Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas.

BACKGROUND: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

RAF-targeted therapy for hepatocellular carcinoma in the regenerating liver.

BACKGROUND: Post-operative liver regeneration may contribute to tumor recurrence. There is a theoretical need for an adjuvant therapy that can suppress tumor growth without adversely affecting post-operative liver regeneration. OBJECTIVE: To evaluate the effect of RAF inhibitor Sorafenib on cell viability and proliferation of hepatoma cells and hepatocytes in vitro and in an in vivo rat model. METHODS: Cell viability, DNA synthesis, and RAF/MAPK kinase activity in the primary hepatocyte and hepatoma cell lines were investigated after Sorafenib exposure. Sequence analysis of the B-RAF gene in hepatic cells was determined. Tumor markers were compared within the rats after 70% hepatectomy with or without daily oral gavages of Sorafenib. Liver regeneration was assessed by liver function tests and proliferation markers. RESULTS: Primary hepatocytes showed higher cell viability, proliferation rate, and stronger RAF/MAPK kinase activity compared with hepatoma cell lines. The in vivo tumor volumes, size, and metastases were significantly decreased (P < 0.05) whereas no significant change in liver regeneration related to Sorafenib exposure was found (P > 0.05). B-RAF V600E mutation was not detected neither in the hepatic cells nor untransformed hepatocytes. CONCLUSIONS: The RAF targeted inhibitor can reduce tumor growth without retarding liver regeneration in this experiment.

Hyperhomocysteinemic subjects have enhanced expression of lectin-like oxidized LDL receptor-1 in mononuclear cells.

An elevated plasma concentration of homocysteine is an independent risk factor for cardiovascular disease. However, the mechanisms are still unclear. Lectin-like oxidized LDL receptor-1 (LOX-1) has ligand specificity for oxidized LDL (oxLDL). We hypothesized that homocysteine's atherogenic effects may involve LOX-1-mediated mechanisms. We examined the effect of folic acid supplementation for 6 wk and 12 mo (5 mg/d for 1 wk, 1 mg/d for 37 wk and 0.4 mg/d for the remaining 14 wk) on LOX-1 mRNA levels and on oxLDL-induced release of tumor necrosis factor alpha from peripheral blood mononuclear cells in hyperhomocysteinemic individuals. Compared with healthy controls, hyperhomocysteinemic subjects had elevated mRNA levels of LOX-1 in mononuclear cells (P < 0.001), and their mononuclear cells released more tumor necrosis factor alpha (TNFalpha) upon oxLDL stimulation (P = 0.01). This oxLDL-stimulated release of TNFalpha correlated with LOX-1 expression (r = 0.57, P = 0.026). Folic acid treatment led to a normalization of homocysteine levels accompanied by a reduction in LOX-1 gene expression (P < 0.02) and in oxLDL-stimulated release of TNFalpha (P < 0.05). These novel findings suggest both that homocysteine exerts its atherogenic effect in part by elevating levels of LOX-1, thereby enhancing oxLDL-induced inflammatory responses, and most important, that folic acid supplementation may downregulate these responses.