Selenium mitigates methotrexate-induced testicular injury: Insights from male NMRI mice model.

BACKGROUND AND AIM: Chemotherapy, particularly with methotrexate (MTX), often elicits testicular toxicity, leading to impaired spermatogenesis and hormone imbalances. This study aimed to investigate the potential protective effects of selenium (Se) against MTX-induced testicular injury. MATERIALS AND METHODS: Male mice were divided into control, MTX, Se, and MTX + Se groups. Histopathological examination involved the preparation of testicular tissue sections using the Johnsen's tubular biopsy score (JTBS) for spermatogenesis evaluation. Biochemical tests included the assessment of testosterone, malondialdehyde (MDA), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to analyze the expression of caspase 3 (casp3), tumor protein 53 (p53), B-cell lymphoma 2 (Bcl2), and Bcl2-associated X protein (Bax) genes. Statistical analysis was performed using ANOVA and Tukey's tests (p < .05). RESULTS: Histopathological analysis revealed significant testicular damage in the MTX group, with decreased spermatogenesis and Leydig cell count, while Se administration mitigated these effects, preserving the structural integrity of the reproductive epithelium. Biochemical analysis demonstrated that MTX led to elevated malondialdehyde (MDA) levels and reduced testosterone, LH, and FSH levels, suggesting oxidative stress and Leydig cell dysfunction. Gene expression analysis indicated that MTX upregulated proapoptotic genes (casp3, p53, and bax) while downregulating the antiapoptotic Bcl2 gene. In contrast, Se treatment reversed these trends, highlighting its potential antiapoptotic properties. CONCLUSION: Our findings underscore the potential of Se as a therapeutic agent to mitigate the reproductive toxicity associated with MTX-induced testicular injury. Se exerts protective effects by regulating oxidative stress, preserving hormone balance, and modulating apoptotic pathways. These results suggest that Se supplementation could be a promising strategy to alleviate chemotherapy-induced testicular damage and preserve male fertility.

Structural analysis of PpSP15 and PsSP9 sand fly salivary proteins designed with a self-cleavable linker as a live vaccine candidate against cutaneous leishmaniasis.

BACKGROUND: Leishmania parasites are deposited in the host through sand fly bites along with sand fly saliva. Therefore, salivary proteins are promising vaccine candidates for controlling leishmaniasis. Herein, two immunogenic salivary proteins, PpSP15 from Phlebotomus papatasi and PsSP9 from Phlebotomus sergenti, were selected as vaccine candidates to be delivered by live Leishmania tarentolae as vector. The stepwise in silico protocol advantaged in this study for multi-protein design in L. tarentolae is then described in detail. METHODS: All possible combinations of two salivary proteins, PpSP15 and PsSP9, with or without T2A peptide were designed at the mRNA and protein levels. Then, the best combination for the vaccine candidate was selected based on mRNA and protein stability along with peptide analysis. RESULTS: At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSP9. At the protein level, the refined three-dimensional models of all combinations were structurally valid; however, local quality estimation showed that the PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position, with low root-mean-square deviation (RMSD), compared with the original proteins. In silico evaluation confirmed the PpSP15-T2A-PsSP9 combination as a good Th1-polarizing candidate in terms of high IFN-γ production and low IL-10/TGF-ß ratio in response to three consecutive immunizations. Potential protein expression was then confirmed by Western blotting. CONCLUSIONS: The approach presented herein is among the first studies to have privileged protein homology modeling along with mRNA analysis for logical live vaccine design-coding multi-proteins.

Antibiotic-Free Nanoplasmids as Promising Alternatives for Conventional DNA Vectors.

DNA vaccines with their extraordinary properties are the best choice as vectors for subunit vaccines but are not in compliance with safety regulations, mainly because of the antibiotic resistance genes on their backbone. New generations of plasmids with minimum bacterial backbones are now developed as promising alternatives to pass the safety rules and be replaced for conventional plasmids. Here we have compared the nanoplasmid (with RNA-out selection system and professional HTLV-1 containing promoter) and the conventionally used pcDNA plasmid, as regards the transfection efficiency. The EGFP gene was cloned in both pcDNA-3.1+ and NTC9385R-MSC and transfected into COS-7 cells for expression evaluation by flow cytometry. Meanwhile, qPCR was used to analyze the EGFP mRNA copy numbers. It was concluded that the nanoplasmid, with its extraordinary properties, can be a tempting alternative to conventional pcDNA in equal or equimolar concentrations for vaccine design. These promising results can put DNA vaccines back into focus, especially regarding diseases controlled by robust cellular immune responses.

Leishmania tarentolae as Potential Live Vaccine Co-Expressing Distinct Salivary Gland Proteins Against Experimental Cutaneous Leishmaniasis in BALB/c Mice Model.

Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.

Effect of fluoxetine treatment on neurotoxicity induced by lysolecithin in male rats.

Demyelination disorder is an unusual pathologic event, which occurs in the central nervous system (CNS). Multiple sclerosis (MS) is an inflammatory demyelinating disease that affects the CNS, and it is the leading cause of disability in young adults. Lysolecithin (LPC) is one of the best toxin-induced demyelination models. In this study, a suitable model is created, and the effect of fluoxetine treatment is examined on this model. In this case, it was assumed that daily fluoxetine treatment had increased the endogenous remyelination in the LPC model. This study was focused on investigating the influence of the fluoxetine dose of 5 or 10 mg/kg per day for 1 and 4 weeks on LPC-induced neurotoxicity in the corpus callosum region. It was performed as a demyelinating model in male Wistar rats. After 3 days, fluoxetine was injected intraperitoneally (5 or 10 mg/kg per day) for 1 and 4 weeks in each group. After completing the treatment course, the corpus callosum was removed to examine the gene expression and histological analysis was performed. The results of the histopathological study of hematoxylin and eosin staining of the corpus callosum showed that in 1 and 4-week treatment groups, fluoxetine has reduced the level of inflammation at the LPC injection site (5 and 10 mg/kg per day). Fluoxetine treatment in the luxol fast blue (LFB) staining of the corpus callosum has been led to an increase in myelination capacity in all doses and times. The results of the genetic study showed that the fluoxetine has significantly reduced the expression level of tumor necrosis factor-α, nuclear factor κß, and induced nitric oxide synthase in comparison with the untreated LPC group. Also, the fluoxetine treatment has enhanced the expression level of the forkhead box P3 (FOXP3) gene in comparison with the untreated group. Fluoxetine has increased the expression level of myelination and neurotrophic genes such as myelin basic protein (MBP), oligodendrocyte transcription factor 2 (OLIG2), and brain-derived neurotrophic factor (BDNF). The outcomes demonstrated that fluoxetine reduces inflammation and strengthens the endogenous myelination in the LPC-induced demyelination model; however, supplementary studies are required for specifying the details of its mechanisms.

Nano-adjuvant based on silk fibroin for the delivery of recombinant hepatitis B surface antigen.

Nanotechnology has a vital role in vaccine development. Nano-adjuvants, as robust delivery systems, could stimulate immune responses. Using nanoparticles (NPs) in vaccine formulations enhances the target delivery, immunogenicity, and stability of the antigens. Herein, silk fibroin nanoparticles (SFNPs) were used as a nano-adjuvant for delivering recombinant hepatitis B surface antigen (HBsAg). HBsAg was loaded physically and chemically on the surface of SFNPs. The HBsAg-loaded SFNPs had a spherical morphology. The in vitro release studies showed that HBsAg had a continuous and slow release from SFNPs during 56 days. During this time, ∼45.6% and 34.1% HBsAg was released from physical-SFNPs and chemical-SFNPs, respectively. HBsAg-loaded SFNPs were also stable for six months with slight changes in the size, surface charge, and morphology. The results of circular dichroism (CD) and fluorescence spectroscopy indicated that the released HBsAg preserved the native secondary and tertiary structures. The quantitative cellular uptake study also showed that physical-SFNPs were taken up more into J774A.1 macrophage cells than chemical-SFNPs. After 28 and 56 days post-injection, the immunogenicity studies showed that the specific total IgG, IgG1, and IgG2a levels against HBsAg were significantly higher in the physically loaded group than in the chemically loaded group and commercial hepatitis B vaccine. IgG2a levels were detected only in mice immunized with physical-SFNPs. However, the low levels of IL-4 and IFN-γ were produced in all vaccinated groups and differences in mean values were not significant compared with control groups. Results indicated an improvement in the levels of anti-HBsAg IgG in mice immunized with the physical-SFNPs group compared to other groups.

Visualization of Leishmania tropica Infection in BALB/c Mice by Bioluminescence Imaging

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.

The outcome of arginase activity inhibition in BALB/c mice hosting Leishmania tropica.

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.

Antileishmanial effect of rapamycin as an alternative approach to control Leishmania tropica infection.

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.

Correction: DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

[This corrects the article DOI: 10.1371/journal.pntd.0007067.].

DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica.

BACKGROUND: The vector-borne disease leishmaniasis is transmitted to humans by infected female sand flies, which transmits Leishmania parasites together with saliva during blood feeding. In Iran, cutaneous leishmaniasis (CL) is caused by Leishmania (L.) major and L. tropica, and their main vectors are Phlebotomus (Ph.) papatasi and Ph. sergenti, respectively. Previous studies have demonstrated that mice immunized with the salivary gland homogenate (SGH) of Ph. papatasi or subjected to bites from uninfected sand flies are protected against L. major infection. METHODS AND RESULTS: In this work we tested the immune response in BALB/c mice to 14 different plasmids coding for the most abundant salivary proteins of Ph. sergenti. The plasmid coding for the salivary protein PsSP9 induced a DTH response in the presence of a significant increase of IFN-γ expression in draining lymph nodes (dLN) as compared to control plasmid and no detectable PsSP9 antibody response. Animals immunized with whole Ph. sergenti SGH developed only a saliva-specific antibody response and no DTH response. Mice immunized with whole Ph. sergenti saliva and challenged intradermally with L. tropica plus Ph. sergenti SGH in their ears, exhibited no protective effect. In contrast, PsSP9-immunized mice showed protection against L. tropica infection resulting in a reduction in nodule size, disease burden and parasite burden compared to controls. Two months post infection, protection was associated with a significant increase in the ratio of IFN-γ to IL-5 expression in the dLN compared to controls. CONCLUSION: This study demonstrates that while immunity to the whole Ph. sergenti saliva does not induce a protective response against cutaneous leishmaniasis in BALB/c mice, PsSP9, a member of the PpSP15 family of Ph. sergenti salivary proteins, provides protection against L. tropica infection. These results suggest that this family of proteins in Ph. sergenti, Ph. duboscqi and Ph. papatasi may have similar immunogenic and protective properties against different Leishmania species. Indeed, this anti-saliva immunity may act as an adjuvant to accelerate the cell-mediated immune response to co-administered Leishmania antigens, or even cause the activation of infected macrophages to remove parasites more efficiently. These findings highlight the idea of applying arthropod saliva components in vaccination approaches for diseases caused by vector-borne pathogens.

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.

Arginase activity in pathogenic and non-pathogenic species of Leishmania parasites.

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.

Delivery systems for Leishmania vaccine development.

Leishmaniasis is a neglected disease and is endemic in tropical and sub-tropical areas worldwide. Lifelong immunity after recovery indicates that vaccination could be a promising approach to overcome the disease. Although different antigens have been successfully tested against all clinical forms, none of them have been shown to fulfill the safety and efficiency requirements for human applications. Hence, strong vehicles are needed to carry antigens of interest and potentiate its presence in the body. So far, various live or chemical carriers have been applied to reinforce the immunological effects of ideal antigens. In the current review, the recent attempts in this field have been summarized.

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice.

Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study.

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus.

Enhanced protective efficacy of nonpathogenic recombinant leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen.

BACKGROUND: Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. CONCLUSION/SIGNIFICANCE: The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.