ABSTRACT
Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell [DC] based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc[IgG] can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I [cross priming]
Methods: DNA construct containing fragment C [Fc] portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli [E. coli] strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining
Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification
Conclusion: Due to successful expression of Foxp3-Fc [IgG], it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means
ABSTRACT
Antifungal drug resistance and few numbers of available drugs limit therapeutic options against fungal infections. The present study was designed to discover new antifungal drugs. This study was carried out in two separate steps, that is, in silico lead identification and in vitro assaying of antifungal potential. A structural data file of a ternary complex of fusicuccin [legend], C terminus of H[+]-ATPase and 14-3-3 regulatory protein [lo9F.pdb file] was used as a model. Computational screening of a virtual 3D database of drug-like molecules was performed and selected small molecules, resembling the functional part of the ligand performing ligand docking, were tested using ArgusLab [4.0.1]. Two lead compounds, 3-Cyclohexan propionic acid [CXP] and 4-phenyl butyric acid [PBA] were selected according to their ligation scores. Standard Strains of Candida albicans and Saccharomyces cerevisiae were used to measure the antifungal potential of the two identified lead compounds against the fungi using micro-well plate dilution assay. Ligation scores for CXP and PBA were -9.33744 and -10.7259 kcal/mol, respectively, and MIC and MFC of CXP and PBA against the two yeasts were promising. The evidence from the present study suggests that CXP and PBA possess potentially antifungals properties