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Background Leishmania tropica is the cause of more than one form of leishmaniasis and lacks a known reservoir animal. This study compares the potential infectivity of recombinant and wild-type L. tropica in BALB/c mice. Methods The potential infectivity of recombinant L. tropicaEGFP or L. tropicaEGFP-LUC by two different, the subcutaneous and intradermal, routes was compared using a range of classical detection methods and bioluminescence imaging (BLI). Results In addition to the results obtained from classical diagnostic approaches, the BLI signals were detected in footpads and ears of L. tropica-infected animals. The BLI revealed that a bioluminescence signal can be observed at the inoculation site. The stability of the BLI remained constant in the footpad, but the signal was detectable for only three months in the pinna due to the decline in infection over time. Conclusion The presented data are a precise verification of the assumption that BALB/c mice could be used as an experimental model for L. tropica infectivity.

Background: Visceral leishmaniasis is the most severe form of leishmaniasis caused by Leishmania (L.) donovani complex. Drug-resistant strains have been developed as a consequence of the current chemotherapeutic interventions, which has increased the need for advanced preventive and therapeutic strategies. A2-CPA-CPB-CTE-recombinant strain of L. tarentolae, which is non-pathogenic to humans, was shown protective in live vaccine as well as its DNA vaccine counterpart in both murine and canine models. Methods: We evaluated the effectiveness of these DNA and live vaccination harboring A2-CPA-CPB-CTE in protecting hamsters against L. infantum infection using prime-boost regimens, namely DNA/DNA and Live/Live (n=9 hamsters per group). Cationic solid lipid nanoparticles (cSLN) were utilized as an adjuvant for DNA priming and electroporation for boosting DNA. At different time points post-challenge, parasite burden and body weight as well as humoral immune responses were measured. Results: Both immunization strategies partially protect hamsters against L. infantum challenge. This protective immunity is associated with remarkable decrease in parasite load in liver and spleen of vaccinated hamsters eight weeks after challenge compared to control group. Conclusion: Both test groups (DNA/DNA and Live/Live) elicited high levels of IgG2 and total IgG as humoral immune responses and lower level of parasite propagation in both liver and spleen.

Background: Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods: Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (CSFM) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results: Diff-Quick staining and epi-fluorescence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion: This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Background The crucial oncogenic role of cancer stem cells (CSCs) in tumor maintenance, progression, drug resistance, and relapse has been clarified in different cancers, particularly in colorectal cancer (CRC). The current study was conducted to evaluate the co-expression pattern and clinical significance of epithelial cell adhesion molecules (EpCAM) and activated leukocyte cell adhesion (CD166 or ALCAM) in CRC patients. Methods This study was carried out on 458 paraffin-embedded CRC specimens by immunohistochemistry on tissue microarray (TMA) slides. Results Elevated expression of EpCAM and CD166 was observed in 61.5% (246/427) and 40.5% (164/405) of CRC cases. Our analysis showed a significant positive association of EpCAM expression with tumor size (P = 0.02), tumor stage (P = 0.007), tumor differentiate (P = 0.005), vascular (P = 0.01), neural (P = 0.01), and lymph node (P = 0.001) invasion. There were no significant differences between CD166 expression and clinicopathological parameters. Moreover, the combined analysis demonstrated a reciprocal significant correlation between EpCAM and CD166 expression (P = 0.02). Interestingly, there was a significant positive correlation between EpCAM/CD166 phenotypes expression and tumor stage (P = 0.03), tumor differentiation (P = 0.05), neural, and lymph node invasion (P =0.01). Conclusions The significant correlation of EpCAM and CD166 expression and their association with tumor progression and aggressive behavior is the reason for the suggestion of these two CSC markers as promising targets to promote novel effective targeted-therapy strategies for cancer treatment in the present study.

Despite successful anti-viral (DAAs) treatment of Hepatitis C virus (HCV) infection, recent data indicated the need for an effective vaccine. Preexisting anti-vector immunity is an obstacle for application of live vectors for antigen delivery and development of effective T-cell based HCV vaccines. Herein, we report construction of recombinant Leishmania tarentolae, a lizard (non-human) parasite, expressing an HCV polytope DNA, PT-NT(gp96), encoding for several immunogenic HCV epitopes and evaluation of its immunogenicity in three different prime/boost immunization groups (G) of BALB/c mice. Homologous prime/boost immunization by L.tarentolae-PT-NT(gp96) either with or without CpG (G1 and G2 respectively) and heterologous immunization with a PT-NT(gp96) encoding-pCDNA plasmid followed by L.tarentolae-PT-NT (G3) was undertaken. Immune responses were measured three and nine weeks (W) post immunization. Splenocytes (cultured with antigen-stimulant) of mice in G1 showed the highest percentage of specific CTL-cytolytic activity compared to G2 and G3 at both short (W3:70.98% versus 41.29% and 13.12%) and long (W9: 50% versus 24.5% and 20%) term periods, accompanied with high levels of secreted IFN-γ. Comparison of IFN-γ, IL-4, IL-17 and TNF-α cytokines levels obtained from the supernatant of antigen-stimulated splenocytes as well as antibodies level (as IgG1/IgG2a ratio; obtained from sera of immunized mice) indicated higher Th1 oriented responses for G1, G2 groups and balanced Th1-Th17 for G3. Results indicated the potential of L.tarentolae (+CpG), as a non-pathogenic live vaccine vector, for delivery and enhancement of immune responses against HCV-polytope antigens.

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4

Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months., Author summary Different strains of Lactococcus lactis can be used as a suitable non-pathogenic vehicle for live vaccination. In this study, our results demonstrated that recombinant L. lactis could express PpSP15, an immunogenic component of Phlebotomus papatasi saliva, on the cell wall. Furthermore, localizing PpSP15 on the cell wall of L. lactis could trigger short (2 weeks) and long (6 months) memory cellular immunity in BALB/c mice, immunized with the recombinant L. lactis. In addition, after challenging the immunized mice with Leishmania major, the cytokine assays indicated that their immune response was shifted to Th1 which controlled the footpad swelling and propagation of L. major in the lymph nodes.

RNA-binding proteins play critical roles in the regulation of gene expression. Among several families of RNA-binding proteins, PUF (Pumilio and FBF) proteins have been the subject of extensive investigations, as they can bind RNA in a sequence-specific manner and they are evolutionarily conserved among a wide range of organisms. The outstanding feature of these proteins is a highly conserved RNA-binding domain, which is known as the Pumilio-homology domain (PUM-HD) that mostly consists of eight tandem repeats. Each repeat recognizes an RNA base with a simple three-letter code that can be programmed in order to change the sequence-specificity of the protein. Using this tailored architecture, researchers have been able to change the specificity of the PUM-HD and target desired transcripts in the cell, even in subcellular compartments. The potential applications of this versatile tool in molecular cell biology seem unbounded and the use of these factors in pharmaceutics might be an interesting field of study in near future.

Two species of Leishmania (L), L. tropica and L. major, are among the main causative agents of cutaneous leishmaniasis. Arginase (ARG) is an essential enzyme for cell growth, thus an attractive drug target. In this study, we tried to survey the inhibitory impact of ARG by nor-NOHA (N-ω-hydroxy-L-nor-arginine) on in vivo infection caused by L. tropica. BALB/c mice were inoculated with L. tropicaEGFP-LUC (Ltrop) or L. majorEGFP-LUC (Lmj) and then were treated by nor-NOHA. ARG inhibitor only indicated a delay in generation of a cutaneous lesion in inoculated footpad with nor-NOHA-Ltrop and nor-NOHA-Lmj. ARG activity has been significantly reduced in nor-NOHA-Ltrop group. In this group, ARG activity inhibition correlated with increased levels of nitric oxide (NO). In both inoculated mice with Ltrop or Lmj, parasite load showed a significant decrease at later steps during the CL course post-treatment. In vivo bioluminescence intensity did not show any ARG's inhibitory effect on treated-Ltrop. The findings verified that the ARG activity may partially control the L. tropica infection in BALB/c mice through reduction of parasite proliferation and parasite killing through NO generation. This effect is dose-dependent.

Cutaneous leishmaniosis (CL) is a parasitic disease in animals and human with no satisfactory treatments and vaccination. Rapamycin is a potent inhibitor of mammalian target of rapamycin (mTOR) with various applications. Here, the effect of rapamycin alone or in combination with two other drugs, namely amphotericin B (AmB) and glucantime, was investigated against Leishmania tropica infection. In vitro viability and electron microscopy evaluation of the parasites showed detrimental changes in their appearance and viability. Treatment with clinically relevant dose of rapamycin (10.2 μg/dose) is able to control the parasite load in BALB/c mice infected with L. tropica. Furthermore, the cytokine profiles showed significant polarization towards Th1 immune response. Surprisingly, combination therapy with either AmB or glucantime was not efficient. Rapamycin is showed an effective alternative therapy against leishmaniosis caused by L. tropica.

PLoS Neglected Tropical Diseases, 13 (1), ISSN:1935-2727, ISSN:1935-2735

Leishmaniasis is a complex vector-borne disease mediated by Leishmania parasite and a strong and long-lasting CD4+ Th1 and CD8+-T cell immunity is required to control the infection. Thus far multivalent subunit vaccines have met this requirement more promisingly. However several full protein sequences cannot be easily arranged in one construct. Instead, new emerging immune-informatics based epitope formulations surpass this restriction. Herein, we aimed to examine the protective potential of a dendritic cell based vaccine presenting epitopes to CD8+ and CD4+-T cells in combination with DNA vaccine encoding the same epitopes against murine cutaneous leishmaniasis. Immature DCs were loaded with epitopes (selected from parasite proteome) in vitro with or without CpG oligonucleotides and were used to immunize BALB/c mice. Peptide coding DNA was used to boost the system and immunological responses were evaluated after Leishmania (L.) major infectious challenge. The pre-challenge response to included epitopes was Th1 polarized which potentially lowered the infection at early time points post-challenge but not at later weeks. Collectively, DC prime-DNA boost was found to be a promising approach for Th1 polarization however the constituent epitopes undoubtedly make a significant contribution in the protection outcome of the vaccine.

Summary Leishmaniasis caused by protozoan parasites of the genus Leishmania. Intracellular infections treatment such as leishmaniasis is frequently hampered by limited access of drugs to infected cells. Moreover, most of the current drugs are confined to some toxic compounds, and there are increasing incidences of development of drug resistance. Hence, production of a new antileishmanial compound is crucial. Paromomycin sulphate (PM) is a promising antileishmanial drug. One strategy to improve drug effectiveness is to use appropriate delivery systems. Solid lipid nanoparticle (SLN) is as an excellent substitute delivery system to other colloidal carrier. In the present study, PM was loaded in solid lipid nanoparticles (PM-SLN) and the in vivo efficacy was studied against Leishmania (L.) major-infected BALB/c mice. For this reason, the footpad swelling was measured and real-time PCR was performed to quantify the parasite load after infectious challenge. The level of cytokines including interleukin-4 (IL-4) and gamma interferon (IFN-γ) and nitric oxide was evaluated. Altogether, this study showed that the PM-SLN formulation is a safe compound and SLN in PM-SLN compound is effective for treatment of leishmaniasis by improving the effectiveness of PM in killing the parasite and switching towards Th1 response.

Leishmaniasis is a serious global health problem affecting many people worldwide. While patients with leishmaniasis can be treated with several agents, drug toxicicty and the emergence of resistant strains render available treatments ineffective in the long run. Inhibitors of the mammalian target of rapamycin (mTOR) have been demonstrated to exert anti-pathogen properties. In this study, we tested the therapeutic efficacy of several mTOR inhibitors in controlling infection with Leishmania major. Rapamycin, GSK-2126458 and KU-0063794 were administered to BALB/c mice, which had received an intrafootpad injection of the parasite. Footpad swelling and parasite burden were assessed, and cytokine production by mouse splenocytes and phenotypic changes in draining lymph node cells were evaluated. Treatment with a clinically relevant dose of rapamycin or with GSK-2126458, but not with KU-0063794, dramatically lowered both the footpad swelling and the parasite load in the draining lymph node. Importantly, the employed dose of rapamycin did not kill the promastigotes in vitro as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and electron microscopy. Moreover, the IL-4 production capacity of splenocytes harvested from infected mice that were treated with rapamycin was significantly reduced. Consequently, the IFN-γ:IL-4 production ratio was elevated, suggesting a T helper-type 1 (Th1)-skewed cytokine profile. Finally, the expression level of CD69, an early activation marker, on splenic and lymph node CD4+ and CD8+ T cells was enhanced in rapamycin-treated mice. Taken together, our findings suggest that select mTOR inhibitors may be used in therapeutic settings for the management of leishmaniasis. We propose that the beneficial effects of such inhibitors stem from their immunomodulatory properties. Therefore, the adjuvanticity of mTOR inhibitors may also be considered in vaccination strategies against Leishmania species., Author summary The lack of effective vaccines and therapies for leishmaniasis along with the well-known resistance of the parasite to available agents prompted us to explore the effects of several mTOR inhibitors, including rapamycin, as potential therapeutics against Leishmania (L.) major, the classic Leishmania parasite strain. We found that systemic treatment with rapamycin and GSK-2126458, but not with KU-0063794, slows the progression of the disease and lowers the parasite burden in infected BALB/c mice. In addition, we observed more pronounced activation of CD4+ and CD8+ T cells in the draining lymph nodes in addition to a T helper 1 (Th1)-biased cytokine profile among the splenic cells of treated mice. Importantly, rapamycin blood levels achieved after in vivo treatment with this agent was far lower than the in vitro doses of rapamycin that killed the promastigote form of the parasite. Therefore, we propose that the impressive therapeutic efficacy of rapamycin, and perhaps GSK-2126458, against L. major are likely owed to the immunomodulatory properties of these agents. Based on our findings, mTOR may be an attractive target for the development of novel and potent anti-Leishmania agents.

Human Neutrophil Peptide 1 (HNP1) produced by neutrophils, is a well-known antimicrobial peptide which plays a role both in innate as well as in adaptive immunity and is under intensive investigation as a potential therapeutic agent. Previous in vitro experiments have indicated the leishmaniacidal effect of recombinant HNP1 on Leishmania major (L. major) promastigotes and amastigotes. In the current study, we further extended the idea to explore the remedial effect of HNP1 in the two modalities of peptide therapy (folded HNP1) and gene therapy in L. major infected BALB/c mice. To this end, mice in five different groups received synthetic folded HNP1 (G1), pcDNA-HNP1-EGFP (G2), pcDNA-EGFP (G3), Amphotericin B (G4) and PBS (G5), which was started three weeks after infection for three consecutive weeks. Footpad swelling was monitored weekly and a day after the therapy ended, IFN-γ, IL-4, IL-10, IL-6 and nitric oxide produced by splenocytes were analyzed together with the parasite load in draining lymph nodes. Arginase activity and dermal histopathological changes were also analyzed in the infected footpads. We demonstrated that both therapeutic approaches effectively induced Th1 polarization and restricted parasite burden. It can control disease progression in contrast to non-treated groups. However, pcDNA-HNP1-EGFP is more promising in respect to parasite control than folded HNP1, but less effective than AmB treatment. We concluded with the call for a future approach, that is, a DNA-based expression of HNP1 combined with AmB as it can improve the leishmaniacidal efficacy., Author summary The outbreak level of cutaneous leishmaniasis is approximated between one and 1.5 million individuals per year. Owning to several disadvantages of current therapies, special attention to expand novel and efficient therapies has been demanded. Among Anti-Microbial Peptides (AMPs), Human Neutrophil Peptide 1 (HNP1) is one of the most potential defensins. Our promising in vitro experiments have shown the leishmaniacidal effect of recombinant HNP1. Here, we displayed the remedial effect of HNP1 in two approaches including peptide therapy and gene therapy in susceptible mice infected with L. major. Our investigation showed that although both approaches could decrease the parasite load and induce Th1 immune response compared to the control group, pcDNA-HNP1-EGFP has a better effect compared to the folded HNP1. Hence, immunotherapy by HNP1 can help elicit proper immunity despite the direct effect on promastigotes and amastigotes forms of parasite.

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.

Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes.

Aim: Several disadvantages about chemotherapy for leishmaniasis has reinforced discovery of novel therapeutic agents especially immunotherapeutics. HNP1, as a member of the mammalian antimicrobial peptides family, is an attractive molecule due to its broad functional spectrum. Here, the in vivo potency of HNP1 in transgenic Leishmania tarentolae as an immunotherapy tool against Leishmania major-infected BALB/c mice was examined. Methods & results: 3 weeks after infection with L. major, the treatment effect of L. tarentolae-HNP1-EGFP was pursued. The results were promising in respect to parasite load control and Th1 immune response polarization compared with controls. Conclusion: Immunotherapy by live L. tarentolae secreting HNP1 can elicit cellular immune response in a susceptible mouse model in order to control L. major infection.

Background Stroke is the second leading cause of death worldwide and is associated with several risk factors with variable risk factor distribution by population. We report the types and frequency of the associated factors of stroke in north Iran. Methods Consecutive patients with stroke were recruited from 2014 to 2015. The two groups of ischemic and hemorrhagic stroke were compared with respect to age, sex, the prevalence, and distribution of the risk factors. Results Among 230 patients (84.3% ischemic stroke) with mean age of 61.2 years, hypertension (73%), diabetes mellitus (53%), cardiovascular disease (51%), and dyslipidemia (47%) were the most frequent risk factors. Hypertension was significantly more prevalent in males compared with females (88% vs. 60%, OR = 4.91, 95% CI: 2.48–9.71). Hypertension, smoking, and opioid consumption were associated with hemorrhagic stroke. Dyslipidemia was significantly higher in ischemic stroke (OR = 2.65, 95% CI: 1.21–5.8). Overall, 84.3% of stroke occurred in patients aged >50 years (92.8% of women vs. 74.5% of the men, OR = 4.43, 95% CI: 1.93–10.16, p = 0.001). Conclusion Stroke was more prevalent in females; hypertension was more prevalent in males. In the age group less than 50 years old, stroke is more prevalent in men.

Proliferation of Leishmania (L.) parasites depends on polyamine availability, which can be generated by the L-arginine catabolism and the enzymatic activity of arginase (ARG) of the parasites and of the mammalian hosts. In the present study, we characterized and compared the arginase (arg) genes from pathogenic L. major and L. tropica and from non-pathogenic L. tarentolae. We quantified the level of the ARG activity in promastigotes and macrophages infected with pathogenic L. major and L. tropica and non-pathogenic L. tarentolae amastigotes. The ARG's amino acid sequences of the pathogenic and non-pathogenic Leishmania demonstrated virtually 98.6% and 88% identities with the reference L. major Friedlin ARG. Higher ARG activity was observed in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae. In vitro infection of human macrophage cell line (THP1) with pathogenic and non-pathogenic Leishmania spp. resulted in increased ARG activities in the infected macrophages. The ARG activities present in vivo were assessed in susceptible BALB/c and resistant C57BL/6 mice infected with L. major, L. tropica and L. tarentolae. We demonstrated that during the development of the infection, ARG is induced in both strains of mice infected with pathogenic Leishmania. However, in L. major infected BALB/c mice, the induction of ARG and parasite load increased simultaneously according to the time course of infection, whereas in C57BL/6 mice, the enzyme is upregulated solely during the period of footpad swelling. In L. tropica infected mice, the footpads' swellings were slow to develop and demonstrated minimal cutaneous pathology and ARG activity. In contrast, ARG activity was undetectable in mice inoculated with the non-pathogenic L. tarentolae. Our data suggest that infection by Leishmania parasites can increase ARG activity of the host and provides essential polyamines for parasite salvage and its replication. Moreover, the ARG of Leishmania is vital for parasite proliferation and required for infection in mice. ARG activity can be used as one of the main marker of the disease severity., Author summary Over the past decades, there has been a significant improvement in the understanding of immune responses against infection with pathogenic Leishmania spp. and the pathogenesis of cutaneous leishmaniasis (CL) in the mouse models. Leishmania parasites infect macrophages that can be activated via two major pathways resulting in classical and alternative activated macrophages which metabolize L-arginine differentially. Classically activated macrophages upregulate the enzyme inducible NO synthase (iNOS) and alternatively activated macrophages upregulate the arginase (ARG). ARG hydrolyzes the conversion of its substrate (L-arginine) to L-ornithine and urea. L-ornithine is a key intermediate substrate for the biosynthesis of polyamines, which are crucial nutrients for cellular processes such as growth, differentiation and proliferation of host cells and Leishmania parasites. Leishmania parasites may partly activate ARGs and inactivate the NO production by the host cells and enhance parasite survival via depletion of the iNOS substrate (L-arginine) and reduce NO levels. In this study, we tested the activity of this enzyme in the promastigote forms of the pathogenic L. major and L. tropica, and non-pathogenic L. tarentolae in vitro and assessed the differences between the levels of ARG activity in THP1 cells infected with different Leishmania spp. Moreover, we investigated the relationship between excessive ARG activity and lesion development in susceptible BALB/c and resistant C57BL/6 mice infected with virulent L. major, L. tropica and non-virulent L. tarentolae parasites and its impact on parasite burden during the development of infection. Our results show that ARG is highly upregulated in all pathogenic promastigotes as compared to non-pathogenic L. tarentolae and had a negative correlation between production of ARG and NO. We showed that during the development of the infection in susceptible BALB/c and resistant C57BL/6 mice, ARG is induced in both strains of mice infected with pathogenic Leishmania but not in non-pathogenic counterparts. These results suggest that ARG activity of Leishmania is essential for parasite survival in vitro and in vivo that directly regulates its growth and replication inside the host cells; therefore, it can be used as a significant marker of disease severity in CL and possible tools for drug designing.

Summary Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of L. major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites’ growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number, or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania’ growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL. This article is protected by copyright. All rights reserved.

Background: The present study was conducted to investigate the optimized timing for macrophages induction and nitric oxide (NO) production against invading Leishmania parasite. Methods: The present study examined the murine macrophage cell line, B10R, in three different states. In the first state, the cells were first infected with L. major and then treated with IFN-γ and LPS as stimulants. In the second state, the cells were infected after stimulation with IFN-γ and LPS. In the third state, the cells were only exposed to stimulants as controls. In all the three states, cell culture supernatants were collected at three points in time (6, 24 and 48 h) and the amount of NO production was measured using Griess assay. Results: The treatment of macrophages with inducers prior to infection with stationary phase parasite led to the secretion of significant amounts of NO, particularly at early time points quit contrary to the cells infected with parasites prior to induction. The amount of NO produced by cells induced after infection was detected significantly lower. Conclusion: The induction of macrophages prior to infection with parasites leads to the production and secretion of greater amounts of NO, resulting in an increased ability to suppress and inhibit parasite proliferation even in the early stages of infection.

Background: Pumilio/fem-3 mRNA binding factor (PUF) proteins can bind RNA in a sequence-specific manner. The deciphered RNA-recognition code of these proteins has enabled researchers to design engineered PUF proteins, capable of binding to any desired target in order to modify its ultimate fate. In this study, a modified Homo sapiens Pumilio 1-homology domain (HsPUM1-HD) was engineered to bind to the internal ribosome entry site (IRES) of hepatitis C virus (HCV) genome to potentially inhibit viral translation. Methods: Based on the RNA-recognition code, required modifications were applied to HsPUM1-HD in order to change its natural recognition sequence to a sequence in the stem-loop III of HCV IRES. RNA protein pull-down assay was performed to assess the sequence specificity of the modified HsPUM1-HD (mHsPUM1-HD). Translational inhibitory effect of mHsPUM1-HD was evaluated in a dual-luciferase reporter assay. Results: The mHsPUM1-HD was found to bind to its cognate RNA in a sequence-specific manner, as a biotinylated target RNA captured mHsPUM1-HD through binding to streptavidin magnetic beads. This protein also reduced HCV IRES-dependent firefly luciferase translation by 40% in HEK293 cells. Conclusions: The present study is the first report of an engineered HsPUM1-HD with potential anti-HCV activity. These findings suggest that PUM-HDs can be engineered to target desired RNAs of infectious agents in order to specifically interrupt protein translation, as an essential step of their life cycle.

Introduction: Leishmania tarentolae (L. tarentolae ) is a nonpathogenic species of Leishmania genus that can be used as an expression system to produce immunogenic proteins or epitopes in vivo acting as an efficient and safe recombinant live vector in vaccinology. Cysteine proteases (CPs) are one group of Family C1 peptidases in Leishmania that are important for survival of the parasite within the host and are introduced as potential vaccine candidates. Two types of cysteine proteases, CPA and CPB, play a critical role in Leishmania . Previously, it has indicated that immunization with two genes or recombinant proteins of CPA and CPB individually or fused together with various adjuvant or combined with liposome are able to elicit a protective immune response against L. major in BALB/c mice. Methods: In this study, for the first time, two lines of recombinant nonpathogenic Leishmania were generated by transfection of a heterologous triple fusion gene encoding cpa/cpb/egfp . For this purpose, two different expressions approaches were taken; episomal expression using rDNA promoter, and integrative expression from rRNA locus of genome. Results: After genotype confirmation, the fluorescence intensity was monitored in different phases of parasite growth by both fluorescence microscopy and FACS analysis.Western blot and RT-PCR analysis showed that cpa/cpb/egfp tri-fused genes were specifically expressed in both lines of transgenic parasites. Discussion: In this study a single fused gene was expressed in both systems and the results showed that the expression of integrated gene was higher than episomal. In addition, the present data suggest that L. tarentolae expressing cpa/cpb/egfp is a promising carrier as vectored vaccine in future research. Vac Res , 2014, 1 (1): 9 Pages

Designing an ideal vaccine against leishmaniasis using a suitable candidate antigen and appropriate antigen delivery system to induce the accurate type of immune response is a process still under investigation. There are some different strategies applied for vaccination against leishmaniasis including conventional and modern (genetically-modified vaccines) approaches. The type of induced immune response for each vaccine depends on the type of vaccine and route of administration, the amount and nature of the antigen/s, as well as adjuvant (if used). Recombinant DNA technology or genetic engineering has accelerated the identification of new types of prophylaxis approaches using crude lysates to single or multi-proteins, epitopes and DNA vaccines that are capable of creating less or more partial protection. At present, a few limited vaccine formulations are licensed in Brazil and Europe just for canines. Vaccine development for humans and even for canines is under serious study. In this review, we focus on c...

The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-γ and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy.

Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail.

Leishmaniasis is a parasitic disease transmitted through the bite of an infected phlebotomine sand fly and caused by protozoan parasites of the genus Leishmania. There is no available vaccine for leishmaniasis in human, and the current chemotherapy approaches are hampered by different clinical problems. Most of available drugs are confined to a limited number of toxic chemical compounds, which some parasite strains have evolved drug resistance against. Hence, drug discovery and production of a new anti leishmanial compound is essential. One promising strategy is using the nanoparticle delivery systems with the aim of accelerating the efficacy of the available treatments. In the present study, paromomycin sulfate (PM) was formulated in solid lipid nanoparticles (SLN) and the in vivo efficacy was investigated against Leishmania tropica in BALB/c mice model. To do so, the increase in footpad thickness was measured and real-time PCR was performed to quantify the parasite load after infectious challenge. The level of nitric oxide and cytokines including interleukin-4 (IL-4) and gamma interferon (IFN -γ) were assessed. Altogether, the results show that PM loaded into SLN is significantly more effective than PM alone in inhibiting the parasite propagation and switching towards Th1 response.

Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail.