In silico and in vitro potentials of crocin and amphotericin B on Leishmania major: Multiple synergistic mechanisms of actions.

A significant barrier to optimal antileishmanial treatment is low efficacy and the emergence of drug resistance. Multiple approaches were used to monitor and assess crocin (a central component of saffron) mixed with amphotericin B (AmpB) potential in silico and in vitro consequences. The binding behavior of crocin and iNOS was the purpose of molecular docking. The results showed that crocin coupled with AmpB demonstrated a safe combination, extremely antileishmanial, suppressed Leishmania arginase absorption, and increased parasite death. This natural flower component is a robust antioxidant, significantly promoting the expression of the Th1-connected cytokines (IL12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (Elk-1, c-Fos, and STAT-1). In comparison, the expression of the Th2-associated phenotypes (IL-10, IL-4, and TGF-ß) was significantly reduced. The leishmanicidal effect of this combination was also mediated through programmed cell death (PCD), as confirmed by the manifestation of phosphatidylserine and cell cycle detention at the sub-GO/G1 phase. In conclusion, crocin with AmpB synergistically exerted in vitro antileishmanial action, generated nitric oxide and reactive oxygen species, modulated Th1, and Th2 phenotypes and transfer factors, enhanced PCD profile and arrested the cell cycle of Leishmania major promastigotes. The main action of crocin and AmpB involved wide-ranging mechanistic insights for conducting other clinical settings as promising drug candidates for cutaneous leishmaniasis. Therefore, this combination could be esteemed as a basis for a potential bioactive component and a logical source for leishmanicidal drug development against CL in future advanced clinical settings.

Antileishmanial potentials of azacitidine and along with meglumine antimoniate on Leishmania major: In silico prediction and in vitro analysis.

This study aimed to investigate the in vitro and in silico antileishmanial activity of azacitidine (AZA) on Leishmania major promastigotes and amastigotes. The in silico method was used to evaluate the possibility of the interaction of AZA into the binding pocket of inducible nitric oxide synthase (iNOS), a leading defensive oxidative metabolite. Following that, in vitro anti-promastigote, and anti-amastigote activity of AZA was determined using an MTT assay and a macrophage model, respectively. Cytotoxic effects of AZA and meglumine antimoniate (MA) were also assessed by MTT assay on murine macrophages. All experiments were performed in triplicate. The results showed that AZA interacted with Ser133, Gln134, and Lys13 amino acids of iNOS, and the molecular docking score was obtained at -241.053 kcal/mol. AZA in combination with MA significantly (P<0.001) inhibited the growth rate of nonclinical promastigote (IC50 247.6±7.3 µM) and 8.5-fold higher of clinical intramacrophage amastigote stage (29.8±5.3 µM), compared to the untreated group. A significant upsurge of Th1 subsets and transcription genes and a meaningful decline in Th2 cytokines subclasses at the equivalent concentrations of AZA and MA was observed (P<0.001). The apoptosis effect of AZA along with MA was significantly induced on L. major in a dose-dependent manner (P<0.001). The present study demonstrated that AZA possesses antileishmanial activity in in vitro and in silico models. However, AZA combined with MA was more effective than AZA alone in inhibiting the growth rate of promastigotes and amastigotes of L. major. This study indicates that AZA in combination with MA demonstrated a potent antileishmanial mechanism, promoting immune response and enhancing an immunomodulatory role toward the Th1 pathway. This experimental study is a basic study for applying more knowledge about the mechanisms of AZA along with MA in animal models in the future.

Nucleoside-Derived Metallohydrogel Induces Cell Death in Leishmania Parasites.

Self-assembled hydrogels by virtue of their unique 3D network and tunability have extensively been explored for bio-medical applications like tissue engineering, delivery and release of therapeutic agents, etc. Herein, we demonstrate for the first-time nucleoside-based biocompatible hydrogels with a remarkable leishmanicidal effect against both Leishmania major promastigotes and amastigotes and no cytotoxic effect on the macrophage cell line. In this work, a series of biocompatible hydrogels have been synthesized by silver ion-driven self-assembly of natural nucleoside and nucleotide-like cytidine and 5'-GMP. The supramolecular metallogel obtained from the assembly of cytidine and boronic acid is capable of inducing apoptotic-like cell death of protozoan parasite by causing damage to the membrane as well as DNA. These hydrogels could find promising applications in combating cutaneous leishmaniasis by topical treatment.

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model.

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC50) values were 21.66 µg/ml, 43.25 60 µg/ml, 52.58 µg/ml, and 70.38 µg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC50 for positive control (glucantime) was 27.03 µg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 µg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

Antileishmanial effects, cellular mechanisms, and cytotoxicity of Elettaria cardamomum essential oil against Leishmania major infection.

Leishmaniasis is an infectious disease with various clinical manifestations. We studied the therapeutic effects of Elettaria cardamomum essential oil (ECEO) against Leishmania major infection. In vitro effects of ECEO against L. major were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and macrophage assays. Nitric oxide (NO) production, infection inhibition in macrophages, and the apoptotic activity of ECEO in treated parasites were also measured. By calculating the 50% cytotoxic concentrations (CC50), we studied the cytotoxicity effects of ECEO on human macrophage cells (THP-1). The efficacy of ECEO for improving cutaneous leishmaniasis (CL) lesions in mice (BALB/c) was determined by evaluating the size of lesions and the number of amastigotes before and after four weeks of treatment. The effects of ECEO on liver and kidney function in the tested mice were also evaluated. ECEO dose-dependently (p<0.001) inhibited the viability and the mean number of promastigotes and amastigote forms of L. tropica. Four weeks of treatment with ECEO at the doses of 2.5 and 5 mg/kg/ day significantly (p<0.001) improved the CL lesions and reduced the number of parasites in the infected mice. ECEO significantly increased NO production, apoptosis induction, and infection rate in parasites. The CC50 value for ECEO and MA was 303.4 µg/mL and 835.2 µg/mL, respectively. In the mice receiving ECEO at the doses of 2.5 and 5 mg/kg/day for 28 days, no significant change was reported between the serum level of liver enzymes and kidney factors when compared with the control group. ECEO displayed promising efficacy in parasite reduction in vitro and in the animal model. ECEO can thus be used as an alternative medicine to treat CL.

Functionally distinct regions of the locus Leishmania major response 15 control IgE or IFNγ level in addition to skin lesions.

Leishmaniasis, a disease caused by parasites of Leishmania spp., endangers more than 1 billion people living in endemic countries and has three clinical forms: cutaneous, mucocutaneous, and visceral. Understanding of individual differences in susceptibility to infection and heterogeneity of its pathology is largely lacking. Different mouse strains show a broad and heterogeneous range of disease manifestations such as skin lesions, splenomegaly, hepatomegaly, and increased serum levels of immunoglobulin E and several cytokines. Genome-wide mapping of these strain differences detected more than 30 quantitative trait loci (QTLs) that control the response to Leishmania major. Some control different combinations of disease manifestations, but the nature of this heterogeneity is not yet clear. In this study, we analyzed the L. major response locus Lmr15 originally mapped in the strain CcS-9 which carries 12.5% of the genome of the resistant strain STS on the genetic background of the susceptible strain BALB/c. For this analysis, we used the advanced intercross line K3FV between the strains BALB/c and STS. We confirmed the previously detected loci Lmr15, Lmr18, Lmr24, and Lmr27 and performed genetic dissection of the effects of Lmr15 on chromosome 11. We prepared the interval-specific recombinant strains 6232HS1 and 6229FUD, carrying two STS-derived segments comprising the peak linkage of Lmr15 whose lengths were 6.32 and 17.4 Mbp, respectively, and analyzed their response to L. major infection. These experiments revealed at least two linked but functionally distinct chromosomal regions controlling IFNγ response and IgE response, respectively, in addition to the control of skin lesions. Bioinformatics and expression analysis identified the potential candidate gene Top3a. This finding further clarifies the genetic organization of factors relevant to understanding the differences in the individual risk of disease.

Evaluation of the Efficacy of Sodium Chloride Nanoparticles on the Vitality of Leishmania Major (in vitro).

Leishmaniasis is one of the most important zoonotic diseases transmitted to humans by sand flies (Phlebotomus spp). Leishmania major promastigote causes Cutaneous Leishmaniasis in humans. The study aimed to investigate the effectiveness of Sodium Chloride nanoparticles (NaCl NPs) on the vitality of Leishmania major promastigote compared with the standard dose of Pentostam under laboratory conditions. Various concentrations of 2, 4, 6, and 8 µg/ml of the NaCl NPs were prepared. These concentrations were tested in vitro on L. major growth by the culture of the parasite in the cell culture microplate. After the fourth day, a different concentration of NaCl NPs was added with three replicates for each concentration. Later, the numbers of promastigotes were counted daily using a Haemocytometer stained by Trypan blue solution stain duration of the study, which continued for four days. The results showed that the Growth Index (GI) rate of L. major promastigote was decreased with increasing NaCl NPs concentration. The Growth Index rates were 1.32×106, 1.31×106, 0.95×106, and 0.78×106 for the mentioned concentrations. These values were compared with the rate of the Pentostam group and control group, which were 1.09×106 and 3.43×106, respectively. The results revealed that the highest inhibition percentage was 92% for 8 µg/ml NaCl NPs after 96 hours, Pentostam group and control group, which were %86 and %0.00 for inhibition promastigote, respectively in the same period. The statistical analysis revealed a significant difference among concentrations at P≤0.05 compared with the Pentostam and control groups. The current study concluded that the NaCl NPs have an excellent biological effect in inhibiting L. major promastigote growth in vitro. These promising results paved the way for employing NaCl NPs to treat human cutaneous leishmaniasis.

[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.

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.

Immunopathological investigation of a gerbil model of cutaneous leishmaniasis.

Leishmaniasis, caused by Leishmania species (intracellular protozoans), is a chronic, systemic disease that causes skin (cutaneous) and internal organ infections (visceral). Its prevalence has increased in recent years. Leishmania species are considered important pathogens that affect public health. After infecting an individual, the pathogen disrupts the immune system, but, there are not enough studies on which immune mechanisms are affected. The aim of this study was to establish a Leishmania major infection model (the causative agent of cutaneous leishmaniasis) in gerbils (Meriones unguiculatus) and to investigate the immune response in this model by examining the expression of important inflammatory genes (IL-1ß, IL-2, IL-6, IFN-É£ and TNF-α). The presence of parasites was confirmed by microscopic examination of samples taken from the lesions and culture studies. The expression of inflammatory cytokine genes was significantly increased in infected gerbils. The changes indicated that both the Th1 and Th2 pathways are activated in cutaneous leishmaniasis infection. Hence, different immunopathological mechanisms should be evaluated in the pathogenesis of the disease.

Ethanolaminephosphate cytidylyltransferase is essential for survival, lipid homeostasis and stress tolerance in Leishmania major.

Glycerophospholipids including phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are vital components of biological membranes. Trypanosomatid parasites of the genus Leishmania can acquire PE and PC via de novo synthesis and the uptake/remodeling of host lipids. In this study, we investigated the ethanolaminephosphate cytidylyltransferase (EPCT) in Leishmania major, which is the causative agent for cutaneous leishmaniasis. EPCT is a key enzyme in the ethanolamine branch of the Kennedy pathway which is responsible for the de novo synthesis of PE. Our results demonstrate that L. major EPCT is a cytosolic protein capable of catalyzing the formation of CDP-ethanolamine from ethanolamine-phosphate and cytidine triphosphate. Genetic manipulation experiments indicate that EPCT is essential in both the promastigote and amastigote stages of L. major as the chromosomal null mutants cannot survive without the episomal expression of EPCT. This differs from our previous findings on the choline branch of the Kennedy pathway (responsible for PC synthesis) which is required only in promastigotes but not amastigotes. While episomal EPCT expression does not affect promastigote proliferation under normal conditions, it leads to reduced production of ethanolamine plasmalogen or plasmenylethanolamine, the dominant PE subtype in Leishmania. In addition, parasites with episomal EPCT exhibit heightened sensitivity to acidic pH and starvation stress, and significant reduction in virulence. In summary, our investigation demonstrates that proper regulation of EPCT expression is crucial for PE synthesis, stress response, and survival of Leishmania parasites throughout their life cycle.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection.

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms' expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-ß expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-ß signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-ß, signaling.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Localized Leishmania major infection disrupts systemic iron homeostasis that can be controlled by oral iron supplementation.

Leishmania parasites are heavily dependent on efficient iron acquisition from a tightly regulated host iron pool for survival and virulence. Prior studies uncovered multiple strategies adopted by the parasite to hijack the iron-regulatory network of macrophages. Despite these extensive studies with infected macrophages, there is limited knowledge of the effect of Leishmania infection on systemic iron homeostasis. This issue is particularly relevant for Leishmania major, which causes localized skin infection with minimal lymphatic spread. We show for the first time that L. major infection in the mouse footpad induced influx of iron at the site of infection through blood with simultaneous upregulation of transferrin receptor 1 and downregulation of phagolysosomal iron exporter Nramp1 expression in the footpad tissue. Interestingly, localized L. major infection had far-reaching effects beyond the infection site triggering anemia-like symptoms. This was evident from depleted physiological iron stores from the liver and bone marrow as well as reduced hemoglobin levels and deformed erythrocytes. The infected mice also developed splenomegaly with signs of splenic stress erythropoiesis as indicated by upregulation of several erythroid-related genes. These observations prompted us to provide oral iron supplementations to the L. major-infected mice, which resulted in a drastic reduction of the parasite load and restoration of iron homeostasis.

Detection of Leishmania major and Leishmania infantum in cats during an outbreak of cutaneous leishmaniosis in Southern Israel.

Prevalence of Leishmania spp. infection was studied in stray cats in two military bases in Southern Israel during a cutaneous leishmaniosis (CL) human outbreak caused by Leishmania major. Human CL cases increased from 0/100 in 2008 to 1.28/100 in 2022 in camp #1, and from 0.17/100 in 2008 to 6.4/100 in 2022, in camp #2. Eight out of 29 cats sampled were Leishmania-seropositive (28 %) and 7/29 (24 %) were internal transcribed spacer 1 (ITS1) PCR-positive, out of which four (14 %) were positive for L. major and three (10 %) for L. infantum. Five positive-cats had skin lesions including ulcers, alopecia and scabs, and five had eye lesions. This is the first report of L. major infection in cats in Israel and one of the first descriptions in felines worldwide. A larger cohort of cats and vector studies are necessary to determine if felids may act as reservoirs or sentinels of human L. major infection.

Delving in folate metabolism in the parasite Leishmania major through a chemogenomic screen and methotrexate selection.

Most of our understanding of folate metabolism in the parasite Leishmania is derived from studies of resistance to the antifolate methotrexate (MTX). A chemical mutagenesis screen of L. major Friedlin and selection for resistance to MTX led to twenty mutants with a 2- to 400-fold decrease in MTX susceptibility in comparison to wild-type cells. The genome sequence of the twenty mutants highlighted recurrent mutations (SNPs, gene deletion) in genes known to be involved in folate metabolism but also in novel genes. The most frequent events occurred at the level of the locus coding for the folate transporter FT1 and included gene deletion and gene conversion events, as well as single nucleotide changes. The role of some of these FT1 point mutations in MTX resistance was validated by gene editing. The gene DHFR-TS coding for the dihydrofolate reductase-thymidylate synthase was the second locus with the most mutations and gene editing confirmed a role in resistance for some of these. The pteridine reductase gene PTR1 was mutated in two mutants. The episomal overexpression of the mutated versions of this gene, but also of DHFR-TS, led to parasites several fold more resistant to MTX than those overexpressing the wild-type versions. Genes with no known link with folate metabolism and coding for a L-galactolactone oxidase or for a methyltransferase were mutated in specific mutants. Overexpression of the wild-type versions of these genes in the appropriate mutants reverted their resistance. Our Mut-seq approach provided a holistic view and a long list of candidate genes potentially involved in folate and antifolate metabolism in Leishmania.

Regiospecific Reduction of 4,6-Dinitrobenzimidazoles: Synthesis, Characterization, and Biological Evaluation.

The regiospecific reduction of 4,6-dinitrobenzimidazole derivatives leading to the corresponding 4-amino-6-nitrobenzimidazoles was studied. The identification of the formed product structures was accomplished by spectroscopic and X-ray diffraction data. The anticancer and antiparasitic activities of the synthesized compounds were examined, and promising activities against Toxoplasma gondii and Leishmania major parasites were discovered for certain 4,6-dinitrobenzimidazoles in addition to moderate anticancer activities of the 4-amino-6-nitrobenzimidazole derivatives against T. gondii cells. However, the tumor cell experiments revealed a promising sensitivity of p53-negative colon cancer cells to these compounds.

Antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized zinc nanoparticles alone and in combined with glucantime against Leishmania major infection.

BACKGROUND: We decided to investigate the antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized Zinc nanoparticles (ZnNPs) alone and combined with glucantime against Leishmania major infection. METHODS: The effect of green synthesized ZnNP on L. major amastigote was studied through macrophage cells. The mRNA expression level of iNOS and IFN-γ followed by the exposure of J774-A1 macrophage cells to ZnNPs was assessed by Real-time PCR. The Caspase-3-like activity of promastigotes exposed to ZnNPs was studied. Effects of ZnNPs alone and combined with glucantime (MA) were studied on cutaneous leishmaniasis in BALB/c mice. RESULTS: ZnNPs displayed the spherical shape with sizes ranging from 30 to 80 nm. The obtained IC50 values for ZnNPs, MA, and ZnNPs + MA were 43.2, 26.3, and 12.6 µg/mL, respectively; indicating the synergistic effects of ZnNPs in combination with MA. CL lesions had completely improved in the mice received with ZnNPs in combination with MA. The mRNA expression level of iNOS, TNF-α, and IFN-γ was dose-dependently (p < 0.01) upregulated; whereas it was downregulated in IL-10. ZnNPs markedly stimulated the caspase-3 activation with no significant toxicity on normal cells. CONCLUSION: Based on these in vitro and in vivo results, green synthesized ZnNPs, mainly along with MA, showed that has the potential to be introduced as a new drug for CL therapy. Triggering of NO production, and inhibition of infectivity rate are revealed as mechanisms of action ZnNPs on L. major. But, supplementary investigations are necessary to clear the efficacy and safety of these agents.

Leishmania major drives host phagocyte death and cell-to-cell transfer depending on intracellular pathogen proliferation rate.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

Tetrahydrobenzo[h]quinoline derivatives as a novel chemotype for dual antileishmanial-antimalarial activity graced with antitubercular activity: Design, synthesis and biological evaluation.

Derivatives with tetrahydrobenzo[h]quinoline chemotype were synthesized via one-pot reactions and evaluated for their antileishmanial, antimalarial and antitubercular activities. Based on a structure-guided approach, they were designed to possess antileishmanial activity through antifolate mechanism, via targeting Leishmania major pteridine reductase 1 (Lm-PTR1). The in vitro antipromastigote and antiamastigote activity are promising for all candidates and superior to the reference miltefosine, in a low or sub micromolar range of activity. Their antifolate mechanism was confirmed via the ability of folic and folinic acids to reverse the antileishmanial activity of these compounds, comparably to Lm-PTR1 inhibitor trimethoprim. Molecular dynamics simulations confirmed a stable and high potential binding of the most active candidates against leishmanial PTR1. For the antimalarial activity, most of the compounds exhibited promising antiplasmodial effect against P. berghei with suppression percentage of up to 97.78%. The most active compounds were further screened in vitro against the chloroquine resistant strain P. falciparum, (RKL9) and showed IC50 value range of 0.0198-0.096 µM, compared to IC50 value of 0.19420 µM for chloroquine sulphate. Molecular docking of the most active compounds against the wild-type and quadruple mutant pf DHFR-TS structures rationalized the in vitro antimalarial activity. Some candidates showed good antitubercular activity against sensitive Mycobacterium tuberculosis in a low micromolar range of MIC, compared to 0.875 µM of isoniazid. The top active ones were further tested against a multidrug-resistant strain (MDR) and extensively drug-resistant strain (XDR) of Mycobacterium tuberculosis. Interestingly, the in vitro cytotoxicity test of the best candidates displayed high selectivity indices emphasizing their safety on mammalian cells. Generally, this work introduces a fruitful matrix for new dual acting antileishmanial-antimalarial chemotype graced with antitubercular activity. This would help in tackling drug-resistance issues in treating some Neglected Tropical Diseases.