Previously, we engineered oncolytic herpes simplex viruses (o-HSVs) retargeted to the HER2 (epidermal growth factor receptor 2) tumor cell specific receptor by the insertion of a single chain antibody (scFv) to HER2 in gD, gH, or gB. Here, the insertion of scFvs to three additional cancer targets—EGFR (epidermal growth factor receptor), EGFRvIII, and PSMA (prostate specific membrane antigen)—in gD Δ6–38 enabled the generation of specifically retargeted o-HSVs. Viable recombinants resulted from the insertion of an scFv in place of aa 6–38, but not in place of aa 61–218. Hence, only the gD N-terminus accepted all tested scFv inserts. Additionally, the insertion of mIL12 in the US1-US2 intergenic region of the HER2- or EGFRvIII-retargeted o-HSVs, and the further insertion of Gaussia Luciferase, gave rise to viable recombinants capable of secreting the cytokine and the reporter. Lastly, we engineered two known mutations in gB; they increased the ability of an HER2-retargeted recombinant to spread among murine cells. Altogether, current data show that the o-HSV carrying the aa 6–38 deletion in gD serves as a platform for the specific retargeting of o-HSV tropism to a number of human cancer targets, and the retargeted o-HSVs serve as simultaneous vectors for two molecules.

Background: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). Methods: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. Results: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. Conclusions: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

Over the past decade, the interest in using 3D cell culture models for studying the mammary gland in biomedical and veterinary fields has increased, but a fully functional in vitro model for domestic species is still lacking. Multiple cellular components, including epithelial cells, vascular endothelial cells, and stromal/stem cells, sustain the secretory mammary gland tissue in a well-organized 3D architecture. Considering the Göttingen Minipigs widely used for translational lactation studies, this work aimed to establish a 3D culture protocol to generate mammary heterogeneous multicellular spheroids composed of three different Göttingen Minipigs primary cells: mammary epithelial cells (mpMECs), aortic endothelial cells (mpAECs), and vascular-wall mesenchymal stem cells (mpVW-MSCs). Cells were cultured with hanging-drop (HD) and ultra-low-adherence plate (ULA) methods, evaluating aggregate formation in both monocultures and co/triple co-cultures. Brightfield area, eccentricity, viability, and cell distribution were analyzed. Results showed mpMECs formed irregular aggregates in both HD and ULA, while more compact and viable spheroids were formed when co-cultured with mpVW-MSCs and mpAECs by ULA. A well-organized cellular distribution was demonstrated by cytokeratin-18, vimentin, and e-NOS immunofluorescence analysis. In conclusion, this study established a stable 3D mammary multicellular spheroid model, representing a promising tool for future studies on hormonal modulation and mammary gland physiology.