis (CL), a disease that has a significant impact on morbidity and mortality. Understanding the immune responses responsible for tissue damage or the process of lesion healing plays a pivotal role in shaping optimal treatment strategies. In this study, we investigated immunological phenotypes for three groups: glucantime treated (n = 30) and untreated (n = 30) CL patients infected with Leishmania tropica (L. tropica), and healthy controls (n = 20). T-lymphocytes (CD4+ and CD8+), and B lymphocytes (CD14+ and CD19+) were isolated using antibody-conjugated microbeads and magnetic field isolation to achieve high purity. A higher significant difference was observed between T-lymphocytes (CD4+ and CD8+), and B-lymphocytes (CD14+ and CD19+) cells in CL-infected groups before and after treatment (p < 0.0001). When compared, there was also a significant difference among T-lymphocytes (CD4+ and CD8+), B lymphocytes (CD14+ and CD19+) p < 0.0001, p < 0.0005, and p < 0.0007, respectively between CL-infected individuals (before and after treatment) to controls. Our findings suggest that an increased proportion of these cells seen in treated patients may mediate healing, while it is also possible that they may contribute to tissue injury. Understanding the immune system and lesion size of CL can help develop immunotherapies and comprehend the evolution of this parasitic disease.

Thymoquinone Effect on Leishmania tropica/infantum and Leishmania-Infected Macrophages.

INTRODUCTION: Leishmania is a parasitic protozoan that tries to enter and amplify within macrophages. Macrophage cells are also immune defense cells that phagocyte many microbes like bacteria, fungi, as well as parasites like Leishmania spp. However, they are unable to kill this parasite that resides in the phagosomes of contaminated macrophages and multiplies in these macrophages, leading to the destruction of contaminated macrophages and the emerging of Leishmania wounds. A large number of current therapies for Leishmania cure have adverse effects, or parasites have developed resistance to some of these therapies, so a better therapy for the cure of Leishmania is required. Thymoquinone is one of the Nigella Sativa ingredients with numerous biological effects, such as antioxidant as well as antimicrobial effects on a variety of microbes, namely fungi, bacteria, as well as parasites like Leishmania spp. The impacts of Thymoquinone on Leishmania tropica and Leishmania infantum, as well as Leishmania-infected macrophages, were examined in this study. METHODS: The impact of various Thymoquinone dosages on L. tropica and L. infantum promastigotes and amastigotes was examined in vitro. Flow cytometry, as well as MTT, was also applied to examine the cytotoxic activity of Thymoquinone on promastigotes of L. tropica and L. infantum, as well as the incidence of apoptosis. The amastigote assay is also utilized to calculate the % of contaminated macrophages as well as the number of the present parasites in each macrophage. RESULTS: The percentage of macrophages contaminated with L. tropica and L. infantum amastigotes after medicating with 20 µM of Thymoquinone was 23% and 19%, respectively. Also, after medicating with 10 µM of Thymoquinone, these percentages were 32% and 31%, respectively. Flow cytometry indicated that Thymoquinone caused 33.9% and 31.4% apoptosis in L. tropica and L. infantum, respectively. As determined by the promastigote assay, the inhibitory concentration (IC50) of Thymoquinone for L. tropica and L. infantum was 9.49 µM and 12.66 µM, respectively. The results of the promastigote and amastigote assay show that with an increase in Thymoquinone doses, its ability to kill Leishmania parasites increases, too. CONCLUSION: According to the results of the study, Thymoquinone has a potentially lethal impact on L. tropica and L. infantum promastigotes as well as amastigotes (within leishmania contaminated macrophages).

Case Report: Extensive Facial Cutaneous Leishmaniasis in a Neonate.

Cutaneous leishmaniasis (CL) is a skin infection caused by various species of the Leishmania parasite and is spread by the bite of an infected female sandfly. In southern Israel, CL caused by Leishmania major is endemic. Cutaneous leishmaniasis is considered a self-limiting disease, characterized by progressive, long-lasting nodulo-ulcerative skin lesions, which usually resolve in several months to years, and leads to scarring, cosmetic disfigurement, and future stigmatization. Although CL is a common disease among children, reports of CL in children younger than 1 year are rare. We present a case of extensive facial CL in an infant whose initial lesions appeared only 25 days after birth. The patient was treated with intravenous liposomal amphotericin B. Two months later, marked improvement was seen, with complete resolution of the inflammation and atrophic scar formation. To our knowledge, this is the earliest age of CL published to date.

[Investigation of Leishmania RNA Virus 2 in Leishmania major and Leishmania tropica Strains Isolated from Cutaneous Leishmaniasis Patients in Türkiye]. / Türkiye'deki Kutanöz Leysmanyazis Hastalarindan Izole Edilen Leishmania major ve Leishmania tropica Izolatlarinda Leishmania RNA Virüsü 2'nin Arastirilmasi.

Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus that is thought to contribute to the severe inflammatory response of the causative Leishmania parasite in the mammalian host by being present in many isolates of Leishmania spp. In our study, it was aimed to obtain data on the presence of Leishmania RNA Virus 2 (LRV2), which is thought to cause a change in the clinical course of leishmaniasis, in Leishmania major and Leishmania tropica isolates isolated from cutaneous leishmaniasis (CL) patients in Türkiye. Leishmania strains stored in liquid nitrogen tank by cryopreservation in Manisa Celal Bayar University Faculty of Medicine Parasite Bank were resuscitated under suitable conditions and cultivated in NNN and RPMI-1640 media. Then, the isolates were allowed to enter the logarithmic phase in a 26ºC incubator and DNA isolations were made using the "High Pure PCR Template Preparation Kit". Real-time polymerase chain reaction (Rt-PCR) melting analyzes were applied to the DNAs obtained by using primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region of Leishmania. After RNA isolation from promastigote suspension, cDNA synthesis was performed by reverse transcription. After gel electrophoresis with PCR amplification products, dsRNA band formation was evaluated in terms of LRV2 positivity under ultraviolet light. Among the 20 examined Leishmania spp. isolates (10 L.tropica and 10 L.major), four (three L.tropica, one L.major) were found to be positive for the presence of LRV2. Although the mechanism of LRV in recent studies has not been fully understood, it is known that it exacerbates the clinic of the disease and even has an effect on the formation of drug resistance by the parasite. It is important to obtain data on the presence of LRV in our country and to contribute to various clinical, drug development, prevalence studies, diagnosis and treatment of the disease in the future.

Leishmanicidal and immunomodulatory activities of the formononetin (a natural isoflavone) against Leishmania tropica.

OBJECTIVE: This work aimed to examine the leishmanicidal, cellular mechanisms and cytotoxicity effects of formononetin (FMN), a natural isoflavone, against Leishmania tropica. We used the MTT assay to determine the leishmanicidal effects of FMN against promastigotes and its cytotoxicity effects on J774-A1 macrophage cells. The Griess reaction assay and quantitative real-time PCR were used to determine the nitric oxide (NO) and the mRNA expression levels of IFN-γ and iNOS in infected J774-A1 macrophage cells. RESULTS: FMN significantly (P < 0.001) decreased the viability and number of promastigotes and amastigotes forms. The 50% inhibitory concentrations value for FMN and glucantime was 9.3 and 14.3 µM for promastigote and amastigote, respectively. We found that the macrophages exposed with FMN especially at concentrations of 1/2 IC50 and IC50 significantly activated the NO release and the mRNA expression levels of IFN-γ, iNOS. The findings of the current research showed the favorable antileishmanial effects formononetin, a natural isoflavone, against various stages of L. tropica through inhibition of infectivity rate of macrophage cells and triggering the NO production and cellular immunity. However, supplementary works are essential to evaluate the ability and safety of FMN in animal model before use in the clinical phase.

Antiprotozoal activity of auranofin on Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii: in vitro and ex vivo study.

BACKGROUND: Three obligate intracellular protozoan parasite species, which are responsible for significant morbidity and mortality and settle in macrophage cells, affect more than one-half of the world's population, namely, Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, which are causative agents of Chagas disease, leishmaniasis and toxoplasmosis, respectively. In the current study, it was aimed to investigate the in vitro and ex vivo antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii. METHODS: The in vitro drug efficacy (IC50) of auranofin was investigated by haemocytometry and the CellTiter-Glo assay methods and the ex vivo drug efficacy (IC50) by light microscopic examination of Giemsa-stained slides. Also, the cytotoxic activity (CC50) of auranofin was examined by the CellTiter-Glo assay. The selectivity index (SI) was calculated for auranofin. RESULTS: According to IC50, CC50 and SI data, auranofin did not exhibit cytotoxic activity on Vero cells, but exhibited antiprotozoal activity on epimastigotes and intracellular amastigotes of T. cruzi, promastigotes and intracellular amastigotes of L. tropica and intracellular tachyzoites of T. gondii (p<0.05). CONCLUSIONS: The detection antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii according to the IC50, CC50 and SI values is considered an important and promising development. This is significant because auranofin may be an effective alternative treatment for Chagas disease, leishmaniasis and toxoplasmosis in the future.