Antimony susceptible Leishmania donovani: evidence from in vitro drug susceptibility of parasites isolated from patients of post-kala-azar dermal leishmaniasis in pre- and post-miltefosine era.

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates (n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates (n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates (n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era (P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro. The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use. IMPORTANCE: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL.

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.

Effect of sodium stibogluconate in recruiting and awakening immune cells in the pleural fluid of pancreatic cancer: preparation for immunotherapy.

Ascites and pleural effusion are recognized complications of pancreatic cancer. These diseases are accompanied by ascites and pleural effusion, and drug treatment is limited by high costs, long hospital stays, and failure rates. Immunotherapy may offer new option, but in most patients with late stages of cancer, immune cells may lose the ability to recognize tumor cells, how to activate their immune cells is a major problem, sodium glucosidate (SSG) is injected into ascites as a protein tyrosine phosphatase inhibitor to wake up immune cells and prepare for immunotherapy. We used single-cell RNA sequencing (scRNA-seq) to investigate whether and how SSG injected into ascites of pancreatic cancer elicits an immune response. Our study showed that the process of SSG fusion treatment of ascites and pleural effusion, the interaction between TandNK cells, MPs cells, monocytes and neutrophils was induced, and large numbers of genes were expressed, resulting in upregulation of immune response, which also approved that SSG is not only used as a protein tyrosine phosphatase inhibitor, but also it works as a protein tyrosine phosphatase inhibitor. It can also be used to regulate immune cell function, recruiting immune cells to the right place with the help of PD-1 or PD-L1 to fight cancer cells in ascites and pleural effusions in cancer patients.

In vitro leishmanicidal activity of two cholesterol derivatives.

We evaluated the leishmanicidal activity of commercially available 5α-cholest-7-en-3ß-ol [5α-chol], (+)-4-cholesten-3-one [(+)-4-chol] and the equimolar mixture of the two of them in promastigotes and amastigotes of two different strains of Leishmania mexicana (LCL) and (DCL). The leishmanicidal effectiveness of these sterols was determined by promastigote growth-kinetic experiments and promastigote viability using the propidium iodide staining procedure. The proliferation test was performed using the CFSE (5-Carboxyfluorescein N-succinimidyl ester) staining of parasites at different time points. To determine the leishmanicidal effectiveness of these sterols in amastigotes, we evaluated parasite killing inside of macrophages at different time points. The trypan blue exclusion test was used to determine cytotoxicity of sterols in uninfected macrophages. We included in all experiments a control group of parasites treated with 2% DMSO (Dimethyl Sulfoxide) and another one treated with the reference drug sodium stibogluconate (Sb). Our results showed that the equimolar mixture at 2000 times lower concentration presented similar leishmanicidal activity as Sb. This mixture was similarly effective at 100 times lower concentration than individual sterols tested separately indicating the existence of a synergistic effect against LCL and DCL parasites. The therapeutic index of the equimolar mixture was 10,000-16,000 times higher than the one recorded by Sb and was not cytotoxic to macrophages. Therefore, the equimolar mixture of 5α-Chol and (+)-4-chol may represent a potential alternative for the treatment of cutaneous leishmaniasis.

Antileishmanial efficacy and tolerability of combined treatment with non-ionic surfactant vesicle formulations of sodium stibogluconate and paromomycin in dogs.

Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.

Immunomodulator mediated changes in plasma membrane calcium ATPase in controlling visceral leishmaniasis.

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.

Comparison of in vitro Resistance of Wild Leishmania Isolates, Which are Resistant to Pentavalent Antimonial Compounds, Against Drugs Used in the Treatment of Leishmaniasis

Objective: Meglumine antimoniate (Glucantime®) and Sodium stibogluconate (Pentostam®) are used for the treatment of cutaneous leismaniasis in Turkey. There is a reported resistance to these drugs in recent years. The aim of the present study was to compare the in vitro sensitivities of resistant Leishmania isolates against Amphotericin B, Miltefosine, Meglumine Antimoniate, Paromomycin and Sodium Stibogluconate. Methods: Five Leishmania isolates of patients with cutaneous leishmaniasis, who showed no clinical recovery despite two consecutive meglumine antimoniate treatments, which were stored in the Parasite Bank in Manisa Celal Bayar University Medical Faculty were selected. They were genotyped with Real-Time PCR using specific primers and probes to ITS1 region. Drug resistance levels of each Leishmania isolate were analysed for Amphotericin B, Miltefosine, Meglumine Antimoniate, Paromomycin, and Sodium Stibogluconate at concentrations of 500, 250, 125, 50, 25 µg/mL with XTT method and hemocytometer. Results: It was observed that the resistant Leishmania tropica isolates showed no resistance to Amphotericin B, and were sensitive to Miltefosine, Sodium Stibogluconate, Paromomycin and Meglumin Antimonate, respectively. In addition, Leishmania tropica (MHOM/AZ/1974/SAF-K27) isolate of the control group could stay viable in none of the drug concentrations of five agents in the study. Conclusion: It was determined that none of the selected resistant L. tropica isolates showed resistance to Amphotericin B and that was also shown statistically (p<0.05). The results of this study are important in guiding clinicians and researchers who conduct studies on drugs and search for new drug molecules.

Transcriptional responses of Leishmania (Leishmania) amazonensis in the presence of trivalent sodium stibogluconate.

BACKGROUND: In the last decade, resistance to antimonials has become a serious problem due to the emergence of drug-resistant strains. Therefore, understanding the mechanisms used by Leishmania parasites to survive under drug pressure is essential, particularly for species of medical-veterinary importance such as L. amazonensis. METHODS: Here, we used RNA-seq technology to analyse transcriptome profiles and identify global changes in gene expression between antimony-resistant and -sensitive L. amazonensis promastigotes. RESULTS: A total of 723 differentially expressed genes were identified between resistant and sensitive lines. Comparative transcriptomic analysis revealed that genes encoding proteins involved in metabolism (fatty acids) and stress response, as well as those associated with antimony resistance in other Leishmania species, were upregulated in the antimony-resistant line. Most importantly, we observed upregulation of genes encoding autophagy proteins, suggesting that in the presence of trivalent stibogluconate (SbIII) L. amazonensis can activate these genes either as a survival strategy or to induce cell death, as has been observed in other parasites. CONCLUSIONS: This work identified global transcriptomic changes in an in vitro-adapted strain in response to SbIII. Our results provide relevant information to continue understanding the mechanism used by parasites of the subgenus Leishmania (L. amazonensis) to generate an antimony-resistant phenotype.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Genomic insights into virulence mechanisms of Leishmania donovani: evidence from an atypical strain.

BACKGROUND: Leishmaniasis is a neglected tropical disease with diverse clinical phenotypes, determined by parasite, host and vector interactions. Despite the advances in molecular biology and the availability of more Leishmania genome references in recent years, the association between parasite species and distinct clinical phenotypes remains poorly understood. We present a genomic comparison of an atypical variant of Leishmania donovani from a South Asian focus, where it mostly causes cutaneous form of leishmaniasis. RESULTS: Clinical isolates from six cutaneous leishmaniasis patients (CL-SL); 2 of whom were poor responders to antimony (CL-PR), and two visceral leishmaniasis patients (VL-SL) were sequenced on an Illumina MiSeq platform. Chromosome aneuploidy was observed in both groups but was more frequent in CL-SL. 248 genes differed by 2 fold or more in copy number among the two groups. Genes involved in amino acid use (LdBPK_271940) and energy metabolism (LdBPK_271950), predominated the VL-SL group with the same distribution pattern reflected in gene tandem arrays. Genes encoding amastins were present in higher copy numbers in VL-SL and CL-PR as well as being among predicted pseudogenes in CL-SL. Both chromosome and SNP profiles showed CL-SL and VL-SL to form two distinct groups. While expected heterozygosity was much higher in VL-SL, SNP allele frequency patterns did not suggest potential recent recombination breakpoints. The SNP/indel profile obtained using the more recently generated PacBio sequence did not vary markedly from that based on the standard LdBPK282A1 reference. Several genes previously associated with resistance to antimonials were observed in higher copy numbers in the analysis of CL-PR. H-locus amplification was seen in one cutaneous isolate which however did not belong to the CL-PR group. CONCLUSIONS: The data presented suggests that intra species variations at chromosome and gene level are more likely to influence differences in tropism as well as response to treatment, and contributes to greater understanding of parasite molecular mechanisms underpinning these differences. These findings should be substantiated with a larger sample number and expression/functional studies.

Treatment outcomes of visceral leishmaniasis in Ethiopia from 2001 to 2017: a systematic review and meta-analysis.

BACKGROUND: Ethiopia has the highest number of visceral leishmaniasis (VL) cases after Sudan in Sub-Saharan Africa. However, there was lack of comprehensive data on VL treatment outcome despite the huge burden of the diseases in the country. Hence, we aimed to perform a systematic review and meta-analysis on this topic to obtain stronger evidence on treatment outcomes of VL from the existing literature in Ethiopia. METHODS: The Cochrane guidelines to conduct meta-analysis following the Preferred Reporting Items for Systematic review and Meta-Analysis statement was used to conduct a computerized systematic search of the PubMed, Google Scholar, and ScienceDirect databases. Random effects model was used to combine studies showing heterogeneity of Cochrane Q P < 0.10 and I2 > 50. Treatment outcomes were assessed at end of treatment and at 6 months follow-up. Subgroup analyses were performed on treatment outcomes based on the different antileishmanial treatment options and patients' HIV status. RESULTS: Fifteen studies were included in the final analyses. At end of treatment, an overall treatment success rate of 82.6% was noticed. At 6 months follow-up, the overall treatment success rate was 72.2%. For patients treated with sodium stibogluconate (SSG), the treatment success rates at the end of treatment and at six-month follow-up were 81.5% and 80.7%, respectively. Multiple doses of liposomal-amphotericin B (L-AMB) had treatment success rates of 96.7 and 71-100% at the end of treatment and at 6 months follow-up, respectively. The combination of SSG with paromomycin (PM) gave treatment success rates of up to 90.1% at the end of treatment. HIV-infected individuals were found to have a higher mortality (odds ratio = 4.77, 95% CI: 1.30-17.43, P = 0.009) rate at 6 months follow-up. CONCLUSIONS: SSG alone has shown lower treatment efficacy in the management of VL when compared to combination of SSG with PM and multiple doses of L-AMB. The combination of SSG with PM gave good treatment success rates with shorter duration of treatment. Hence, the combination of SSG with PM should be used preferentially over SSG monotherapy. Multiple doses of L-AMB showed great efficacy especially among patients with complications, severe disease, HIV co-infection, and intolerance to the adverse effects of antimonials. HIV-infected individuals had a worse prognosis than their HIV-negative counterparts.

Evaluation of In vitro and In vivo Drug Efficacy Over Leishmania tropica: A Pilot Study.

OBJECTIVE: Two pentavalent antimonials, meglumine antimoniate (Glucantime®, France) and sodium stibogluconate (Pentostam®, England), are used to treat cutaneous leishmaniasis (CL) in Turkey. The present study, serving as a guidebook for young researchers, aims to provide basis for conducting drug resistance tests and active ingredient scanning in in vitro and in vivo models. METHODS: A CL isolate kept in liquid nitrogen was initially thawed and genotyped by real-time polymerase chain reaction (PCR) using ITS1 prob. In vitro and in vivo tests were conducted to determine drug resistance against meglumine antimoniate and sodium stibogluconate. Hemocytometry and XTT (sodium 3,39-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate) methods were used to investigate in vitro drug resistance. CL mouse models were used to analyze in vivo drug resistance. RESULTS: The isolate was determined as Leishmania tropica by genotyping by PCR on the internal transcribed spacer 1 (ITS1) gene region. In in vitro drug resistance tests, sodium stibogluconate was observed to be more effective than meglumine antimoniate, but there was no statistically significant difference between the two (p > 0.05). It was observed that the footpad lesions of the animals started to shrink afterward the 5th week of infection following treatment with these agents, and parasitologic recovery was observed at the end of 3 months. CONCLUSIONS: With an aim to be used as a guidebook for young researchers, active ingredient scanning and drug resistance tests in both in vitro and in vivo models were presented in the current study.

Antimony susceptibility of Leishmania isolates collected over a 30-year period in Algeria.

BACKGROUND: In Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies. METHODOLOGY/PRINCIPAL FINDINGS: Within the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 µg/mL and 15.91 µg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade. CONCLUSION/SIGNIFICANCE: Our results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains.

Decanethiol functionalized silver nanoparticles are new powerful leishmanicidals in vitro.

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC50's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.

Integrating genomics and proteomics permits identification of immunodominant antigens associated with drug resistance in human visceral leishmaniasis in India.

Resistance of human pathogens like Leishmania to drugs is a growing concern where the multidrug-resistant phenotype renders chemotherapy ineffective. The acquired resistance of Leishmania to antimony has promoted intense research on the mechanisms involved but the question has not been resolved yet. In this study we have explored host-pathogen- drug interactions leading to identification of pharmacological determinants of host macrophages that resist the sodium antimony gluconate (SAG) mediated intracellular parasite killing. mRNA profiling of mammalian host stage amastigotes of sodium antimony gluconate (SAG) 'sensitive' and 'resistant' parasite lines was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Patient sera was used to identify immunogenic proteins by two-dimensional gel analysis (2DE) and mass spectrometric analysis (LC-MS/MS). Immunofluorescence microscopy confirmed the identities on 'sensitive' and 'resistant' parasite lines. A total of nine immunogenic proteins whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins are altered in expression levels in the 'resistant' parasites. Global expression profiling using microarrays revealed this regulation was not reflected by changes in the levels of the cognate mRNAs. Following identification of proteins by mass spectrometry, one such regulated protein, enolase, was chosen for more detailed analysis. Immunofluorescence microscopy employing antisera against this enzyme confirmed that its level was differentially regulated in the 'resistant' isolate. We show that high serum level of immunoreactive protein is associated with 'resistant' phenotype. Differentially expressed proteins with immunomodulatory activities were found to be associated with the 'resistant phenotype'.

A Telomeric Cluster of Antimony Resistance Genes on Chromosome 34 of Leishmania infantum.

The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu(2+) challenge but not under sodium arsenite, Cd(2+), or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity.

INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9µg/mL, whereas that of MA was 60µg/mL. Sb-FEL showed an IC50 value of 0.2µg/mL, whereas that of free Sb was 9µg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

In-vitro sensitivity of Pakistani Leishmania tropica field isolate against buparvaquone in comparison to standard anti-leishmanial drugs.

In this study, in vitro anti-leishmanial activity of buparvaquone was evaluated against promastigotes and intracellular amastigotes of Pakistani Leishmania tropica isolate KWH23 in relation to the current standard chemotherapy for leishmaniasis (sodium stibogluconate, sodium stibogluconate, amphotericin B and miltefosine). For buparvaquone, mean % inhibition in intracellular amastigotes at four different concentrations (1.35 µM, 0.51 µM, 0.17 µM and 0.057 µM) was 78%, 44%, 20% and 14% respectively, whereas, against promastigotes it was 89%, 77%, 45% and 35% respectively. IC50 values calculated to estimate the anti-leishmanial activity of buparvaquone against intra-cellular amastigotes and promastigotes was 0.53 µM (95% C.I. = 0.32-0.89) and 0.15 µM (95% C.I. = 0.01-1.84) respectively. Amphotericin B was the most potent in-vitro drug tested, with an IC50 of 0.075 µM (95% C.I. = 0.006-0.907) against promastigotes, and 0.065 µM (95% C.I. = 0.048-0.089) against intra-cellular amastigotes. Amphotericin B was more cytotoxic against THP1 cells, with an IC50 of 0.15 µM (95% C.I. = 0.01-0.95) and an apparent in-vitro therapeutic index of 2.0, than was buparvaquone, with an IC50 of 12.03 µM (95% C.I. = 5.36-26.96) against THP1 cells and a therapeutic index of 80.2. The study proposes that buparvaquone may be further investigated as a candidate drug for treatment of cutaneous leishmaniasis.

Co-administration of glycyrrhizic acid with the antileishmanial drug sodium antimony gluconate (SAG) cures SAG-resistant visceral leishmaniasis.

Since there are very few affordable antileishmanial drugs available, antimonial resistance has crippled antileishmanial therapy, thereby emphasising the need for development of novel therapeutic strategies. This study aimed to evaluate the antileishmanial role of combined therapy with sodium antimony gluconate (SAG) and the triterpenoid glycyrrhizic acid (GA) against infection with SAG-resistant Leishmania (GE1F8R). Combination therapy with GA and SAG successfully limited infection with SAG-resistant Leishmania in a synergistic manner (fractional inhibitory concentration index <1.0). At the same time, mice infected with SAG-resistant Leishmania and co-treated with GA and SAG exhibited a significant reduction in hepatic and splenic parasite burden. In probing the mechanism, it was observed that GA treatment suppressed the expression and efflux activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), two host ABC transporters responsible for antimony efflux from host cells infected with SAG-resistant parasites. This suppression correlated with greater intracellular antimony retention during SAG therapy both in vitro and in vivo, which was reflected in the reduced parasite load. Furthermore, co-administration of GA and SAG induced a shift in the cytokine balance towards a Th1 phenotype by augmenting pro-inflammatory cytokines (such as IL-12, IFNγ and TNFα) and inducing nitric oxide generation in GE1F8R-infected macrophages as well as GE1F8R-infected mice. This study aims to provide an affordable leishmanicidal alternative to expensive antileishmanial drugs such as miltefosine and amphotericin B. Furthermore, this report explores the role of GA as a resistance modulator in MRP1- and P-gp-overexpressing conditions.