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.publishedVersionPeer reviewe

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity

Comparative assessment of recombinant and native immunogenic forms of Fasciola hepatica proteins for serodiagnosis of sheep fasciolosis

Laboratory diagnosis of sheep fasciolosis is commonly performed by coprological examinations; however, this method may lead to false negative results during the acute phase of the infection. Furthermore, the poor sensitivity of coprological methods is considered to be a paradox in the chronic phase of the infection. In this study, we compared the immunoreactivity of native and recombinant forms of Fasciola hepatica excretory/secretory antigens and determined their capabilities for the development of F. hepatica-specific immunoassays. Immunoreactivity and specificity of recombinant and native forms of F. hepatica antigens, including fatty acid binding protein (FABP), glutathione-S-transferase (GST), and cathepsin L-1 (CL1), in parallel with native forms of FABP and GST, were studied for serodiagnosis of the chronic form of sheep fasciolosis, individually or in combination with each other by enzyme-linked immunosorbent assays (ELISA). The correlation of the findings was assessed by receiver-operator characteristic (ROC); furthermore, the specificity and sensitivity were assessed by Youden's J. Serologic cross-reactivity was evaluated using samples from healthy sheep (n = 40), Fasciola-infected sheep (n = 30), and sheep with other parasitic infections (n = 43). The FABPs were determined to be greater than 95% sensitive for F. hepatica serodiagnosis. The most desirable diagnostic recombinant antigen was rCL1, which showed 100% sensitivity and 97% specificity in ELISA and was capable of discriminating the positive and negative samples by maximum Youden's J results. We conclude that rCL1 can be used for routine serodiagnosis of chronic fasciolosis. Thus, it could be advantageous in development of immunoassays for screening of ovine herds in fasciolosis-endemic areas and as a reliable agent for detection of fasciolosis in non-endemic regions

Identification and characterization of the first fish parvalbumin-like protein data from a pathogenic fungal species, Trichophyton violaceum

Parvalbumins are the most important fish allergens, which are heat-stable, classified in the family of calcium-binding EF-hand proteins, and contain one magnesium binding site. The functional connection between calcium and parvalbumin gives fish the high-speed swimming ability because of high concentration of Ca2+-binding parvalbumin in fish white muscles. Although parvalbumins are widely studied and conceivably play crucial roles in the physiology and swimming pattern of fishes, still no report is available about their presence in microbes, such as pathogenic fungal species. We detected a DNA sequence in the genome of Trichophyton violaceum and used in silico and polymerase chain reaction (PCR) technique with a designed pair of primers to identify it as parvalbumin-coding gene.publishedVersionPeer reviewe

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity

Promising effects of formononetin, a natural isoflavone derived from herbs, against Toxoplasma gondii

Introduction: Formononetin (FMN) is a natural isoflavone found in many plants. This work examined the anti-Toxoplasma effects and cytotoxicity properties of FMN on Toxoplasma gondii. Methods: Effects of FMN (2-64 µg/mL) on tachyzoites forms were measured by cell viability assay for 48 hours. The effects of different concentrations of FMN on infectivity rate, intracellular parasites, and nitric oxide (NO) in macrophage cells (J774-A1) were also evaluated. Results: FMN markedly (P<0.001) reduced the viability rate of tachyzoites forms with an IC50 value of 9.85 μg/mL. FMN also declined the rate of intracellular tachyzoites whereas, FMN increased the FMN production in macrophage cells. Conclusion: The results of the present in vitro study revealed the favorable anti-Toxoplasma effects of FMN against tachyzoites and intracellular forms of T. gondii. Although the accurate anti-Toxoplasma mechanisms of FMN are not clear, our results showed that triggering the NO production might be considered one of the main mechanism actions of FMN for controlling and eliminating T. gondii. However, further surveys are mandatory to assess the effects of FMN in animal models and to evaluate its accurate mechanism actions before its use in clinical phase

Molecular-Based Detection of Leishmania infantum in Human Blood Samples in a New Focus of Visceral Leishmaniasis in Lorestan Province, Iran

Background: The fatal form of leishmaniasis is visceral form (VL), found in some of the countries in the world. Visceral leishmaniasis has been reported sporadically from all provinces in Iran, including Lorestan. This study aimed to characterize parasite species in DAT positive and some of the DAT negative human blood samples of Delfan district, Lorestan Province, central Iran. Methods: Blood samples were collected from different geographical areas of Delfan. Serum was used for DAT test and remained part of molecular study. DNA was extracted by using DNG-plus extracted kit (Cinagen, Iran). Poly­merase chain reaction amplification of Leishmania kDNA and PCR-RFLP of ITS1 was done to identify Leishmania species. Some amplicons were sequenced, submitted to GenBank and analyzed by BLASTn. Results: Expected band of kDNA for L. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-Leishmania antibody at different titers or were negative serologically. Using BLASTn, 93% similarity with L. infantum has been shown. The rDNA-ITS1 was amplified only in 9 samples (4.7%). RFLP pattern was similar to what expected for L. infantum. Conclusion: A new emerging hypo-endemic focus, caused by L. infantum, is going to be established in Delphan District, Lorestan Province. Further studies on vector and reservoirs are necessary for the region and other parts of Lorestan Province

Characterization of a multi-epitope peptide with selective MHC-binding capabilities encapsulated in PLGA nanoparticles as a novel vaccine candidate against Toxoplasma gondii infection

No effective human vaccine against Toxoplasma gondii (T. gondii) has yet been developed; however, a protective vaccine using immunogenic peptides in a safe delivery vehicle system offers promise. Here, we employed bioinformatics to design a multimeric recombinant T. gondii vaccine using predicted T and B cell epitopes of SAG1, AMA1, ROP2, and GRA4 proteins based on their binding capabilities to common major histocompatibility complex (MHC) molecules. Furthermore, we encapsulated the expressed protein in poly lactic-co-glycolic acid (PLGA) nanoparticles as a delivery vehicle and also used alum as an adjuvant to determine the vaccine potency of this multimeric antigen. BALB/c mice were vaccinated and then challenged with T. gondii RH strain, and the survival rate and cytokine profiles were studied. Mice vaccinated with the multi-epitope-based vaccine, both with and without PLGA, had greater Th1 immune responses, survival rates, specific antibody titers, and IFN-γ and IL-2 levels than controls, while the alum-adsorbed vaccine stimulated a Th2-type humoral immune respons