S. Ohtsuka

Prevention of primary islet isograft nonfunction in mice with pravastatin

BACKGROUND Nonspecific inflammatory damage in the early stages of transplantation is the major cause of primary islet graft nonfunction. Using murine isografts, we attempted to prevent this islet graft damage by treating recipients with pravastatin (Pravacol), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor. Nicotinamide was also tested to determine the synergistic effect of both agents. METHODS Unpurified newborn BALB/c islets, ranging in number from 1800 to 2500, were transplanted into the left renal subcapsular space of a syngeneic adult mouse made diabetic with streptozotocin. Recipient mice were divided into the following four groups, based on treatment protocols: treatment with 40 mg/kg pravastatin (group 1), 500 mg/kg nicotinamide (group 2), 40 mg/kg pravastatin and 500 mg/kg nicotinamide (group 3), and vehicle alone (group 4). Pravastatin and nicotinamide were administered orally every day for 14 days, starting on the day of transplantation (day 0). Nonfasting blood glucose levels, urine glucose levels, and the intravenous glucose tolerance test were used to monitor the diabetic state. The reversal of diabetes was defined by normoglycemia and negative urine glucose maintained for more than 7 days. RESULTS After islet transplantation, levels of blood and urine glucose were significantly lower in groups 1 and 3, compared with those in group 4. K-values of an intravenous glucose tolerance test performed on day 14 were significantly higher in groups 1 and 3 than those of group 4. Reversal of diabetes had occurred in 63% of mice in group 1 and 67% in group 3, levels that were higher than those in group 2 (17%) and group 4 (0%) (P<0.02, groups 1 and 3 vs. group 4). Histological examination of grafts, biopsied on day 21, revealed well preserved islets with little sign of inflammation in groups 1 and 3, whereas grafts in groups 2 and 4 contained broken, smaller islets surrounded by severe fibrosis and mononuclear cell infiltration. CONCLUSION Our results in mice have shown the effectiveness of pravastatin for protecting islets from nonspecific inflammatory damage. Nicotinamide did not show a synergistic effect with pravastatin at the dosage used in this study. These results indicate that pravastatin may be a useful agent for clinical islet transplantation.

Increased islet viability by addition of beraprost sodium to collagenase solution.

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 n M was added to the collagenase solution. After purification, the islet yield was 434,561 ± 35,691 islet number expressed as 150 &mgr;m equivalent size (IEQ)/pancreas or 8,799 ± 345 IEQ/g of pancreas in the BPS group and 349,987 ± 52,887 IEQ/pancreas or 7,998 ± 1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 ± 0.7% in the BPS group and 82.0 ± 0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number × % viability) was 384,586 ± 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 ± 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 ± 38.5 versus 92.3 ± 25.6 &mgr;U/IEQ;P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves &bgr; cell function.

Clinical islet transplantation experience of the University of California Islet Transplant Consortium

BACKGROUND The University of California Islet Transplant Consortium was formed to evaluate the feasibility of performing clinical islet transplantation at different transplant centers by using a single centralized islet isolation laboratory. METHODS From July 1992 through February 1995 seven adult islet transplantations were performed, six allografts and one autograft. Once procured, human pancreata were brought to the UCLA-VA Islet Core Laboratory for islet isolation and purification, which were then transported to different centers for transplantation. Patients 1 through 3 received their transplants in Los Angeles, patient 4 received her islet transplant in Torrance, and patients 5 through 7 received their transplants in San Francisco. RESULTS Although none of these patients achieved insulin independence, four of seven had functioning grafts longer than 6 months as indicated by circulating C-peptide level greater than 0.7 ng/ml. Furthermore, improved glucose control as shown by a decreased insulin requirement was seen in 57% (four of seven patients) of these patients. The ability to isolate islets at a single laboratory and transport them long distances to different centers was shown in patients 4 through 7. CONCLUSIONS Islet transplantation can be performed with improvements in blood glucose control, and islets can be isolated at a centralized location and successfully transported to different centers for transplantation.

Improved Recovery of Cryopreserved Canine Islets by Use of Beraprost Sodium

Cryopreservation of pancreatic islets provides many advantages for clinical transplantation. Unfortunately, the freezing and thawing processes lead to a significant loss of islets. In this study, an attempt was made to increase the yield and viability of islets after cryopreservation and thawing. By using canine islets, we evaluated whether beraprost sodium (BPS), a stable prostacyclin analog, protects islets during the freeze-thaw processes and improves the recovery of frozen-thawed islets. Canine islets were frozen and thawed by the procedures used routinely for storage of human islets. In this study, we deliberately used islets of lower purity (60+/-3.6%), which is undesirable for cryopreservation. The recovery of viable islets after thawing is poorer with islets of lower purity than with highly purified islets. BPS was added to both the cryopreservation solutions containing dimethyl sulfoxide (DMSO) and the thawing solution containing sucrose. After thawing, the islet recovery (islet number after thawing divided by islet number before freezing) was 71.1+/-12.7% with 1 nM BPS, 77.8+/-5.6% with 10 nM BPS, 79.3+/-6.7% with 100 nM BPS, and 69.2+/-7.2% in control preparations without BPS. Islet viability assessed by supravital staining was 57.5+/-5.6%, 64.7+/-7.0%, 67.5+/-6.5%, and 57.7+/-4.9% with 1 nM, 10 nM, and 100 nM BPS and controls, respectively. Both islet recovery and viability were significantly better with 10 nM and 100 nM BPS than with the controls (p<0.03). After 3 days in culture, islet numbers in the 10 nM and 100 nM BPS groups were significantly higher and showed better insulin-release responses than those from the 1 nM BPS and control groups. Histologically, islet structure was well preserved in the 10 nM and 100 nM BPS groups, whereas many islets of the control group were smaller and fragmented. Electron microscopic examination revealed that 10 nM and 100 nM BPS preserved the microstructure of islet cells, and signs of apoptosis or necrosis were rare. It was concluded that BPS improved the recovery and viability of canine islets after cryopreservation and thawing. BPS would be a useful agent for improving the recovery of cryopreserved human islets for clinical transplantation.

Prolongation of islet allograft survival in mice by combined treatment with pravastatin and low-dose cyclosporine.

Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is known to have suppressive effects on immune and inflammatory cells. We have previously shown in mice and dogs that this agent prevents primary nonfunction of islet iso- and autografts by reducing inflammation at the graft site. The present study was designed to further investigate whether pravastatin has a synergistic effect with cyclosporine (Cs) to prolong islet allograft survival in mice. Unpurified 3000 BALB/c newborn islets were transplanted under the renal capsule of a streptozotocin-diabetic C57BL/6 mouse. Pravastatin and Cs were administered for 10 days starting on the day of grafting (day 0). Five groups were set up based on the treatment protocol: group 1, treatment with 40 mg/kg pravastatin; group 2, 30 mg/kg Cs; group 3, 50 mg/ kg Cs; group 4, 40 mg/kg pravastatin and 30 mg/kg Cs; group 5, vehicle alone. Graft survival was indicated by blood glucose levels sustained at <200 mg/dl, and graft rejection by >250 mg/dl for 2 consecutive days. Hyperglycemia persisted in six of the eight (75%) mice and grafts were rejected in 3.6 ± 0.5 days (mean ± SD) in group 5. In group 1, grafts were also rejected in 3.8 ± 0.8 days, but blood glucose was transiently <200 mg/dl in three of the five mice. Despite Cs, grafts were rejected between 7 and 15 days (10.3 ± 2.4 days) in group 2. Among six mice in group 3, one maintained euglycemia for >60 days, the other rejected the graft on day 15, and the remaining four died with functioning grafts between 9 and 13 days due to Cs toxicity. A combination of a low dose of Cs and pravastatin (group 4) prolonged graft survival for >19 days in five of the eight mice, and for 7–13 days in the remaining three mice. Histological examination of the grafts in this group showed significantly reduced local inflammation. Results indicate a synergistic effect of pravastatin and Cs on prevention of islet allograft rejection.