Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.

Naloxone/alum mixture a potent adjuvant for HIV-1 vaccine: induction of cellular and poly-isotypic humoral immune responses.

In the present study we used a fusion peptide from HIV-1 p24 and Nef as vaccine model and adjuvant activity of Naloxone/alum mixture was evaluated in a peptide vaccine model. HIV-1 p24-Nef fusion peptide was synthesized. Female BALB/c mice were divided into five groups. The first group immunized subcutaneously with the p24-Nef fusion peptide adjuvanted with Naloxone/alum mixture and boosted with same protocol. The second was immunized with fusion peptide adjuvanted in alum. The control groups were injected with NLX (Group 3), Alum (Group 4), or PBS (Groups 5) under the same conditions. To determine the type of induced immune response, sera and splenocytes were analyzed by commercial ELISA method for total IgG and isotypes and cytokine secretion (IL-4 & IFN-γ), respectively. We have also used the ELISPOT assay to monitor changes in the frequency of IFN-γ-producing T cells. The proliferation of T cells was assessed using Brdu method and T-cell cytotoxicity was assessed with CFSE method. Immunization of mice with HIV-1 p24-Nef fusion peptide formulated in Naloxone/alum mixture significantly increased lymphocyte proliferation and shifted cytokine responses toward Th1 profile compared to all other groups. Analysis of humoral immune responses revealed that administration of HIV-1 p24-Nef fusion peptide with Naloxone/alum mixture significantly increased specific IgG responses and also increased IgG1,IgG2a, IgG2b, IgG3, and IgM vs. alum-adjuvanted vaccine groups. Naloxone/alum mixture as an adjuvant could improve cellular and humoral immune response for HIV vaccine model and this adjuvant maybe useful for HIV vaccine model in human clinical trial.