Previous studies in rodent and canine animal models have suggested a detrimental impact on islet recovery and function when pancreas excision is preceded by in situ vascular flushing with cold preservation solutions. We studied the efficacy of islet isolation from 19 consecutive cadaver pancreases procured alternately by initial pancreatectomy before in situ flush (group 1, our standard procurement technique, n = 9) or pancreas removal following in situ vascular flushing with cold University of Wisconsin solution (group 2, n = 10). Once procured, pancreases were weighed, the main pancreatic duct was cannulated, and 150 ml of collagenase solution was injected. The pancreases were transported to the isolation laboratory and processed within 2 hr. Islets were counted and sized after dithizone staining, and the islet equivalents were calculated. Aliquots of isolated islets were cryopreserved using established techniques. Islet function of both freshly isolated and frozen-thawed islets was assessed using a glucose-stimulated perifusion system. Significantly more pancreas was harvested after University of Wisconsin flush (90.6 +/- 6.9 g for group 1 versus 66.7 +/- 4 for group 2, P < 0.05). The quantity of islets per gram of processed pancreas released during enzymatic digestion from each of the experimental groups did not differ significantly (4.5 +/- 0.6 x 10(3) islet equivalents per gram for primary pancreatectomy versus 4.0 +/- 0.4 x 10(3) University of Wisconsin flush). Similarly, following Ficoll purification, the overall yields of islets did not differ significantly. Total islet yield in the primary pancreatectomy group was 181 +/- 25 x 10(3) islet equivalents (2.7 +/- 0.3 x 10(3) IE/g) versus 217 +/- 41 x 10(3) for the University of Wisconsin flush group (2.9 +/- 0.8 x 10(3) islet equivalents/g; P not significant). No differences were observed in in vitro viability. Perifusion stimulation indexes (peak/basal insulin release) were 5.9 +/- 1.3 for group 1 and 7.1 +/- 1.5 for group 2. These results conflict with published results in animal models and indicate that large numbers of viable islets can be recovered from cadaver pancreas utilizing either procurement technique. The decreased operating time, simplicity, and safety favor the use of total pancreatectomy after limited in situ vascular flushing as the method of choice for pancreas procurement for subsequent islet isolation.

Cadaver↗

PubMed1994

BCG immunotherapy prevents recurrence of diabetes in islet grafts transplanted into spontaneously diabetic NOD mice.

Islet transplantation can provide a therapeutic option for patients who suffer from type 1 diabetes mellitus, especially if current aggressive immunosuppression could be eliminated. In vitro immunomodulation has achieved this goal for islet allografts--however, strategies must be developed to prevent B cell lysis secondary to the chronic autoimmune process of diabetes. Previously, we have found that adjuvant therapy with CFA was effective in preventing the onset of diabetes and the recurrence of B cell lysis post-islet transplantation. In this study we evaluated the efficacy of BCG, a more clinically relevant immunoadjuvant, to prevent recurrent autoimmune damage to islets grafted into diabetic NOD mice. Highly purified islets were isolated from either 5-7-week-old prediabetic NOD mice or 5-8-week-old CBA/J mice using standard islet isolation techniques. Four hundred purified islets were transplanted into the kidney capsule of diabetic NOD recipients. A single dose of BCG was administered in recipients of syngeneic (gp 1) and allogeneic islets (gp 2). In gp 1, 8 of 10 BCG-treated syngeneic recipients remained normoglycemic for > 100 days posttransplant. In untreated recipients of syngeneic islets (gp 3) the graft failed at 19 days (n = 16) (P value, gp 1 vs. gp 3 < 0.001). In untreated allograft recipients, islet grafts functioned for 11 days (n = 8), which did not differ significantly (P = NS) from the BCG-treated allografts (gp 4) (14 days, n = 7). In 6 mice with long-term graft function, a second syngeneic graft implanted into the contralateral kidney maintained normoglycemia for 50 days following the removal of the first islet graft. Histological examination of the grafts from gp 1 mice showed granulated B cells with periinsular lymphocytes, while those of the control animals showed lytic B cells and marked lymphocytic infiltration. We conclude that adjuvant therapy with a single dose of BCG can prevent the recurrent autoimmune insulitis, but not allograft rejection in islets transplanted into NOD mice, and that a state of unresponsiveness is induced to allow acceptance of a second syngeneic graft. These data suggest that adjuvant therapy may be useful to sustain the function of transplanted islets in the type 1 diabetic.

Animals↗

PubMed1994

Improved survival of in vitro cultured canine islet allografts.

Pure islets transplanted from single donors survive in outbred dogs when CsA is administered at high doses (15-20 mg/kg/day), but not report has documented prolonged function with less CsA. In this study, we investigated survival of canine islets that were immunomodulated with in vitro culture and transplanted with minimal CsA. Highly purified islets from single donors (n = 17) were cultured in vitro at 22 degrees C for 7 days. Four groups of apancreatic, outbred mongrel dogs received islets into the spleen: group 1 (n = 4), fresh islet controls; group 2 (n = 5), cultured islet controls; group 3 (9033 +/- 994 islets/kg, n = 12), culture and CsA; group 4 (9050 +/- 1091 islets/kg, n = 8), fresh islet and CsA. CsA was administered in doses of 5-8 mg/kg/day to maintain whole blood trough levels of 300-500 ng/ml. At 30 days after implant, the CsA was stopped. After 7-day culture, 80% of the endocrine mass was recovered. All grafts restored plasma glucose to < 150 mg/dl. Group 1 and 2 dogs became hyperglycemic at 5 +/- 1 (+/- SE) and 6 +/- 1 days. Three grafts in group 3 failed when CsA did not reach target blood levels. Of the 9 dogs with CsA levels of 300-500 ng/ml, 1 died at 9 days while normoglycemic, and the remainder were normoglycemic for 35.5 +/- 8.3 days (median 38 days). Seven group 4 dogs with these CsA levels achieved normoglycemia for 7.7 +/- 1.9 days (median 9 days, survival vs. group 3, P = 0.03, by Mann-Whitney test). In vitro tissue culture of mass quantities of single-donor allogeneic islets enhances their survival with low dose immunosuppression in outbred diabetic dogs.

Animals↗

PubMed1994

Human pancreatic islet beta-cell destruction by cytokines is independent of nitric oxide production.

The inflammatory cytokines, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma are cytotoxic to human islet beta-cells in vitro. To determine the possible role of nitric oxide (NO) as a mediator of cytokine-induced islet beta-cell destruction, we studied the relationships between NO production and destruction of human pancreatic islet cells incubated with cytokines in vitro. The cytokine combination of interleukin-1 beta (50 U/mL), tumor necrosis factor-alpha (10(3) U/mL), and interferon-gamma (10(3) U/mL) induced a significant increase in NO production and significant decreases in DNA and insulin contents of the islet cell cultures after a 48-h incubation. L-NG-Monomethyl arginine, an inhibitor of NO synthase, completely prevented cytokine-induced NO production during incubations of 18, 36, 60, and 84 h. Cytokine-induced decreases in DNA and insulin contents of the islet cell cultures, however, were unaffected by the NO synthase inhibitor. Conversely, nicotinamide prevented cytokine-induced islet beta-cell destruction without inhibiting NO production. We conclude that cytokine-induced NO production in human islet cells may be neither necessary nor sufficient to destroy the islet beta-cells and that cytotoxic mechanisms, independent of NO, exist and can be inhibited by nicotinamide.

Arginine↗

PubMed1994