Promastigote Growth Research Articles

CYP5122A1 encodes an essential sterol C4-methyl oxidase in Leishmania donovani and determines the antileishmanial activity of antifungal azoles

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes.

Identifying Rab2 Protein as a Key Interactor of Centrin1 Essential for Leishmania donovani Growth.

Previously, we have demonstrated that deletion of a growth-regulating gene (LdCen1) in the Leishmania donovani parasite (LdCen1-/-) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1-/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1-/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1-/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Anti-Leishmania amazonensis Activity of Morolic Acid, a Pentacyclic Triterpene with Effects on Innate Immune Response during Macrophage Infection.

Leishmaniasis is a group of infectious diseases transmitted to humans during vector bites and caused by protozoans of the genus Leishmania. Conventional therapies face challenges due to their serious side effects, prompting research into new anti-leishmania agents. In this context, we investigated the effectiveness of morolic acid, a pentacyclic triterpene, on L. amazonensis promastigotes and amastigotes. The present study employed the MTT assay, cytokine analysis using optEIATM kits, an H2DCFDA test, and nitric oxide dosage involving nitrite production and Griess reagent. Morolic acid inhibited promastigote and axenic amastigote growth forms at IC50 values of 1.13 µM and 2.74 µM, respectively. For cytotoxicity to macrophages and VERO cells, morolic acid obtained respective CC50 values of 68.61 µM and 82.94 µM. The compound causes damage to the parasite membrane, leading to cellular leakage. In the infection assay, there was a decrease in parasite load, resulting in a CI50 of 2.56 µM. This effect was associated with immunomodulatory activity, altering macrophage structural and cellular parasite elimination mechanisms. Morolic acid proved to be an effective and selective natural compound, making it a strong candidate for future in vivo studies in cutaneous leishmaniasis.

Activity of the Di-Substituted Urea-Derived Compound I-17 in Leishmania In Vitro Infections.

Protein synthesis has been a very rich target for developing drugs to control prokaryotic and eukaryotic pathogens. Despite the development of new drug formulations, treating human cutaneous and visceral Leishmaniasis still needs significant improvements due to the considerable side effects and low adherence associated with the current treatment regimen. In this work, we show that the di-substituted urea-derived compounds I-17 and 3m are effective in inhibiting the promastigote growth of different Leishmania species and reducing the macrophage intracellular load of amastigotes of the Leishmania (L.) amazonensis and L. major species, in addition to exhibiting low macrophage cytotoxicity. We also show a potential immunomodulatory effect of I-17 and 3m in infected macrophages, which exhibited increased expression of inducible Nitric Oxide Synthase (NOS2) and production of Nitric Oxide (NO). Our data indicate that I-17, 3m, and their analogs may be helpful in developing new drugs for treating leishmaniasis.

Approved drugs successfully repurposed against Leishmania based on machine learning predictions.

Drug repurposing is a promising approach towards the discovery of novel treatments against Neglected Tropical Diseases, such as Leishmaniases, presenting the advantage of reducing both costs and duration of the drug discovery process. In previous work, our group developed a Machine Learning pipeline for the repurposing of FDA-approved drugs against Leishmania parasites. The present study is focused on an in vitro validation of this approach by assessing the antileishmanial effects of 10 predicted drug candidates. First, we evaluated the drugs' activity against promastigotes from two strains of L. infantum and one of L. major, which caused distinct clinical manifestations, using an MTT assay. The standard anti-Leishmania drug Amphotericin B was used as a positive control. Five molecules demonstrated anti-Leishmania effects, out of which Acebutolol, Prilocaine and Phenylephrine are described herein for the first time. When tested on promastigote growth, Acebutolol displayed IC50 values ranging from 69.28 to 145.53 µg/mL. Prilocaine exhibited IC50 values between 33.10 and 45.81 µg/mL. Phenylephrine, on the other hand, presented IC50 values >200 µg/mL. The two remaining drugs, Dibucaine and Domperidone, exhibited significantly low IC50 values varying between 0.58 and 1.05 µg/mL, and 6.30 and 8.17 µg/mL, respectively. Both compounds were previously described as anti-Leishmania agents in vivo. All five compounds demonstrated no notable cytotoxic effects on THP-1-derived macrophages at the IC50 concentrations, allowing for their testing on the intracellular form of L. major and L. infantum parasites. Interestingly, all compounds exhibited antileishmanial activity on amastigotes with enhanced IC50 values compared to the corresponding promastigotes. Noticeably, Dibucaine and Domperidone displayed IC50 values of at most 1.99 µg/mL. Acebutolol, Prilocaine and Phenylephrine showed IC50 values ranging from 13.84 to 66.81 µg/mL. Our previously published Computer-Aided repositioning pipelines of FDA-approved drugs as antileishmanial agents identified Dibucaine and Domperidone as candidates in support of previous in vivo studies. This study consolidates such findings through the in vitro validation against 2 Leishmania species, highly prevalent in Africa and Middle East, and reveals Acebutolol, Prilocaine, and Phenylephrine as novel anti-Leishmania effectors, confirming the relevance of our approach and calling for further investigations.

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.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Monomethylsulochrin isolated from biomass extract of Aspergillus sp. against Leishmania amazonensis: In vitro biological evaluation and molecular docking.

Leishmaniasis represents a serious world health problem, with 1 billion people being exposed to infection and a broad spectrum of clinical manifestations with a potentially fatal outcome. Based on the limitations observed in the treatment of leishmaniasis, such as high cost, significant adverse effects, and the potential for drug resistance, the aim of the present study was to evaluate the leishmanicidal activity of the compounds pseurotin A and monomethylsulochrin isolated from the biomass extract of Aspergillus sp. The chromatographic profiles of the extract were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV), and the molecular identification of the pseurotin A and monomethylsulochrin were carried out by electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS) and nuclear magnetic resonance (NMR). Antileishmanial activity was assayed against promastigote and intracellular amastigote of Leishmania amazonensis. As a control, cytotoxicity assays were performed in non-infected BALB/c peritoneal macrophages. Ultrastructural alterations in parasites were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential were determined by flow cytometry. Only monomethylsulochrin inhibited the promastigote growth (IC50 18.04 ± 1.11 µM), with cytotoxicity to peritoneal macrophages (CC50 5.09 91.63 ± 1.28 µM). Activity against intracellular amastigote forms (IC50 5.09 ± 1.06 µM) revealed an increase in antileishmanial activity when compared with promastigotes. In addition to a statistically significant reduction in the evaluated infection parameters, monomethylsulochrin altered the ultrastructure of the promastigote forms with atypical vacuoles, electron-dense corpuscles in the cytoplasm, changes at the mitochondria outer membrane and abnormal disposition around the kinetoplast. It was showed that monomethylsulochrin leads to a decrease in the mitochondrial membrane potential (25.9%, p = 0.0286). Molecular modeling studies revealed that monomethylsulochrin can act as inhibitor of sterol 14-alpha-demethylase (CYP51), a therapeutic target for human trypanosomiasis and leishmaniasis. Assessed for its drug likeness, monomethylsulochrin follows the Lipinski Rule of five and Ghose, Veber, Egan, and Muegge criteria. Furthermore, monomethylsulochrin can be used as a reference in the development of novel and therapeutically useful antileishmanial agents.

An insight into differential protein abundance throughout Leishmania donovani promastigote growth and differentiation

Leishmania donovani causes anthroponotic visceral leishmaniasis, responsible for about 50,000 annual deaths worldwide. Current therapies have considerable side effects. Drug resistance has been reported and no vaccine is available nowadays. The development of undifferentiated promastigotes in the sand fly vector’s gut leads to the promastigote form that is highly infective to the mammalian host. Fully differentiated promastigotes play a crucial role in the initial stages of mammalian host infection before internalization in the host phagocytic cell. Therefore, the study of protein levels in the promastigote stage is relevant for disease control, and proteomics analysis is an ideal source of vaccine candidate discovery. This study aims to get insight into the protein levels during the differentiation process of promastigotes by 2DE-MALDI-TOF/TOF. This partial proteome analysis has led to the identification of 75 proteins increased in at least one of the L. donovani promastigote differentiation and growth phases. This study has revealed the differential abundance of said proteins during growth and differentiation. According to previous studies, some are directly involved in parasite survival or are immunostimulatory. The parasite survival–related proteins are ascorbate peroxidase; cystathionine β synthase; an elongation factor 1β paralog; elongation factor 2; endoribonuclease L-PSP; an iron superoxide dismutase paralog; GDP-mannose pyrophosphorylase; several heat shock proteins—HSP70, HSP83-17, mHSP70-rel, HSP110; methylthioadenosine phosphorylase; two thiol-dependent reductase 1 paralogs; transitional endoplasmic reticulum ATPase; and the AhpC thioredoxin paralog. The confirmed immunostimulatory proteins are the heat shock proteins, enolase, and protein kinase C receptor analog. The potential immunostimulatory molecules according to findings in patogenic bacteria are fructose-1,6-diphophate aldolase, dihydrolipoamide acetyltransferase, isocitrate dehydrogenase, pyruvate dehydrogenase E1α and E1β subunits, and triosephosphate isomerase. These proteins may become disease control candidates through future intra-vector control methods or vaccines.

Synthesis and characterization of zinc derivatized 3, 5-dihydroxy 4', 7-dimethoxyflavone and its anti leishmaniasis activity against Leishmania donovani.

This study reports the synthesis and characterization of zinc derivatized 3,5-dihydroxy 4', 7- dimethoxyflavone (DHDM-Zn) compound for the development of new antileishmanial agents. The interaction studies of DHDM with zinc were carried out by UV spectra and fluorescence spectra analysis. Characterization of the complex was further accomplished by multi-spectroscopic techniques such as FTIR, Raman, HRMS, NMR, FESEM-EDX. The morphological and topographical studies of synthesized DHDM-Zn were carried out using FESEM with EDX. Further, it was demonstrated that DHDM-Zn exhibited an excellent in vitro antagonistic effect against the promastigote form of L. donovani. In addition, the possible mechanisms of promastigote L. donovani cell death, by involvement of derivatized compound in arrest of the cell cycle in the G1 phase and residual cell count reduction were investigated. Promastigote growth kinetics performed in the presence of the derivatized compound revealed a slow growth rate. The combination of growth kinetics and cell cycle analysis, made it possible to interpret and classify the cause of leishmanial cell death accurately. These results support that zinc derivatized complex (DHDM-Zn) might work as a lead compound for designing and developing a new antileishmanial drug.

Chalcone-rich extracts from Lonchocarpus cultratus roots present in vitro leishmanicidal and immunomodulatory activity.

This study aimed to evaluate the in vitro anti-Leishmania activity of chalcone-rich three extracts (LDR, LHR and LMR) from Lonchocarpus cultratus (Vell.) A.M.G. Azevedo & H.C. Lima against L. amazonensis. Also, the immunomodulatory and antioxidant capacity was assessed. Successive extraction with hexane, dichloromethane and methanol were performed to obtain LHR, LDR and LMR extracts from L. cultratus roots, which were characterized by 1H NMR. Promastigotes, amastigotes and peritoneal macrophages were exposed to crescent concentrations of the three extracts, and after incubation, the inhibition rates were determined to both types of cells, and morphological analyses were performed on the parasite. The immunomodulatory activity was determined against stimulated macrophages. LDR, LHR and LMR inhibited promastigote cell growth (IC50 0.62 ± 0.3, 0.94 ± 0.5 and 1.28 ± 0.73 µg/ml, respectively) and reduced the number of amastigotes inside macrophages (IC50 1.36 ± 0.14, 1.54 ± 0.26 and 4.09 ± 0.88 µg/ml, respectively). The cytotoxicity against murine macrophages resulted in a CC50 of 13.12 ± 1.92, 92.93 ± 9.1 and >300 µg/ml, resulting in high selectivity index to promastigotes and amastigotes. The extracts also inhibited the nitric oxide secretion in RAW 264.7 macrophages. The antioxidant capacity resulted in a higher scavenger LMR ability. These results suggest that L. cultratus extracts have anti-Leishmania potential, are non-toxic, and immunosuppress macrophages in vitro.

In vitro antileishmanial and immunomodulatory activities of the synthetic analogue riparin E

Leishmaniases are infectious diseases caused by protozoa of the genus Leishmania, that may have different clinical manifestations. First line drugs used in the treatment of leishmaniosis are high costly, and are very aggressive requiring medical monitoring. Thus new therapeutic alternatives are needed and, in this context, natural products have been considered as a source of new antileishmania agents. Riparins are alkamides found in the unripe fruits of Aniba riparia. Several biological activities are described for this group of compounds, such as antimicrobial and antiparasitic potential. The objective of this work was to evaluate the anti-leishmania activity riparin E (Rip-E) in vitro, against promastigotes and internalized amastigotes of Leishmania amazonensis. Rip-E was able to inhibit promastigote cell growth (IC50 4.7 μg/ml) and to reduce the percentage of macrophages infected with amastigotes, reducing its infectivity (survival index) (IC50 1.3 μg/ml). The cytotoxicity against BALB/c murine macrophages was also assessed (CC50 50.6 μg/ml) and the selectivity index was 38.9. Rip-E also demonstrated immunomodulatory activity, evidenced by the increase of the phagocytic capacity and lysosomal activity. However, Rip-E did not affect directly the production of nitric oxide. These results suggest that Rip-E has antileishmania potential, by both its direct inhibitory effect and its ability to activate macrophages.

Safety and Efficacy of Prosopis juliflora Leaf Extract as a Potential Treatment against Visceral Leishmaniasis in Balb/c Mice.

Background:Visceral Leishmaniasis caused by Leishmania donovani is a major health problem in the tropics and sub-tropic regions where it is endemic. We aimed in testing the leishmanicidal activity and toxicity of Prosopis juliflora leaf extract in BALB/c mice and in vitro test systems respectively.Methods:In the year 2017 until 2019, BALB/c mice of mixed sexes aged between 6 and 8 weeks in groups of 8 were used. Group I treated with 100 mg/kg of P. juliflora extract, Group II -1 mg/kg of Sodium stibogluconate (SSG) and Group III treated with normal saline. All mice were anaesthized and sacrificed to obtain blood, spleen samples for antibody measurements, and determination of parasite loads.Results:There was significant inhibitory effect (P<0.05) exhibited by P. juliflora leaf extract on promastigote growth during the in vitro test whereby up to 98% parasites were killed at the highest concentrations of 100 μg/Ml of the extract as compared to SSG, which showed less inhibitory effect on promastigotes. P. juliflora exhibited a higher splenic antiamastigote effect after 21 days of administration as compared to SSG. P. juliflora methanolic leaf extract induced a higher total IgG level as compared to the reference drug which could be attributed to higher titer in IgG2a subtype in mice treated with the extract, which was not induced in mice, treated with SSG.Conclusion:P. juliflora exhibited higher inhibitory effects against L. donovani promastigotes as well as amastigotes and induced significantly higher IgG antibody levels as compared to SSG (P<0.05). Furthermore, it was safer than SSG on Vero E6 cells.

A comparison of the efficiency of molecular and conventional methods in the diagnosis of cutaneous leishmaniosis.

Leishmaniosis is one of the most important vector borne diseases. Among different forms of the disease, cutaneous leishmaniosis (CL) is the most common. Determining the method of definitive diagnosis for the disease has been the aim of various studies. Therefore this study afforded an opportunity to investigate this subject. To diagnose CL in 150 suspected patients referred to Mehran and Dehloran health centers during June 2018 to November 2019, two polymerase chain reaction (PCR) methods were performed and compared with the in vitro culture and microscopic evaluation of stained slides. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For semi-nested PCR and PCR-RFLP, the tissue and serosity from the lesions were used for DNA extraction. The semi-nested PCR technique using minicircle kDNA gene showed the highest positivity rates among all diagnostic assays with 114/150 (76%) of the samples and was used as reference standard, followed by the PCR-RFLP test using ITS1 gene with 112/150, (74.7%) positivity rates, microscopy with 101/150 (67.3%) and then culture 72/150 (48%). microscopy and culture methods together improved overall positivity rates to 68.7% (103/150). The all positive samples using molecular technique were identified as Leishmania major. The highest sensitivity (98.3%), specificity (100%), accuracy (98.8%), negative predictive value (94.7%) and κ coefficient (0.96=almost perfect) was observed by comparing PCR-RFLP and semi-nested PCR. kDNA-semi-nested PCR and ITS1-PCR-RFLP presented an interesting alternative to conventional methods for the identification of CL and improved its diagnostic value significantly in suspected patients with negative direct smears.

Antileishmanial activity of cordiaquinone E towards Leishmania (Leishmania) amazonensis

Leishmaniasis is caused by several protozoan species of Leishmania, and being endemically present in 98 countries around the world, it is also a severe public-health problem. The available antileishmanial drugs are toxic and yet present risks of recurrent infection. Efforts to find new, effective, and safe oral agents for the treatment of leishmaniasis are continuing throughout the world. This work aimed to evaluate the antileishmania activity of cordiaquinone E (CORe), isolated from the roots of Cordia polycephala (Lam.) I. M. Johnston. Cytotoxicity, and possible mechanisms of action against promastigote and amastigote forms of Leishmania amazonensis were examined. CORe was effective in inhibiting promastigote (IC50 4.5 ± 0.3 µM) and axenic amastigote (IC50 2.89 ± 0.11 µM) growth in concentrations found non-toxic for the host cell (CC50 246.81 ± 14.5 µM). Our results revealed that CORe presents direct activity against the parasite, inducing cell death by apoptosis. CORe present greater activity against intracellular amastigotes (EC50 1.92 ± 0.2 µM), yet with much higher selectivity indexes than the reference drugs, being respectively more benign towards RAW 264.7 macrophages than meglumine antimoniate and amphotericin B, (respectively by 4.68 and 42.84 fold). The antiamastigote activity was associated with increased TNF-α, IL-12, NO, and ROS levels, as well as decreased IL-10 levels. These results encourage the progression of studies on this compound for the development of new leishmanicidal agents.

Vitamin D increases killing of intracellular Leishmania amazonensis in vitro independently of macrophage oxidative mechanisms.

Vitamin D has been reported to activate macrophage microbicidal mechanisms by inducing the production of antimicrobial peptides and nitric oxide (NO), but conversely has been shown to contribute to a greater susceptibility to Leishmania amazonensis infection in mice. Thus, this study aimed to evaluate the role of vitamin D during intracellular infection with L. amazonensis by examining its effect on macrophage oxidative mechanisms and parasite survival in vitro. Vitamins D2 and D3 significantly inhibited promastigote and amastigote growth in vitro. Vitamin D3 was not able to induce NO and reactive oxygen species (ROS) production in uninfected macrophages or macrophages infected with L. amazonensis. In addition, vitamin D3 in combination with interferon (IFN)-γ did not enhance amastigote killing and in fact, significantly reduced NO and ROS production when compared with the effect of IFN-γ alone. In this study, we demonstrated that vitamin D directly reduces parasite growth in infected macrophages (approximately 50-60% at 50 μm) but this effect is independent of the activation of macrophage oxidative mechanisms. These findings will contribute to a better understanding of the role of vitamin D in cutaneous leishmaniasis.

Synthesis and in vitro Leishmania promastigote growth inhibition efficacy of novel 4(3H)-quinazolinone derivatives

Synthesis and in vitro Leishmania promastigote growth inhibition efficacy of novel 4(3H)-quinazolinone derivatives

Phenotype evaluation of human and canine isolates of Leishmania infantum

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.

Comparison of camel, dog and the laboratory animals' sera with the fetal calf serum (FCS) for cultivation of Leishmania major.

The best-known serum for Leishmania spp. cultivation is the fetal calf serum (FCS), which is very expensive with ethical concerns. This study was conducted to compare various laboratory (BALB/c mice, rat, rabbit, hamster and guinea pig) and non-laboratory (dog and camel) animals' sera as a substitute for FCS in L. major culture. L. major, MRHO/IR/75/ER strain, was cultivated in RPMI-1640 medium enriched with different percentages of mentioned animal's sera. Parasite growth was checked constantly. The rate of growth and survival of parasites were compared with a control medium enriched with FCS. As well, biochemical (albumin, globulin AST, ALT, ALP, Bil, BUN, Crea, Ca, P, Na, K, Fe, TIBC, Mg, zinc, Chol, HDL, LDL, TG, BS, uric acid, LDH, CPK) analysis of all sera was performed and compared with FCS. The most promastigote growth rate is considered in 10% BALB/c, guinea pig and hamster sera on the 6th day of cultivation. Also, on the 8th day, parasites showed viability in all animal sera. The promastigote growth in culture media enriched with the camel and the dog sera in comparison with laboratory animals was considered very low. Differences between 10% FCS and 10% cocktail serum were not significant (p > 0.05) but with other sera were significant (p < 0.05). Also, differences between BALB/c with hamster and guinea pig sera were not significant, respectively (p = 0.07 and p = 0.09). According to the biochemical analysis of all sera, the higher content of iron was detected in the hamster, guinea pig, BALB/c and fetal calf sera. The magnesium and zinc content of guinea pig and BALB/c serum was found to be more than the others and comparable with FCS. The promastigote growth decreased by camel, dog and ratsera orderly. In this study, a rapid increase in parasite growth in media supplemented with hamster, BALB/c and guinea pig sera was considered. It could be suggested to use these sera as a suitable alternative for FCS in molecular biology researches.

Leishmania infantum isolates exhibit high infectivity and reduced susceptibility to amphotericin B.

Leishmaniasis is a neglected disease caused by a protozoan parasite of the Leishmania species in over 98 countries in five continents. Visceral leishmaniasis is one of the main forms of the disease and is mainly caused by Leishmania infantum, whose main vector is the dipteran Lutzomyia longipalpis. The presence of the vector in Uruguay was recorded for the first time in 2010 and an autochthonous outbreak of canine visceral leishmaniasis occurred in the northern locality of the country in 2015. We report the isolation in blood-free FBS-supplemented defined media of five isolates responsible for the referred outbreak, and characterize them in terms of their growth as promastigotes, infectivity and replication in human derived monocytes and drug resistance. Results indicate similar promastigote growth among the strains, enhanced infectivity and replication for the five strains isolated from the Uruguayan outbreak when compared with reference strains from South America, equivalent drug susceptibility for miltefosine and nifurtimox and a significant difference in IC50 values for amphotericin B between the Uruguayan strains, 3-4 fold higher than the reference strain.