Immunogenic properties of empty pcDNA3 plasmid against zoonotic cutaneous leishmaniasis in mice.

BACKGROUND: Leishmania (L) parasite, the causative agent of zoonotic cutaneous leishmaniasis (ZCL), effectively stimulates the mammalian cells to mount strong humoral responses by enhancing T-helper-2 (Th2)-associated cytokines for its survival. The best strategy to decrease the intensity of infection in the host is induction of cellular immunity. METHODS: We evaluated the effects of the empty bacterial pcDNA3 plasmid on mice infected with L. major and quantified the immune mediators including IFN-γ, IL-4, IL-10, IgG2a, IgG1, arginase activity and nitric oxide (NO) in the mice. Moreover, the footpad lesion size and parasite load were assessed. RESULTS: We observed that pcDNA3 could modulate the immune responses in favor of host cells and decrease the disease severity. Th2- associated mediators, including arginase, IL-4, and IL-10 are downregulated, while cellular responses are upregulated in line with an increase in the levels of nitric oxide (NO) and interfero-gamma (IFN-γ). Interestingly, pcDNA3 induced specific Th1-associated antibodies, IgG2a isotype; however, it suppressed the production of humoral IgG1. The stimulation of the immune response by the empty pcDNA3 is able to shift the immune function to predominant cellular responses caused by Th1, and it had a positive effect on the treatment of zoonotic cutaneous leishmaniasis (ZCL). CONCLUSIONS: Altogether, we introduced the pcDNA3 as a potential interfering factor in the modulation of the immune system against ZCL. Since this vector has been widely used as a control group in different studies, we suggest that the potential function of the empty vector should be deeply assessed, as it exerts anti-parasitic effects on mice infected with L. major.

Arginase/nitric oxide modifications using live non-pathogenic Leishmania tarentolae as an effective delivery system inside the mammalian macrophages.

Recombinant live delivery system based on chemokine IFN-γ-inducible protein-10 kDa (CXCL 10 or IP-10), as a suitable immunotherapy tool, have been used for the treatment of Leishmania infections. This chemokine can defeat Leishmania spp. infection via producing nitric oxide (NO) for parasite killing. This study was performed to investigate the effects of IP-10 on the infected human macrophages by L. tarentolae expressing IP-10. We also quantified the arginase activity and NO production in the co-cultured human macrophages with L. tarentolae expressing IP-10 as compared with wild L. tarentolae. The results elucidate that in the infected cells with L. tarentolae expression of IP-10 the arginase activity decreased, and inversely, NO production intensely increased. Altogether, L. tarentolae expressing IP-10 shows a favorable therapeutic tool to improve the treatment of Leishmania infection. This work suggests that L. tarentolae expressing IP-10 cause specific effects on the metabolic pathways of the macrophage host, which might enable the host cells in killing of parasites and decreasing the survival of them against Leishmania infection.

Sambucus ebulus extract stimulates cellular responses in cutaneous leishmaniasis.

This is the first study aiming to determine the therapeutic effects of the Sambucus ebulus aquatic extract as an antileishmanial herbal drug and evaluate the immune responses in Leishmania major major infected BALB/c mice. The antileishmanial activity of S ebulus aquatic extract was evaluated using MTT test as well as parasite rescue and transformation assay. Footpad swelling and parasite load of infected mice were measured by several techniques. The immune responses were evaluated by measuring the levels of IFN-γ, IL-4, nitric oxide and arginase. The results indicated that S. ebulus can significantly decrease L. major promastigotes and amastigotes viability, but it was not toxic to macrophages. The lesion size, parasite burden and the level of ARG decreased in the treated infected mice, while the IFN-γ-to-IL-4 ratio and the level of NO increased significantly. Altogether, the S. ebulus extract is an effective compound for killing Leishmania parasite without excessive toxicity to the host cells and can cure the CL by switching the host immune responses towards Th1 response. Thus, it may be a perfect therapeutic option for CL treatment.

Effect of elaidic acid on ABCA1 expression in raw 264.7 cells. Is it through PPAR-gamma?

In recent years, Trans Fatty Acids have shown a strong correlation with cardiovascular disease. However, the mechanisms explaining their atherogenicity are still unclear. ABCA1, which is involved in the reverse cholesterol transport pathway, has been considered as a new therapeutic target for cardiovascular disease. In vitro studies of the effects of PPAR-γ on lipid homeostasis in macrophage cells suggested a role for PPAR-γ in the regulation of ABCA1-dependent cholesterol efflux to apoA-I pathway. Thus, in this study we examined the effect of elaidic acid (EA) as the most abundant TFA on expression of ABCA1 and PPAR-γ in RAW 264.7 mouse macrophage cell line. Accordingly, after determining appropriate concentrations of EA using MTT, RAW 264.7 cells were treated with different concentrations of EA, and at the end, gene expression was assayed by Real-Time PCR. Our results shown that the expression of ABCA1 decreased in the treated group in comparison with the control group by 1.7, 2.3, and 5.1 fold, after 12 h treatment for 0.5, 1, and 2 mM EA concentration respectively. In addition, after 24 h treatment with EA, the rate of decreasing ABCA1 expression was 2.1, 2.6, 5.7 fold, respectively (P < 0.01). However, EA had no significant effect on PPAR-γ mRNA expression. Therefore, it could be concluded that the atherogenic effect of EA may be mediated by reducing ABCA1 expression in RAW 264.7 cells; however, this reduction has not mediated through altering PPAR-γ expression.

Human Neutrophil Peptide 1 as immunotherapeutic agent against Leishmania infected BALB/c mice.

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.

Live Leishmania tarentolae secreting HNP1 as an immunotherapeutic tool against Leishmania infection in BALB/c mice.

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.