In addition to the immature edible flower heads, the cultivation of globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori) generates substantial quantities of by-products, including leaves, stems, and roots, which constitute potential sources of bioactive compounds and prebiotic dietary fiber. Preserving agricultural biodiversity and promoting socioeconomic development are essential for enhancing domestic production and fostering innovation. In the search for new biomolecules with antioxidant properties, this research focused on a globe artichoke landrace at risk of genetic erosion, still cultivated in the northern part of the Lazio region, known as the “Carciofo Ortano”. To investigate the antioxidant properties of various globe artichoke tissues from the “Carciofo Ortano” landrace, methanolic extracts were prepared from the immature main and secondary flower heads, stems, and leaves of representative genotypes of this landrace. Additionally, extracts were obtained from the same tissues of four landraces/clones included in the varietal platform of the PGI “Carciofo Romanesco del Lazio”, which served as reference genotypes: Campagnano, Castellammare, C3, and Grato 1. The antioxidant properties of these extracts were assessed using FRAP, ABTS, DPPH assays, and total phenolic content (TPC). The stem and secondary flower head extracts of two representative “Carciofo Ortano” genotypes and the Grato 1 clone, which have higher phenolic content, demonstrated the highest antioxidant activity. These extracts were therefore studied for their chemical profile using HPLC-DAD and SPME-GC/MS analysis. Additionally, the same extracts were investigated in vitro for their antioxidant capacity in differentiated SH-SY5Y cells, assessing their effects on ROS levels and the restoration of GSH levels. Furthermore, the in vivo beneficial effects of counteracting oxidative stress were evaluated in high sucrose-fed Drosophila melanogaster, as oxidative stress is a typical hallmark of hyperglycemic status. Overall, the results indicated that the edible immature inflorescences of the “Carciofo Ortano” landrace, along with the byproducts of its cultivation, are sources of raw materials containing biomolecules whose properties can be exploited for further applications in the pharmaceutical and medical sectors.

The C1858T PTPN22 variant is strongly associated with Type 1 diabetes and autoimmune thyroid disease. Current treatment is the substitutive hormonal administration which does not target the disease pathogenetic mechanism. Background/Objectives: We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes in order to halt autoimmune disease progression. The objective of this study was to optimize lipoplexes formulations functionalized with F9-PEG (a Siglec-10's ligand) to facilitate targeted delivery, by testing their physical and chemical properties to warrant best performance in in vivo studies. Methods: The effectiveness of siRNA-liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to methods of F9-PEG addition and ATTO740 fluorescent labelling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS, DELS), and high-performance liquid chromatography (HPLC). A preliminar investigation of lipoplexes biodistribution was conducted by fluorescent tagging in C57BL/6 mice. Results: The optimal charge ratio of +2 /-1 (lipid/siRNA) ensured greater stability of lipoplexes. Stability was improved by functionalization with F9-PEG further by the addition of ATTO740-dye. HPLC confirmed the integrity of siRNA after preparation. Injection of PKH26-labelled lipoplexes in mice showed selective accumulation in target tissues within 48 hours. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.

Background: The C1858T PTPN22 variant is strongly associated with type 1 diabetes and autoimmune thyroid disease. Current treatment is substitutive hormonal administration, which does not target the disease pathogenetic mechanism. We previously implemented a novel immunotherapy, employing siRNA directed against the C1858T variant of PTPN22 delivered via functionalized lipoplexes, in order to halt autoimmune disease progression. Objectives: The objective of this study was to optimize lipoplex formulations functionalized with F9-PEG (a Siglec-10’s ligand) to facilitate targeted delivery by investigating their physical and chemical properties to warrant the best performance in in vivo studies. Methods: The effectiveness of siRNA liposome binding was evaluated by varying the relative lipid/siRNA charge ratio and analyzing the stability of the different formulations with respect to the methods of F9-PEG addition and ATTO740 fluorescent labeling by electrophoresis, dynamic and dielectrophoretic light scattering (DLS and DELS), and high-performance liquid chromatography (HPLC). Results: The optimal charge ratio of +2/−1 (lipid/siRNA) ensured a greater stability of lipoplexes and complete complexation of siRNA. Stability was improved by selecting a protocol of preparation that envisages functionalization with F9-PEG and the addition of ATTO740 lipid in the lipid film preparation step. HPLC confirmed the integrity of siRNA after preparation. Conclusions: The results of this study lead to formulations of F9-PEG lipoplexes with optimal properties that could be used for biodistribution and safety/efficacy studies in mice. Lipoplexes functionalized with F9-PEG could therefore represent a promising personalized nanotherapeutic platform for targeted siRNA delivery in endocrine C1858T patients.

Central tolerance is provided by the autoimmune regulator (AIRE) expressing medullary thymic epithelial cells through high-avidity recognition of self-antigens. Peripheral mechanisms regulate adaptive immunity by deleting autoreactive T-cells that escape thymic selection, or by inducing their functional unresponsiveness through interaction with antigen-presenting cells, exposing cognate antigens. Multiple types of extrathymic AIRE-expressing cells residing in secondary lymphoid organs have been consistently described. We investigated whether AIRE binds to promoters of known autoantigens in human peripheral blood mononuclear cells by chromatin immunoprecipitation from four healthy donors using an anti-AIRE monoclonal antibody. Quantitative real-time PCR was used to detect AIRE occupancy at promoters of selected autoantigens. Accordingly, we revealed promoter amplicons of all tested autoantigen genes, and their corresponding transcripts. Expression of AIRE was further confirmed at both transcriptional and protein levels. Overall, although the role of AIRE in regulating autoantigen expression in thymic epithelial cells has been well reported, our study provides preliminary descriptive evidence of AIRE occupancy at promoters of known autoantigens in human bulk peripheral blood mononuclear cells, suggesting the presence of AIRE-mediated regulatory events in peripheral blood. Our data provide a rationale for future investigations aimed at elucidating the underlying molecular and immunological mechanisms.

The C1858T PTPN22 (R620W) variant has been implicated in the pathogenesis of several autoimmune disorders and represents a promising immunotherapeutic target for Type 1 diabetes. We have been implementing a novel immunotherapeutic approach based on the use of lipoplexes that deliver siRNA duplexes. Efficacy and safety of lipoplexes halting variant expression was demonstrated in the peripheral blood of patients in vitro. According to regulatory authorities in Europe preclinical safety and efficacy must be ascertained in vivo in appropriate animal models before undertaking clinical investigations. In the light of the foregoing the aim of this study was to verify that lipoplexes against the murine Ptpn22-R619W, equivalent to the human PTPN22-R620W, could be used for animal experimentation. The murine fibroblast cell line L929 was transfected with the PF62-pLentiPtpn22-R619W plasmid. We designed siRNA duplexes specific for the Ptpn22-R619W allele and formulated them into cationic lipoplexes in order to halt the variant expression in the transfected L929 cell line. Transfection of fibroblasts expressing R619W using lipoplexes resulted in efficient silencing at 100 pmol siRNA after 48 hours post-transfection reaching higher significant knock‑down after 72 hours. Lipoplexes efficiently suppress the pathogenic Ptpn22 variant expression in vitro, supporting the feasibility of a pre‑clinical platform for in vivo lipoplexes testing in CRISPR‑engineered NOD/ShiLtJ mice carrying the R619W mutation.

Eugenia Belcastro was not included as an author in the original publication