Mitochondrial DNA as a target for analyzing the biodistribution of cell therapy products.

Biodistribution tests are crucial for evaluating the safety of cell therapy (CT) products in order to prevent unwanted organ homing of these products in patients. Quantitative polymerase chain reaction (qPCR) using intronic Alu is a popular method for biodistribution testing owing to its ability to detect donor cells without modifying CT products and low detection limit. However, Alu-qPCR may generate inaccurate information owing to background signals caused by the mixing of human genomic DNA with that of experimental animals. The aim of this study was to develop a test method that is more specific and sensitive than Alu-qPCR, targeting the mitochondrial DNA (mtDNA) sequence that varies substantially between humans and experimental animals. We designed primers for 12S, 16S, and cytochrome B in mtDNA regions, assessed their specificity and sensitivity, and selected primers and probes for the 12S region. Human adipose-derived stem cells, used as CT products, were injected into the tail vein of athymic NCr-nu/nu mice and detected, 7 d after administration, in their lungs at an average concentration of 2.22 ± 0.69 pg/µg mouse DNA, whereas Alu was not detected. Therefore, mtDNA is more specific and sensitive than Alu and is a useful target for evaluating CT product biodistribution.

A Novel Therapeutic Reagent, KA-1002 for Alleviating Lysophosphatidic Acid-Mediated Inflammation Related Gene Expression in Swine Macrophages.

Stresses and various infectious reagents caused multiple inflammatory diseases in swine in a livestock industrial environment. Therefore, there is a need for an effective therapeutic or preventive agent that could alleviate chronic and acute inflammation. We found that lysophosphatidic acid (LPA), a stress-induced potent endogenous inflammatory molecule, causes a broad range-regulation of inflammation related genes inflammation in swine macrophages. We further investigated the genome scaled transcriptional regulatory effect of a novel LPA-signaling antagonist, KA-1002 on swine macrophages, inducing the alleviated LPA-mediated inflammation related gene expression. Therefore, KA-1002 could potentially serve as a novel therapeutic or preventive agent to maintain physiologically healthy and balanced conditions of pigs.

Metallothionein-III increases ADAM10 activity in association with furin, PC7, and PKCα during non-amyloidogenic processing.

A-disintegrin and metalloproteinase 10 (ADAM10) is involved in the generation of amyloid-ß (Aß) during amyloid precursor protein (APP) processing, and has a protective effect against Aß neurotoxicity. We explored how metallothionein-III (MT-III) is regulated in the non-amyloidogenic pathway to generate soluble APPα (sAPPα). MT-ІІІ increased sAPPα levels and reduced Aß peptide levels, but did not affect ADAM10 expression. However, MT-III increased the activity of ADAM10. MT-ІІІ-induced sAPPα secretion, and Aß peptide formation was blocked by specific inhibitors of furin, proprotein convertase7 (PC7), and PKCα. These results demonstrate that MT-ІІІ increases the amount of active ADAM10 in association with furin, PC7 and PKCα.