In silico Analysis of a 29 kDa Echinococcus granulosus Protoscolex Protein (P29) as a Vaccine Candidate against Cystic Echinococcosis.

Vaccination can be a key step in controlling hydatid cyst infection in humans and livestock in endemic areas of the disease. The aim of the Present study was to determine some of the basal biochemical properties followed by prediction and screening of B-cell and MHC-binding epitopes of EgP29 protein in silico. Some of the basic physico-chemical properties along with antigenicity, allergenicity, solubility, post-translational modification (PTM) sites, subcellular localization, signal peptide, transmembrane domain, secondary and tertiary structures followed by refinement and validations were computationally determined for this protein. Also, B-cell epitopes were predicted and screened using various web servers, while MHC-binding and CTL epitopes were predicted using IEDB and NetCTL servers, respectively. The protein is a 238-residue, 27 kDa molecule, with high thermotolerance (aliphatic: 71.81) and hydrophilicity (negative GRAVY). There were several glycosylation and phosphorylation sites in the sequence, without a transmembrane domain and signal peptide. Moreover, several B-cell and MHC-binding epitopes were found in the EgP29 protein, which could be further used in multi-epitope vaccines. In conclusion, results of the present study can be a promising sign for achieving effective approaches to the preparation of a multi-epitope vaccines against echinococcosis. So, it is necessary that the effectiveness of the protein and its epitopes be evaluated in vitro and in vivo.

Evaluation of murine lung epithelial cells (TC-1 JHU-1) line to develop Th2-promoting cytokines IL-25/IL-33/TSLP and genes Tlr2/Tlr4 in response to Aspergillus fumigatus.

OBJECTIVE: The aims of this study were to determine the role of live and heat-killed Aspergillus fumigatus conidia in releasing interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP) and to express Toll-like receptor (Tlr)2 and Tlr4 genes. MATERIALS AND METHODS: Murine lung epithelial cells were incubated with live and heat-killed A. fumigatus conidia at 37°C for 6, 24 and 48h. After treatments, ELISA was performed to measure the concentrations of IL-25, IL-33 and TSLP in the supernatants. Quantitative real-time PCR (qPCR) was performed to assess the expression levels of Tlr2 and Tlr4 genes. RESULTS: The concentrations of IL-25 and IL-33 significantly increased after exposure to live and heat-killed conidia for various times when compared with untreated control (P<0.05). The secretion of TSLP at different concentrations of heat-killed conidia was significantly higher than both live conidia and untreated control (P<0.05). qRT-PCR results indicated a up-regulation from 1.08 to 3.60-fold for Tlr2 gene expression and 1.20 to 1.80-fold for Tlr4 gene expression exposed to heat-killed conidia. CONCLUSION: A. fumigatus has a potential ability to stimulate murine lung epithelial cells to produce IL-25/IL-33/TSLP, as well as to express Tlr2/Tlr4 genes, indicating an important role of lung epithelial cells in innate immune responses to A. fumigatus interaction.

The killing in vitro effect of Half-Wave Rectified Sine electricity plus silver nanoparticle on Leishmania major promastigotes and BALB/C mice skin leishmanial lesion healing.

In this experimental study, first the killing effect of silver nanoparticles alone or in combination with 3mA of Half-Wave Rectified Sine current was assessed in promastigote culture for 10 minutes. The survival rate of infected promastigotes was evaluated by Flow cytometery. In the second step, BALB/c mice were infected experimentally with L. major, followed by silver nanoparticles injected inter-lesion and simultaneously 3 mA a Half-Wave Rectified Sine current induction was applied directly into the wound. Finally, the lesion size and the mice body weight changes were measured during 5 weeks. Results indicated that simultaneous use of nanoparticle and electricity increased the mortality of promastigotes significantly. However, when 3 mA of HWRS and 160 µm/ml nanosilver were used alone in medium culture only 73.4% and 32% of promastigotes were killed respectively but the combined use destroys the promastigotes completely. The diameter of the lesions after six weeks in the control group; group treated with meglumine antimoniate and the group treated with HWRS increased to 6.01, 0.02 and 0.52 mm but in group treated with HWRS plus Nanosilver was reduced to -0.14 mm. The results showed that, when silver nanoparticles with HWRS current electricity were used in mice, the skin lesions were reduced in size but like Glucantime, complete healing was not achieved.

Molecular Cloning and Expression of EG95 Gene of Iranian Isolates of Echinococcus granulosus.

BACKGROUND: Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein, against E. granulosus, has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector. METHODS: Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces, and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step, the subcloned gene was expressed in CHO as eukaryotic cell. RESULTS: EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot. CONCLUSION: Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.