Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Electrochemical approach for the analysis of DNA degradation in native DNA and apoptotic cells.

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

Effect of the Teeth Whitening Procedure on the Mineral Composition of Oral Fluid.

The basis of modern tooth whitening systems is the use of a whitening gel, which usually contains hydrogen peroxide or carbamide peroxide. The study included 81 patients aged 22 to 35 years with a tooth color A2 and a darker color on the Vita Classic scale. The purpose of our research was to identify a new approach to whitening teeth to improve safety and gentleness. To perform this, we assessed the effect of the tooth whitening procedure on the mineral composition of the oral fluid. A new approach to the teeth whitening procedure was to use a mouth retractor and a tool for aspirating the whitening gel, which we developed. Before the procedure, a protective film-forming aerosol, which included sodium ascorbate, was applied. After the tooth whitening procedure, the enamel was remineralized with a sealing liquid for 14 days. The concentrations of calcium and phosphorus in the oral fluid were determined using a spectrophotometer with a set of reagents (Human). The results obtained indicate that the new approach to the teeth whitening procedure contributed to less pronounced changes in the concentrations of calcium (+29.07, p < 0.001) and phosphorus (-14%, p < 0.001) in the oral fluid immediately after the procedure and in combination with the standard procedure for teeth whitening; immediately after this procedure, the calcium concentration increased by 74.4% (p < 0.001), and the phosphorus concentration decreased by 23.07% (p < 0.001). The use of remineralizing agents led to a faster recovery of the initial levels of calcium and phosphorus in the oral fluid.

Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

The maturation, development, and function of regulatory T cells (Tregs) are under the control of the crucial transcription factor Forkhead Box Protein 3 (FoxP3). Through alternative splicing, the human FoxP3 gene produces four different splice variants: a full-length variant (FL) and truncated variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant) or a deletion of both exons (∆2∆7 variant). Their involvement in the biology of Tregs as well as their association with autoimmune diseases remains to be clarified. The aim of this work was to induce a single FoxP3 splice variant in human Tregs by splice switching oligonucleotides and to monitor their phenotype and proliferative and suppressive activity. We demonstrated that Tregs from peripheral blood from patients with multiple sclerosis preferentially expressed truncated splice variants, while the FL variant was the major variant in healthy donors. Tregs with induced expression of truncated FoxP3 splice variants demonstrated lower suppressive activity than those expressing FL variants. Reduced suppression was associated with the decreased expression of Treg-associated suppressive surface molecules and the production of cytokines. The deletion of exons 2 and/or 7 also reduced the cell proliferation rate. The results of this study show an association between FoxP3 splice variants and Treg function and proliferation. The modulation of Treg suppressive activity by the induction of the FoxP3 FL variant can become a promising strategy for regenerative immunotherapy.