Yukihide Yonekawa

Analyses of Peripheral Blood Mononuclear Cells in Operational Tolerance After Pediatric Living Donor Liver Transplantation

Operational tolerance (graft acceptance in an immunosuppression (IS)‐free environment) after living‐donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vδ1/Vδ2 γδT‐cells ratio in operationally tolerant patients (Gr‐tol; n = 12), compared with those from age‐matched volunteers (Gr‐vol; n = 24) or patients on IS (Gr‐IS; n = 19). The frequency of NK cells was decreased in Gr‐tol, compared with those in Gr‐IS or Gr‐vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr‐vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.

Insulin independence after living-donor distal pancreatectomy and islet allotransplantation

Rising demand for islet transplantation will lead to severe donor shortage in the near future, especially in countries where cadaveric organ donation is scarce. We undertook a successful transplantation of living-donor islets for unstable diabetes. The recipient was a 27-year-old woman who had had brittle, insulin-dependent diabetes mellitus for 12 years. The donor, who was a healthy 56-year-old woman and mother of the recipient, underwent a distal pancreatectomy. After isolation, 408 114 islet equivalents were transplanted immediately. The transplants functioned immediately and the recipient became insulin-independent 22 days after the operation. The donor had no complications and both women showed healthy glucose tolerance. Transplantation of living-donor islets from the distal pancreas can be sufficient to reverse brittle diabetes.

Successful islet transplantation from nonheartbeating donor pancreata using modified ricordi islet isolation method

Background. Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). Methods. First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. Results. These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469±36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5±0.4% at transplant to 5.1±0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2±3.3 units at transplant to 11±4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. Conclusion. This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.

Modified two-layer preservation method (M-Kyoto/PFC) improves islet yields in islet isolation.

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.

Impact of Right Lobe with Middle Hepatic Vein Graft in Living-Donor Liver Transplantation

Technical improvements in adult‐to‐adult living‐donor liver transplantation (LDLT) have led to the use of right‐lobe grafts to overcome the problems encountered with ‘small‐for‐size grafts’. The major controversy remains that the venous drainage from anterior segment substantially depends on tributaries of the middle hepatic vein (MHV), and deprivation of such tributaries may critically influence the postoperative graft function. Right‐lobe grafts with MHV could resolve the potential problem of congestion in anterior segment. From December 2000 to January 2004, we performed 217 right‐lobe LDLTs for adult patients. Of these, 40 patients received a right lobe with MHV graft (18.4%). The overall cumulative 3‐year graft survival rate of a right lobe with (n = 40) and without MHV (n = 177) was 86.2% and 74.8% (p = NS). The proximal side of the MHV and the drainage vein of segment IV to the MHV (the left medial superior vein) were preserved in 24 patients. All of them needed venous interposition graft for anastomosis. All patients had a patent right hepatic vein (RHV) and MHV anastomosis during the follow‐up period. We adopted the right lobe with MHV graft in 40 LDLT cases. Vein graft is essential for safe MHV anastomosis in cases which preserve proximal side of the MHV.

Cell Permeable Peptide of JNK Inhibitor Prevents Islet Apoptosis Immediately After Isolation and Improves Islet Graft Function

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 NH2‐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 cells and lead to improvement in the outcomes for pancreatic islet transplantation.

Evaluation of Islet Transplantation from Non‐Heart Beating Donors

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 HbA1C 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 β‐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.

PDX-1 protein is internalized by lipid raft-dependent macropinocytosis

PDX-1 plays a central role in regulating insulin gene transcription and differentiation of insulin-producing cells. It was previously reported that, due to its own Antennapedia-like protein transduction domain (PTD), exogenous PDX-1 protein can permeate cells and induces insulin gene expression in pancreatic ducts, thought to be islet progenitor cells. These data suggest that PDX-1 protein transduction could be a safe and valuable strategy for facilitating differentiation of progenitor cells into insulin-producing cells without requiring gene transfer technology. Here it is shown that after an initial ionic cell–surface interaction, PDX-1 proteins are rapidly internalized by lipid raft-dependent macropinocytosis. HeLa cells were treated with both FITC-conjugated PDX-1 PTD and FM 4–64, a general fluorescent marker of endocytosis. A punctate cytoplasmic distribution of PDX-1 PTD, which colocalized with FM 4–64, was observed in treated cells. Because expression of dominant-negative dynamin-1 did not block PDX-1 PTD uptake, PDX-1 protein transduction is independent on phagocytosis and clathrin- or caveolar-mediated endocytosis. Cells were pretreated with amiloride, a specific inhibitor of the Na+/H+ exchange required for macropinocytosis, or cytochalasin D, an F-actin elongation inhibitor. Treatment of cells with both macropinosome inhibitors resulted in the reduction in PDX-1 PTD transduction into vesicles, suggesting that PDX-1 PTD-mediated cellular entry occurs by lipid raft-mediated macropinocytosis. Taken together, these observations provide the mechanism of PDX-1 protein transduction and suggest that the protein transduction system could work for experimental and therapeutic strategies.

Effective Islet Isolation Method with Extremely High Islet Yields from Adult Pigs

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase™ HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

Follow-up study of the first successful living donor islet transplantation.

Background. Islet transplantation has become an option for the treatment of insulin-dependent diabetes mellitus and is usually performed using brain-dead heartbeating donors. However, we have very limited number of such donors in Japan; therefore, it is not allowed to perform islet transplantation with brain-dead donors. In order to perform islet transplantation in Japan, we need to seek new donor resources. Methods. We performed the first successful living-donor islet transplantation. In this case, the recipient had brittle diabetes with hypoglycemic unawareness. The donor was deemed qualified after undergoing both metabolic and preoperative assessments. Distal pancreatectomy was performed using open laparotomy and more than 400,000 islets were isolated and transplanted immediately. Results. The recipient has been insulin independent posttransplant with positive C-peptide for more than one year. She no longer suffers from hypoglycemic unawareness and displayed a substantial improvement in hemoglobulin (Hb) A1C. The donor’s clinical course was uneventful, which allowed her to return to her job within one month. She maintained normal fasting C-peptide and HbA1C levels during follow-up period. Conclusion. In our first case of living donor islet transplantation, both the donor and the recipient have been maintaining excellent glycemic control with no untreatable complications for more than one year.