A study of the glycoantigens of neonatal porcine islet-like cell clusters using a lectin microarray.

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for clinical xenotransplantation. Two types of islet transplantation are: adult pig islets and neonatal porcine islet-like cell clusters (NPCC). However, besides a-Gal expression, differences in glycosylation and xenoantigenicity between both types were not clear so fat to date. In this study, we performed lectin microarray analyses of NPCCs cultured for 1, 5, or 9 days. METHODS: We studied differences in gycoantigens among several kinds of wild-type NPCCs isolated from 1- to 3-day-old neonatal wild-type pigs (Large White/Landrace × Duroc) and cultured for 1, 5 and 9 days in Ham's 10 in the presence of nicotinamide, using a previously published technique. After sonication and centrifugation, supernatant proteins from each islet were labeled with Cy3, applied to a lectin array and scanned with an SC-Profiler for evaluation using an Array Pro Analyzer. RESULTS: The overall signals of NPCC at days 5 and 9, showed almost the same values to most lectins, whereas those on day 1 showed differences, suggesting that the NPCC on day 1 contain immature cells that gradually turn to mature NPCCs in culture.

Natural history of lymph pumping pressure after pelvic lymphadenectomy.

Lower limb lymphedema is difficult to prevent and diagnose early because its natural history is unclear. Therefore, the aim of this study was to clarify its pathogenesis and to identify risk factors that may lead to early diagnosis. In 29 patients, aged 25 to 74 years with cervical, uterine, or ovarian cancer who underwent pelvic lymphadenectomy, indocyanine green fluorescence lymphangiography was performed with an infrared camera system, and lymph pumping pressure was measured indirectly preoperatively, and one, two, three, and six months postoperatively. Of these 29 patients, 22 (75.9%) completed the examinations. In the non-lymphedema group, the average lymph pumping pressure did not change significantly at postoperative follow-up compared with preoperative values. On the other hand, lymph pumping pressure increased at various time points in five patients who developed early lymphatic changes with dermal diffusion at the level of the proximal femur. An increase in lymph flow path resistance due to pelvic lymphadenectomy resulted in an initial increase in lymph pumping pressure, followed by a subsequent decrease, in the early lymphatic changes group. This trend in the pressure change signifies that the lymph vessels became dysfunctional as they were overwhelmed by the overload condition and this feature may be a clinically useful signal for the early diagnosis of developing lymphedema.

Effect of JNK inhibitor during islet isolation and transplantation.

Although islet transplantation has been remarkably improved by the Edmonton protocol, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases; many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hours. In this study, we show the effect of a JNK inhibitor during islet isolation and transplantation. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet cell apoptosis and improves islet graft function. These findings suggest that inhibition of JNK could prevent the impairment of islet cells and improve outcomes after pancreatic islet transplantation.

Continuous, but not occasional, oral ethanol intake reduces the success of intraportal transplanted islets of Langerhans: an experimental study.

BACKGROUND: Islet transplantation is gradually gaining acceptance for the treatment of type 1 diabetes mellitus. One of the unknown questions is alcohol intake; we have prohibited alcohol intake after islet transplantation although there is no solid evidence to support this. MATERIALS AND METHODS: In this study, we employed a mouse model to determine the effect of oral ethanol intake on transplanted islets. Either 500 or 150 islets were infused selectively into the right liver lobe of chemically induced diabetic mice. After transplantation, mice were orally administered either water (as a control) or various concentrations of ethanol for 14 consecutive days occasionally (once per day) or continuously (all intake was alcohol). Blood glucose levels were monitored and oral glucose tolerance tests (OGTT) performed. RESULTS: After 500 islets had been transplanted, all mice were cured from diabetes, but the continuous alcohol intake group showed significantly prolonged time to diabetes reversal and significantly lower glucose clearance rates by OGTT compared with the control group. After 150 islet transplantations, the diabetes cure rate in the continuous alcohol intake group was significantly lower than the control group (continuous alcohol vs control: 3/8 vs 11/12, P < .05). However, the occasional alcohol intake group showed no difference from the control group, even with as few as 150 islets transplanted per mouse. CONCLUSION: The present results demonstrated that continuous but not occasional alcohol intake reduced the success of intraportal 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 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 beta-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.

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.

Kyoto islet isolation method: the optimized one for non-heart-beating donors with highly efficient islet retrieval.

The availability of pancreata for clinical cadaveric islet transplantation is restricted to non-heart-beating donors (NHBDs) in Japan. This forced us to modify the current standard islet isolation protocol that was made up for brain-dead donors and make it suitable for NHBDs. The Kyoto islet isolation method is the one with induction of several steps based on the ideas both already reported literally and invented originally by ourselves. Using this islet isolation method, we isolated islets from 13 human pancreata of NHBDs and transplanted 11 preparations to six type-1 diabetic patients. The rate to meet release criteria of Edmonton protocol was 84.6%. Establishment of this method allowed us to begin a clinical islet transplantation program in Japan and to continue to perform the preparation of islets from NHBDs with high rate to meet the release criteria of the Edmonton protocol.

In situ cooling of pancreata from non-heart-beating donors prior to procurement for islet transplantation.

BACKGROUND: The specific aim of this study was to develop an effective technique for pancreas procurement for islet transplantation from a non-heart-beating donor (NHBD). METHODS: Between January 2004 and November 2004, 11 human pancreata were procured and processed for islet isolation at a cell processing center. After confirmation of brain-death status, a double-balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for kidney procurement. RESULTS: Warm ischemic time was controlled with the modified in situ regional cooling system at 6.0 +/- 0.9 minutes (mean +/- SE). The operations for procurement of the kidneys and pancreata lasted 48.1 +/- 3.6 minutes and 9.9 +/- 4.8 minutes, respectively. The islet yield per isolation was 396,767 +/- 142,842 IE (islet equivalents). Ten of the 11 cases met the criteria for pancreatic islet transplantation based on the Edmonton protocol. CONCLUSIONS: We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from an NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

Insulin independence of unstable diabetic patient after single living donor islet transplantation.

BACKGROUND: Current success in islet transplantation will lead to a donor shortage. Living donor islet transplantation could be an alternative approach to expand the potential donor pool. In this study we describe the first successful living donor islet transplantation for unstable diabetes, performed at Kyoto University Hospital on January 19, 2005. METHODS: The donor was a healthy 56-year-old woman and mother of the recipient. The recipient was a 27-year-old woman with insulin-dependent diabetes since the age of 15 years. She experienced frequent hypoglycemic unawareness episodes. Her blood glucose concentration was difficult to control and C-peptide level was negative after glucagon stimulation. She needed an average 28 of units of insulin per day. The donor underwent a distal pancreatectomy and islets were isolated from the resected pancreas graft. The total islet yield was 408,114 islet equivalents and isolated islets were immediately transplanted into the recipient's liver. RESULTS: After transplant, the blood glucose level of the recipient was tightly controlled without hypoglycemic episodes. She was discharged on day 37 with a normal oral glucose tolerance test (OGTT). The recipient remained insulin-independent for >3 months, since day 22 posttransplant. The donor's postoperative clinical course was uneventful. She was discharged on postoperative day 18 and returned to her job within 1 month. CONCLUSIONS: We report the first successful living donor islet transplantation for the treatment of unstable diabetes. We believe that living donor islet transplantation may become an option in the treatment of insulin-dependent diabetes.

Simple evaluation of engraftment by secretory unit of islet transplant objects for living donor and cadaveric donor fresh or cultured islet transplantation.

BACKGROUND: Evaluation of engraftment is important to assess the success of islet transplantation. Recently we developed secretory unit of islet transplant objects (SUITO) index for simple evaluation of engraftment. Assuming that normal subjects aged <40 years have 100% pancreatic beta-cell function, SUITO index was calculated by the formula: 1500 x fasting C-peptide immunoreactivity [ng/dL]/(fasting blood glucose [mg/dL] - 63). In this study, we compared the efficacy of islet transplantation from cadaveric and living donors using the SUITO index. METHODS: We performed eight islet transplantations with non-heart-beating donors (NHBDs) into five patients. Two patients received fresh islets once, one patient received fresh islets twice, one patient received cultured islets once, and one patient received cultured islets twice plus fresh islets once. In addition, one patient received fresh islets from a living donor. We calculated the SUITO index from postoperative days 3 to 30 for each case. RESULTS: Mean SUITO index after one fresh islet transplant was 11.7 +/- 1.0, after two fresh islet transplants was 28.5 +/- 3.4, after one cultured islet transplant was 2.1 +/- 0.4, after two cultured islet transplant was 12.1 +/- 1.9, and after two cultured islet transplant plus one fresh islet transplant was 26.7 +/- 1.7. The mean SUITO index after single living donor islet transplant was 40.7 +/- 2.6, which was significantly higher compared with all other groups. Insulin independence was obtained when the SUITO index was >26, which might reflect that 26% beta-cell mass was required for insulin independence. CONCLUSION: SUITO index is useful to evaluate islet engraftment and to predict the possibility of insulin independence.

Analysis of large-scale nonhuman primate islet isolations.

AIMS: It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. METHODS: Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. RESULTS: Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. CONCLUSIONS: TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.

Partial hepatectomy and subsequent radiation facilitates engraftment of mouse embryonic stem cells in the liver.

For liver-targeted regenerative medicine, embryonic stem (ES) cell-derived hepatocyte-like cells proffer great expectation. In vitro exposure to a combination of various growth factors, such as hepatocyte growth factor and fibroblast growth factor-4, as well as cytokines, leads to differentiation of ES cells into hepatocyte-like cells. We sought to determine the in vivo environment that allowed engraftment of ES cells transplanted to the liver. Thus, we examined the effect of partial hepatectomy (50%) (PHT) and subsequent radiation (RT) of the male Balb/c mouse host liver on ES cell engraftment. ES cells (5 x 10(6)) derived from 129Sv mice were transplanted into the residual liver. The controls were ES cells transplanted into a normal liver. Bromo-deoxy-residine (BrdU)-uptake was performed to evaluate the effect of hepatectomy and RT on hepatocyte regeneration. Mouse ES cells engrafted, forming teratomas in the normal liver without showing any mononuclear infiltration. A liver modified by PHT and RT facilitated engraftment of mouse ES cells compared with a normal liver. Hepatic RT significantly suppressed hepatocytic uptake of BrdU.

Transduction of human islets with the lentiviral vector.

Establishment of an efficient gene delivery system for human pancreatic beta cells is important for the development of diabetes-targeted cell therapies. The human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector is well documented to be an effective gene transfer tool for various types of cells. Thus, we examined the efficiency of lentivirus-mediated gene delivery for human islets. Human islets were isolated using defined protocols for enzymatic dissociation and purification using discontinuous Ficoll gradients with a refrigerated Cobe 2991 machine. Isolated islets were shipped to Japan, cryopreserved for 3 months, and then subjected to transduction. A vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector LtV-NLS/LacZ was produced in 293T cells under the Fugene6 method. 804G extracellular matrices were applied for monolayer formation of islets. Detection of NLS/LacZ expression was performed using X-gal staining. Lentiviral transduction was effective in these monolayer islets.

Active magnetic compensation composed of shielding panels.

Magnetically shielded rooms (MSRs) with materials of high permeability and active shield systems have been used to shield magnetic noise for biomagnetic measurements up to now. However, these techniques have various disadvantages. Therefore, we have developed a new shielding system composed of shielding panels using an active compensation technique. In this study, we evaluated the shielding performance of several unit panels attached together. Numerical and experimental approaches indicated that the shielding factor of a cubic model composed of 24 panels was 17 for uniform fields, and 7 for disturbances due to car movement. Furthermore, the compensation space is larger than that of an ordinary active system using large coils rather than panels. Moreover, the new active compensation system has the important advantage that panels of any shape can be assembled for occasional use because the unit panels are small and light.