Ex Vivo Host and Parasite Response to Antileishmanial Drugs and Immunomodulators

Background

Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses.

Methodology

To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 μg Sb^V/mL) or miltefosine (2, 4, 8, 16 and 32 μM HePC). Concentrations of 4 μM of miltefosine and 8 μg Sb^V/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays.

Principal Findings

Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL -10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine.

Conclusions and Significance

Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.     

UCP2 Deficiency Helps to Restrict the Pathogenesis of Experimental Cutaneous and Visceral Leishmaniosis in Mice

Background

Uncoupling protein 2 (UCP2) is a mitochondrial transporter that has been shown to lower the production of reactive oxygen species (ROS). Intracellular pathogens such as Leishmania upregulate UCP2 and thereby suppress ROS production in infected host tissues, allowing the multiplication of parasites within murine phagocytes. This makes host UCP2 and ROS production potential targets in the development of antileishmanial therapies. Here we explore how UCP2 affects the outcome of cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL) in wild-type (WT) C57BL/6 mice and in C57BL/6 mice lacking the UCP2 gene (UCP2KO).

Methodology and Findings

To investigate the effects of host UCP2 deficiency on Leishmania infection, we evaluated parasite loads and cytokine production in target organs. Parasite loads were significantly lower in infected UCP2KO mice than in infected WT mice. We also found that UCP2KO mice produced significantly more interferon-γ (IFN-γ), IL-17 and IL-13 than WT mice (P<0.05), suggesting that UCP2KO mice are resistant to Leishmania infection.

Conclusions

In this way, UCP2KO mice were better able than their WT counterparts to overcome L. major and L. infantum infections. These findings suggest that upregulating host ROS levels, perhaps by inhibiting UPC2, may be an effective approach to preventing leishmaniosis.     

Direct Comparison of the Efficacy and Safety of Oral Treatments with Oleylphosphocholine (OlPC) and Miltefosine in a Mouse Model of L. major Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis (CL) represents a range of skin diseases caused by infection with Leishmania parasites and associated with tissue inflammation and skin ulceration. CL is clinically widespread in both the Old and New World but lacks treatments that are well tolerated, effective and inexpensive. Oleylphosphocholine (OlPC) is a new orally bioavailable drug of the alkylphosphocholine family with potent antileishmanial activity against a broad range of Leishmania species/strains.

Methodology/principal findings

The potential of OlPC against Old World CL was evaluated in a mouse model of Leishmania (L.) major infection in BALB/c mice. Initial dose-response experiments showed that an oral daily dose of 40 mg/kg of OlPC was needed to impact time to cure and lesion sizes. This dose was then used to directly compare the efficacy of OlPC to the efficacy of the antileishmanial drugs miltefosine (40 mg/kg/day), fluconazole (160 mg/kg/day) and amphotericin B (25 mg/kg/day). OlPC, miltefosine and fluconazole were given orally for 21 days while amphotericin B was administered intraperitoneally for 10 days. Ulcer sizes and animal weights were followed up on a weekly basis and parasitemia was determined by means of a real-time in vivo imaging system which detects luminescence emitted from luciferase-expressing infecting L. major parasites. Amphotericin B and OlPC showed excellent efficacy against L. major lesions in terms of reduction of parasitic loads and by inducing complete healing of established lesions. In contrast, treatment with miltefosine did not significantly affect parasitemia and lesion sizes, while fluconazole was completely ineffective at the dose regimen tested.

Conclusions/Significance

Given the data showing the outstanding efficacy and tolerability of OlPC, our results suggest that OlPC is a promising new drug candidate to improve and simplify current clinical management of L. major CL.     

In vitro and in vivo efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites

Background

The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro.

Methodology/Principal Findings

We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance.

Conclusions/Significance

Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.     

Nelfinavir,an HIV-1 Protease Inhibitor,Induces Oxidative Stress–Mediated,Caspase-Independent Apoptosis in Leishmania Amastigotes

Background

Visceral leishmaniasis has now emerged as an important opportunistic disease in patients coinfected with human immunodeficiency virus type-1 (HIV-1). Although the effectiveness of HIV-1 protease inhibitors, such as nelfinavir, in antiretroviral therapies is well documented, little is known of the impact of these drugs on Leishmania in coinfected individuals.

Methodology and Principal Findings

Here, we show that nelfinavir generates oxidative stress in the parasite, leading to altered physiological parameters such as an increase in the sub-G1 DNA content, nuclear DNA fragmentation and loss of mitochondrial potential, which are all characteristics of apoptosis. Pretreatment of axenic amastigotes with the caspase inhibitor z-VAD-fmk did not inhibit the increase in sub-G1 DNA content in nelfinavir-treated parasites, suggesting therefore that this antiviral agent does not kill Leishmania amastigotes in a caspase-dependent manner. Furthermore, we observed that the mitochondrial resident protein endonuclease G is involved. We also demonstrate that parasites overexpressing GSH1 (the rate limiting enzyme of glutathione biosynthesis) were more resistant to nelfinavir when compared to untransfected controls.

Conclusions and Significance

These data suggest that nelfinavir induces oxidative stress in Leishmania amastigotes, culminating in caspase-independent apoptosis, in which DNA is degraded by endonuclease G. This study provides a rationale for future, long-term design of new therapeutic strategies to test nelfinavir as a potential antileishmanial agent as well as for possible future use in Leishmania/HIV-1 coinfections.     

Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival

Background

Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs) as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.

Methodology/Principal Findings

An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.

Conclusions/Significance

Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.     

Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

Background

Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.

Methodology/Principal Findings

We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4).

Conclusion/Significance

Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.     

Potency, efficacy and durability of DNA/DNA, DNA/protein and protein/protein based vaccination using gp63 against Leishmania donovani in BALB/c mice

Background

Visceral leishmaniasis (VL) caused by an intracellular protozoan parasite Leishmania, is fatal in the absence of treatment. At present there are no effective vaccines against any form of leishmaniasis. Here, we evaluate the potency, efficacy and durability of DNA/DNA, DNA-prime/Protein-boost, and Protein/Protein based vaccination against VL in a susceptible murine model.

Methods and Findings

To compare the potency, efficacy, and durability of DNA, protein and heterologous prime-boost (HPB) vaccination against Leishmania donovani, major surface glycoprotein gp63 was cloned into mammalian expression vector pcDNA3.1 for DNA based vaccines. We demonstrated that gp63 DNA based vaccination induced immune responses and conferred protection against challenge infection. However, vaccination with HPB approach showed comparatively enhanced cellular and humoral responses than other regimens and elicited early mixed Th1/Th2 responses before infection. Moreover, challenge with parasites induced polarized Th1 responses with enhanced IFN-γ, IL-12, nitric oxide, IgG2a/IgG1 ratio and reduced IL-4 and IL-10 responses compared to other vaccination strategies. Although, vaccination with gp63 DNA either alone or mixed with CpG- ODN or heterologously prime-boosting with CpG- ODN showed comparable levels of protection at short-term protection study, DNA-prime/Protein-boost in presence of CpG significantly reduced hepatic and splenic parasite load by 10⁷ fold and 10¹⁰ fold respectively, in long-term study. The extent of protection, obtained in this study has till now not been achieved in long-term protection through HPB approach in susceptible BALB/c model against VL. Interestingly, the HPB regimen also showed marked reduction in the footpad swelling of BALB/c mice against Leishmania major infection.

Conclusion/Significance

HPB approach based on gp63 in association with CpG, resulted in robust cellular and humoral responses correlating with durable protection against L. donovani challenge till twelve weeks post-vaccination. These results emphasize the potential of DNA-prime/Protein-boost vaccination over DNA/DNA and Protein/Protein based vaccination in maintaining long-term immunity against intracellular pathogen like Leishmania.     

The Cure Rate after Placebo or No Therapy in American Cutaneous Leishmaniasis: A Systematic Review and Meta-Analysis

Introduction

There are few drugs with proven efficacy in cutaneous leishmaniasis (CL), and pentavalent antimonial derivatives are still the main first-line therapeutic agents worldwide, despite their recognized high toxicities. Randomized controlled clinical trials assessing the efficacy and safety of new therapeutic modalities are of high priority, and the definition of the design of such trials raises debate about the use of placebo as a comparator. To support the use of placebo as a comparator, two main points need to be addressed: 1- the cure rate without any therapeutic intervention and 2- the damage caused by CL and its impact on patients.

Objective

The aim of this study was to systematically assess the spontaneous cure rate for American CL and to broaden the discussion about placebo use in CL trials.

Methods

The PRISMA guidelines for systematic reviews and the Cochrane manual were followed. The sources used were the PubMed and LILACS databases. Studies were included if they reported cure rates using placebo or no treatment in American CL.

Results

Thirteen studies of a total of 352 patients were ultimately included in this review. The summarized global cure rates for all Leishmania species according to the intention-to-treat analyses performed at approximately three (“initial cure”) and nine (“definitive cure”) months after “no treatment” or placebo use were 26% (CI95%: 16 to 40%) and 26% (CI95%:16 to 38%), respectively. Notably, a significantly lower cure rate was observed for L. braziliensis infection (6.4%, CI95%:0.2 to 20%) than for L. mexicana infection (44%, CI95%:19 to 72%), p = 0.002. Of note, relapse occurred in 20% of patients with initial healing (CI95%:9.2 to 38.9%).

Conclusion

These results clearly demonstrate a low spontaneous cure rate following no-treatment or placebo use, confirming that this strategy for the control group in CL studies expose patients to greater morbidity, especially for CL caused by L. braziliensis. Therefore, from this point, the crucial questionto consider regarding placebo use isthe seriousness of the suffering caused by this disease.     

Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis

Background

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied.

Methodology/Principal Findings

In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC₅₀ for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential.

Conclusions/Significance

The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.     

Intrachromosomal Amplification,Locus Deletion and Point Mutation in the Aquaglyceroporin AQP1 Gene in Antimony Resistant Leishmania (Viannia) guyanensis

Background

Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.

Methodology/Principal Findings

Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.

Conclusions/Significance

This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.     

Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures

Background

Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses.

Methods

We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses.

Results

All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses.

Conclusions

The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our results highlight the existence of divergent evolutionary pressures leading to CpG dinucleotide depletion among small ds-DNA viruses infecting vertebrate hosts.     

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

Background

Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited.

Methodology/Principal Findings

This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types.

Conclusions/Significance

The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person–to-person transmission as the main dynamic of transmission of CL.     

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia)

Background

Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.

Methodology

Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime – modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.

Results

Heterologous prime – boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.

Conclusions

Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.     

Involvement of Cytochrome P450 1A1 and Glutathione S-Transferase P1 Polymorphisms and Promoter Hypermethylation in the Progression of Anti-Tuberculosis Drug-Induced Liver Injury: A Case–Control Study

Background

Anti-tuberculosis (anti-TB) drug-induced liver injury (ADLI) is one of the most common adverse effects associated with TB treatment. Cytochrome P450 (CYP) 1A1 and glutathione S-transferase (GST) P1 are important phase I/II metabolizing enzymes involved in drug metabolism and detoxification. Genetic polymorphism and CpG island methylation have been reported as factors influencing the expression of CYP1A1 and GSTP1.

Objective

This study aimed to determine the potential relationships of CYP1A1 and GSTP1 polymorphisms and CpG island methylation with ADLI risk.

Design

This was a population-based one-to-one matched case–control study.

Setting

The subjects were patients with TB receiving treatment in China from December 2010 to June 2013.

Patients

In total, 127 patients with TB and ADLI (case group) and 127 patients with TB but without liver injury (control group) were included in this study. Subjects were matched in terms of sex, age, and therapeutic regimen.

Methods

The general condition of each patient was assessed using questionnaires. The CYP1A1 MspI and GSTP1 Ile105Val polymorphisms as well as methylation status were detected by polymerase chain reaction (PCR)–restriction fragment length polymorphism and the methylation-specific PCR method.

Results

We found no significant difference in GSTP1 and CYP1A1 genotypes between the two groups, probably because the sample size was not large enough; however, patients with ADLI had significantly higher GSTP1 and CYP1A1 promoter methylation rates than control subjects [odds ratio (OR) = 2.467 and 2.000, respectively]. After adjusting for drinking, which significantly differed between the groups as per univariate analysis, we found that hypermethylation of GSTP1 and CYP1A1 promoters was associated with ADLI (OR = 2.645 and 2.090, respectively).

Conclusion

Hypermethylation of CpG islands of GSTP1 and CYP1A1 promoters may thus play important roles in the development of ADLI and provide evidence of being used as novel markers for ADLI risk prediction.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

Background

Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.

Methodology/Principal Findings

Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 µM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 µM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function.

Conclusions/Significance

The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.