Background: G47∆ is a triple-mutated oncolytic herpes simplex virus type 1 (HSV-1) recently approved as a new drug for malignant glioma in Japan. As the next-generation, we develop armed oncolytic HSV-1 using G47∆ as the backbone. Because oncolytic HSV-1 elicits specific antitumor immunity, interleukin 12 (IL-12) can function as an effective payload to enhance the efficacy. Methods: We evaluate the optimal methods for expressing IL-12 as a payload for G47∆-based oncolytic HSV-1. Two new armed viruses are generated for evaluation by employing different methods to express IL-12: T-mfIL12 expresses murine IL-12 as a fusion peptide, with the genes of two subunits (p35 and p40) linked by bovine elastin motifs, and T-mIL12-IRES co-expresses the subunits, with the two genes separated by an internal ribosome entry site (IRES) sequence. Results: T-mfIL12 is significantly more efficient in producing IL-12 than T-mIL12-IRES in all cell lines tested, whereas the expression methods do not affect the replication capabilities and cytopathic effects. In two syngeneic mouse subcutaneous tumor models of Neuro2a and TRAMP-C2, T-mfIL12 exhibits a significantly higher efficacy than T-mIL12-IRES when inoculated intratumorally. Furthermore, T-mfIL12 shows a significantly higher intratumoral expression of functional IL-12, causing stronger stimulation of specific antitumor immune responses than T-mIL12-IRES. Conclusions: The results implicate that a fusion-type expression of IL-12 is a method superior to co-expression of separate subunits, due to higher production of functional IL-12 molecules. This study led to the creation of triple-mutated oncolytic HSV-1 armed with human IL-12 currently used in phase 1/2 trial for malignant melanoma.

Objective: Endomyocardial biopsy is the gold standard method for the diagnosis of cardiac allograft rejection. However, it causes damage to the heart. In this study, we developed a noninvasive method for quantification of granzyme B (GzB) in vivo by targeted ultrasound imaging, which detects and provides quantitative information for specific molecules, for acute rejection assessment in a murine cardiac transplantation model.Methods: Microbubbles bearing anti-GzB antibodies (MB_Gzb) or isotype antibodies (MBcon) were prepared. Hearts were transplanted from C57BL/6J (allogeneic) or C3H (syngeneic) donors to C3H recipients. Target ultrasound imaging was performed on Days 2 and 5 post-transplantations. A pathologic assessment was performed. The expression of granzyme B and IL-6 in the heart was detected by Western blotting.Results: After MB injection, we observed and collected data at 3 and 6 min before and after the flash pulse. Quantitative analysis revealed that the reduction in peak intensity was significantly higher in the allogeneic MB_Gzb group than in the allogeneic MB_con group and the isogeneic MB_con group at PODs 2 and 5. In the allogeneic groups, granzyme B and IL-6 expression levels were higher than those in the isogeneic group. In addition, more CD8 T cells and neutrophils were observed in the allogeneic groups.Conclusion: Ultrasound molecular imaging of granzyme B can be used as a noninvasive method for acute rejection detection after cardiac transplantation.

Background: Human epithelial cell sheets (ECSs) are used to clinically treat epithelial conditions such as burns, corneal blindness, middle ear cholesteatoma and vitiligo. As a widely used material in clinic, there is little information on the biobanking of ECSs and its repair effect after storage. Results: Two methods for biobanking foreskin ECSs were compared in a short term (7 days): 4-degree storage and programmed cryopreservation. Cell sheet integrity, viability, apoptosis, immunogenicity, mechanical properties and function were evaluated. In vivo, ECSs were directly transplanted to skin defect models and histological examination was performed at 1 week postoperatively. We successfully extracted human foreskin-derived primary epithelial cells and fabricated them into ECSs. Compared with 4-degree storage, programmed cryopreservation preserved the ECS structural integrity, enhanced the mechanical properties, decreased HLA-I expression, and increased cell viability and survival. An increased proportion of melanocytes with proliferative capacity remained in the cryopreserved sheets, and the undifferentiated epithelial cells were comparable to those of the fresh sheets. In vivo, cryopreserved ECSs could reduce inflammatory cell infiltration and promote connective tissue remodeling, epithelial cell proliferation and vascular regeneration. Conclusions: Programmed cryopreservation of ECSs was superior and more feasible than 4-degree storage and the cryopreserved ECSs achieved satisfying skin wound healing in vivo. We anticipate that the off-the-shelf ECSs could be quickly used, such as, to repair human epithelial defect in future. Graphical

Kidney transplantation is a primary therapy for end-stage renal disease (ESRD) all the time. But it does not mean that we have fully unraveling the mystery of kidney transplantation and confer every patient favorable prognosis. Immune rejection has always been a stumbling block when we try to increase the success rate of kidney transplantation and improve long-term outcomes. Even if the immune rejection is effectively controlled in acute phase, there is a high possibility that the immune response mediated by chronically activated antibodies will trigger chronic rejection and ultimately lead to graft failure. At present, immunosuppressive agent prepared chemically is mainly used to prevent acute or chronic rejection, but it failed to increase the long-term survival rate of allografts or reduce the incidence of chronic rejection after acute rejection, and is accompanied by many adverse reactions. Therefore, many studies have begun to use immune cells to regulate the immune response in order to control allograft rejection. This article will focus on the latest study and prospects of more popular regulatory myeloid cells in the direction of renal transplantation immunotherapy and introduce their respective progress from experimental research to clinical research.

Background: Presenilin enhancer gamma-secretase subunit (PSENEN), the straight target of metformin, is highly expressed in several cancers. The role of PSENEN in kidney renal clear cell carcinoma (KIRC) has not been reported. Methods: PSENEN expression in KIRC specimens was investigated in The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, as well as by immunohistochemical analysis and qPCR assay. The relationship between PSENEN expression and patient survival was discussed. The biological function of PSENEN in KIRC and its correlation with immune infiltration of KIRC were then investigated, and possible cellular mechanisms were again analyzed. The effects of metformin on KIRC cell proliferation, migration and invasion were discussed in cellular experiments. Results: PSENEN was found to be highly expressed in KIRC. The high PSENEN expression was an adverse factor in KIRC. Several immune-related pathways were enriched including immune response, complement and coagulation cascade reactions, and neutrophil extracellular trap formation, as evidenced by enrichment analyses. Immune infiltration analysis revealed that PSENEN expression correlated positively with regulatory T cells. Gene set variation analysis suggested that PSENEN expression correlated positively with oxidative phosphorylation. In addition, a certain concentration of metformin was found to inhibit the proliferation, migration and invasion of KIRC cells, in which PSENEN down-regulation, AMPK up-regulation and mTOR down-regulation were also observed. Conclusions: PSENEN may be involved in regulating the immune microenvironment of KIRC, and oxidative phosphorylation may also be a pathway for its involvement in cancer development. PSENEN is a novel prognostic marker for KIRC.