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Islet allotransplantation can achieve insulin independence in patients with type I diabetes. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. However, UW solution has several disadvantages, including the inhibition of Liberase activity. In this study, we investigated the features of a new solution, designated M-Kyoto solution. M-Kyoto solution contains trehalose and ulinastatin as distinct components. Trehalose has a cytoprotective effect against stress, and ulinastatin inhibits trypsin. In porcine islet isolation, islet yield was significantly higher in the M-Kyoto/PFC group compared with the UW/PFC group. There was no significant difference in ATP content in the pancreas between the two groups, suggesting that different islet yields are not due to their differences as energy sources. Compared with UW solution, M-Kyoto solution significantly inhibited trypsin activity in the digestion step; moreover, M-Kyoto solution inhibited collagenase digestion less than UW solution. In conclusion, the advantages of M-Kyoto solution are trypsin inhibition and less collagenase inhibition. Based on these data, we now use M-Kyoto solution for clinical islet transplantation from nonheart-beating donor pancreata.

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Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH 2 -terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet † These authors contributed equally to this study. cells and lead to improvement in the outcomes for pancreatic islet transplantation.

We evaluated islet transplantation from non-heart beating donors (NHBDs) with our Kyoto Islet Isolation Method. All patients had positive C-peptide after transplantation. The average HbA 1C levels of the five recipients significantly improved from 7.8 ± 0.4% at transplant to 5.2 ± 0.2% currently (p < 0.01). Three patients with no or a single autoantibody became insulin independent while the other two patients with double autoantibodies reduced their insulin requirement but did not become insulin independent. C-peptide in patients who became insulin-independent gradually increased after each transplantation whereas C-peptide in patients who did not become insulin-independent from 3 months after the first transplantation to the next transplantation dramatically decreased. The b -score of the three patients who became insulin independent was the best of eight. In conclusion, our method makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.Abbreviations: CIT, cold ischemic time; NHBD, nonheart beating donor; PFC, perfluorochemical; WIT, warm ischemic time

For islet transplantation, it is important to obtain an available islet mass adequate for diabetes reversal from a single donor pancreas. A recent report demonstrated that the use of M-Kyoto solution instead of UW solution improved islet yields in the two-layer method for pancreas preservation. The present study investigated whether the ductal injection of a large volume of preservation solution (UW and M-Kyoto solution) before pancreas storage improves islet yields. Islet yield both before and after purification was significantly higher in the ductal injection (+) group compared with the ductal injection (-) group. TUNEL-positive cells in the ductal injection (+) group were significantly decreased in comparison to the ductal injection (-) group. The ductal injection of preservation solution increased the ATP level in the pancreas tissue and reduced trypsin activity during the digestion step. Annexin V and PI assays showed that the ductal injection prevents islet apoptosis. In a transplant model, the ductal injection improved islet graft function. These findings suggest that the ductal injection of preservation solution, especially the M-Kyoto solution, leads to improved outcomes for pancreatic islet transplantation. Based on these data, this technique is now used for clinical islet transplantation from non-heart-beating donor pancreata or living donor pancreas.

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