Multiple Sclerosis Gene Therapy Using Recombinant Viral Vectors: Overexpression of IL-4, IL-10 and Leukemia Inhibitory Factor in Wharton's Jelly Stem Cells in The EAE Mice Model

Objective: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. Materials and Methods: In this experimental study, a DNA construction containing IL-4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2x10(5) rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. Results: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with > 90% efficiency. Recombinant viruses were produced and results of titration showed 2-3x10(7) infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. Conclusion: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model.

OBJECTIVES: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. MATERIALS AND METHODS: In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×105 rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. RESULTS: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×107 infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. CONCLUSIONS: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Designing, Implementation and Evaluation of Bacteriology Courses in Active-Collaborative Learning by Using the Complete-Form Workbook

Background & Objectives: One of the most important elements in SPICES model is student-centered education. Taking notes is one of the simplest ways to increase student interaction and participation in learning. The purpose of this project was to design, implement, and evaluate an active-interactive education model in teaching the bacteriology course. Materials & Methods: This project was carried out in three stages including design, implementation and evaluation. At the design stage, the outline of the program was determined through meetings with faculty members from Microbiology and Infectious Diseases departments; the curriculum-based workbook was designed based on a mind map form; then, teaching was based on the workbook and student participation in the classes. Finally, project evaluation was done by completing the evaluation form by students and then statistical analysis was performed. Results: Statistical results comparing the students of Medicine and Laboratory Science showed the highest student satisfaction with using book and suggestion of using book for other courses, and least satisfaction with graphical suitability and structural design in both groups. Significant differences between the two groups were found to be statistically significant for the students of Medicine compared to the students of the Laboratory Science (p = 0.049). Conclusion: The use of filling-out form textbooks has an important role in enhancing students' interaction and participation in Medical Bacteriology education. It is suggested that a similar structure to this book be explored to teach other Microbiology courses

Multiple alignment of the P7 protein sequences in HCV strains.

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S2 Fig -

Linear (A and C) and Discontinuous (B and D) B-cell epitopes of the construct 1 (A and B) and construct 2 (C and D) vaccines (colored spheres). (DOCX)</p

Helper T Lymphocyte (HTL) epitopes of the NS3 protein.

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The 3D view of the final system conformations.

The interface residues between two proteins TLRs (orange cartoon) and vaccines (magenta cartoon) residues (orange and magenta sticks) are labeled. Hydrogen bonds and hydrophobic contacts are presented as green dashed line and arc with spokes radiating, respectively. A and B indicate TLR4-construct 1 and TLR3-construct 2 complexes, respectively.</p

Various features of multi-epitope vaccines.

Various features of multi-epitope vaccines.</p

Multiple alignment of the NS5B protein sequences in HCV strains.

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Multiple alignment of the NS3 protein sequences in HCV strains.

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