Liver Expression of a MiniATP7B Gene Results in Long-Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice.

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Tempranillo-derived grape seed extract induces apoptotic cell death and cell growth arrest in human promyelocytic leukemia HL-60 cells.

Although grape seed extract (GSE) has proven to be effective against various cancers, few studies have investigated the effects of GSE on human leukemia. In this study, we analysed the mechanisms involved in the apoptotic effects induced by GSE on human promyelocytic leukemia HL-60 cells. Thus, GSE treatment succeeded in activating caspase-3 (P < 0.05), the activation being dose-dependent and time-dependent. Activation of caspase-3 induced by GSE was accompanied by mitochondrial membrane depolarization (P < 0.05). Moreover, disruption of mitochondrial integrity caused by GSE treatment subsequently led to activation of caspase-9 (P < 0.05), and also produced a slight increase in ROS levels (P < 0.05). Cytotoxic effects elicited by GSE treatment ultimately resulted in extensive S-phase arrest (P < 0.05) and a substantial increase in the intrinsic rate of apoptosis (P < 0.05). Our findings suggest that the GSE induces apoptotic cell death and cell growth inhibition in human leukemic HL-60 cells, which seems to be dependent on mitochondrial damage. Therefore, the GSE obtained from Tempranillo cultivars could be an effective approach to restrain uncontrolled cell proliferation and survival in leukemia cells.

Urinary 6-sulfatoxymelatonin and total antioxidant capacity increase after the intake of a grape juice cv. Tempranillo stabilized with HHP.

Red grapes contain elevated amounts of antioxidant compounds (polyphenols) that may potentially prevent cell aging, cardiovascular disease and oxidation-related disorders. Since functional drinks are presently one of the most dynamic sectors of the market, the present work was aimed at evaluating the possible antioxidant effect of an experimental grape juice in terms of urinary 6-sulfatoxymelatonin (aMT6-s) and total antioxidant capacity in young (20 ± 10 yr-old), middle-aged (45 ± 10 yr-old) and elderly (75 ± 10 yr-old) individuals. Grapes (Vitis vinifera cv. Tempranillo) were de-stemmed, racked and pressed. The juice was subsequently stabilized by high hydrostatic pressure (HHP). Participants consumed 200 mL of grape juice twice a day (as the lunch and dinner desserts) for 5 days. First-void morning urines were collected before treatment (basal values), the day immediately after the last ingestion of juice (assay), and one day afterwards (post-assay). aMT6-s and total antioxidant capacity were quantified using commercial ELISA and colorimetric assay kits, respectively. The intake of grape juice cv. Tempranillo induced a significant increase of urinary aMT6-s and total antioxidant capacity in the three groups of age analyzed as compared to their corresponding basal and post-assay values. These functional/nutraceutical properties may be of interest for a prospective commercialization of the grape juice. The novel technology used for juice stabilization may be suitable for introducing into the market a product with high sensory and nutritional quality, as it has been shown in this study.