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Biomedical subjects

G L Warnock

Publications and source records attributed to G L Warnock.

At least 109 records · Page 6Linked to original sources
PubMed1989

Viable purified islets of Langerhans from collagenase-perfused human pancreas.

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 x 10(3), distributed as 76 x 10(3) large (greater than 100-micron) and 85 x 10(3) small (less than 100-micron) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 microliter, with purity of 70-90% (large islets) and 20-40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.

Adult↗
PubMed1989

Insulin sensitivity and glucose effectiveness in long-term islet-autotransplanted dogs.

Parameters determining glucose tolerance were quantified with Bergman's minimal-model method applied to frequently sampled intravenous glucose tolerance tests in five normal and five islet-autotransplanted mongrel dogs 8-10 mo posttransplantation. Despite normal fasting glucose and insulin levels, glucose tolerance was reduced in the islet-transplanted dogs (1.5 +/- 0.4 vs. 4.2 +/- 0.4%/min in normal controls, P less than .002). The reduction in glucose tolerance was due to a reduced insulin response to glucose injection (4 +/- 1 vs. 32 +/- 5 microU/ml, P less than .001) and reduced glucose effectiveness (1.7 +/- 0.5 vs. 5.3 +/- 0.6 x 10(2) min-1, P less than .005) but not to a reduction in insulin sensitivity (8.4 +/- 0.6 vs. 7.8 +/- 0.7 x 10(4) min-1.microU-1.ml-1, P greater than .5). Our results suggest that reduced insulin secretory response as a result of islet transplantation may result in a defect in glucose's ability to promote its own disposal but not necessarily in a defect in insulin sensitivity.

Animals↗
PubMed1989

Comparison of sites for transplantation of canine pancreatic microfragments.

The ideal site to transplant pancreatic islets has yet to be determined. To evaluate this problem, we have compared the efficacy of pancreatic microfragments transplanted into the splenic, intrahepatic and renal subcapsular sites. Function was assessed by plasma glucose (PG) and intravenous glucose tolerance test (ivGTT). Portal pressures and coagulation profiles (PT, PTT, FDP, Platelet count) were measured following intrasplenic and intraportal transplants. Liver enzymes (LDH, Alk. Phos., SGOT), serum bilirubin, BUN and creatinine were assessed serially in all groups. Nine of 10 dogs that received islets refluxed into the spleen were normoglycemic (mean PG = 94.5 mg/dL) at 1 mo with one dog failing four days following implantation (PG = 350 mg/dL). Following intraportal embolization, two of six dogs remained normoglycemic (PG = 95.7 mg/dL) at 1 mo. One dog died due to mesenteric venous thrombosis, two died suddenly within 24 h of engraftment without obvious cause, and a fourth died six days following transplantation (PG = 678 mg/dL). None of the six renal subcapsular transplants induced normoglycemia (mean PG = 430 mg/dL) at 1 mo. Mean rise in portal pressure (cmH2O +/- SEM) during intrasplenic and intraportal transplantation of islets was 1.7 +/- 0.6 and 31.2 +/- 3.3 respectively (p less than 0.001). Following intrasplenic and intrahepatic engraftment, significant coagulation abnormalities did not occur. Elevation of liver enzymes occurred 24 h following implantation to all three recipient sites but returned to preoperative values by 1 mo. Bilirubin was not affected. Renal function tests were not significantly altered in any groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Studies of the isolation and viability of human islets of Langerhans.

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.

Cell Separation↗
PubMed1988

Critical mass of purified islets that induce normoglycemia after implantation into dogs.

Twenty grafts of highly purified islets of Langerhans (mean +/- SE vol 0.98 +/- 0.3 ml, islet diam 122 +/- 5 micron) were autoimplanted into the spleen or liver of totally pancreatectomized dogs. Portal venous pressure did not change significantly. Incremental doses of islets of 1000-3000 (n = 3), 3000-4000 (n = 6), 4000-5000 (n = 5), 5000-6000 (n = 3), and 6000-8000 (n = 3) per kilogram body weight resulted in corresponding fasting plasma glucose (PG) of 258 +/- 18, 163 +/- 13, 158 +/- 17, 138 +/- 15, and 108 +/- 6 mg/dl. In 3 apancreatic control dogs, PG was 338 +/- 9 mg/dl. One (normoglycemic) dog died of wound complications, and follow-up PG at 1 mo was 89 +/- 5 mg/dl in 6 of 10 dogs that received 3000-5000 islets/kg and 91 +/- 6 mg/dl in all 6 that received greater than 5000 islets/kg. K values 1 mo after surgery during glucose tolerance tests were 1.8 +/- 0.3 for 6 spleen dogs and 1.6 +/- 0.3 for 6 liver dogs. Six months after splenic implantation, PG was 75 +/- 4 mg/dl and rose to greater than 350 mg/dl after splenectomy. These data define the critical number of purified dog islets of known size that is necessary to induce prolonged normoglycemia. Sufficient pure islets can be collected from 1 dog pancreas to correct diabetes after autoimplantation into the liver or spleen.

Animals↗
PubMed1988

Pancreatic islet cell transplantation: past, present and future.

Experimental islet cell transplantation is very successful. In syngeneic strains of rodents isolated and purified islets from multiple donors infused into the portal vein of a diabetic recipient can normalize glucose and prevent microvascular complications. The techniques used to isolate and purify islets in rodents could not be adapted to the fibrous mammalian pancreas. It was not possible to harvest a sufficient volume of isolated islets from one pancreas to restore metabolic function in a diabetic recipient. A partial solution to the problem was to eliminate the steps of purification used in rodents and transplant dispersed pancreatic fragments consisting of islet cells as well as contaminating exocrine, endothelial, ductal and specific immune cells. Initial clinical trials using impure pancreatic fragment grafts were unsuccessful and risky dampening enthusiasm for islet transplantation in general. It was during this same time period that Lacy and Lafferty demonstrated that deletion of donor immune cells from a graft preparation of isolated islets in rodents prolonged allograft survival without immunosuppression. This exciting discovery revived efforts to isolate high yields of pure islets from the mammalian pancreas. The major objective of experimental studies at present is directed at finding a practical and efficient means of separating the endocrine component of the pancreas from contaminating exocrine and immunogenic cells. Escalating success in this area has permitted clinical trials of islet transplantation to resume with optimism for success.

Animals↗
PubMed1988

Normoglycemia after implantation of purified islet cells in dogs.

A major problem that limits the effectiveness of clinical trials of islet transplantation is the inability to isolate sufficient pure viable islets from a single pancreas. The authors have evaluated this problem, using dogs rendered diabetic by total pancreatectomy. An average of 123 +/- 11 X 10(3) highly purified islets (mean graft weight 0.75 +/- 0.1 g) were implanted as splenic allografts (seven dogs), splenic autografts (six dogs) or liver autografts (six). In the autograft recipients, fasting normoglycemia was maintained during follow-up to 10 months; onset of hyperglycemia was delayed in three liver recipients and one splenic autograft recipient at 1.5, 2, 8 and 10 months respectively. The K values (decline in glucose levels in %/min) during intravenous glucose tolerance testing were more than 1.0. Six recipients of allografts and cyclosporine (CsA) were normoglycemic when CsA trough serum levels were greater than 300 micrograms/L, although the fasting plasma glucose level was higher than that in autograft recipients. These data show that purified islets isolated from a large mammal will maintain fasting normoglycemia for prolonged periods after auto- or alloimplantation.

Animals↗
PubMed1988

Reversal of diabetes by transplantation of cryopreserved rat islets of Langerhans to the renal subcapsular space.

Intraportal transplantation of cryopreserved islets of Langerhans has been shown to reverse streptozotocin-induced diabetes in rats. This study demonstrates the feasibility of transplanting 3,000 cryopreserved islets beneath the kidney capsule of syngeneic streptozotocin-diabetic Wistar-Furth rats. The clinical indices of plasma glucose, urine volume, and urine glucose returned to baseline values following implantation of fresh and cryopreserved islets. The clinical indices post-transplant and the K values (decline in plasma glucose concentration, percent/min) during intravenous glucose tolerance tests were not significantly different in rats receiving freshly isolated or cryopreserved islet grafts. The rapid return to the diabetic state after nephrectomy proved conclusively that the reversal of diabetes was due to the grafted islets. This was confirmed by immunocytochemical localization of well confirmed by immunocytochemical localization of well granulated islets beneath the kidney capsule. We conclude that cryopreserved islets reverse streptozotocin-induced diabetes after renal subcapsular implantation and that the clinical response is similar to fresh isografts.

Animals↗
PubMed1987

Survival of cryopreserved isolated adult human pancreatic islets of Langerhans.

Human islets of Langerhans were isolated from the pancreata of 13 adult organ donors, cultured for 24 h, cryopreserved, stored in liquid nitrogen at -196 degrees C for 6-88 days, thawed, and then cultured again. The number of islets recovered after this procedure was 80% of that present at the beginning. The viability of cultured/cryopreserved islets was then compared with that of islets from the same donor cultured for 24 hr, and was assessed by supravital staining, insulin response to glucose, and survival after implantation under the kidney capsule of nude rats. Supravital staining showed more nonviable cells in cryopreserved islets than in their cultured counterparts. Significant response to glucose was seen before and after cryopreservation in 2 of 4 sets of islets. Xenografts of 200 cultured islets (from 13 donors) were implanted in 15 nude rats under the kidney capsule, and a further 15 rats had cryopreserved islets (from the same 13 donors) similarly implanted beneath the kidney capsule. Two weeks later tissue was visible at the site of implantation in all 30 rats. Histological examination of both groups showed the tissue to have the morphology of islets, confirmed by immunohistochemical chemical localization of insulin. The insulin content of kidneys bearing 200 cultured islets was 7.88 +/- 1.6 mU (n = 13) versus 6.84 +/- 1.43 mU (n = 13) for kidneys bearing cryopreserved islets. Thus this technique for cryopreservation of isolated adult human islets enables a high recovery of endocrine tissue that survives after transplantation to nude rats, but some evidence of damage was apparent from insulin secretion studies and electron-microscopic studies.

Animals↗
PubMed1987

Prolongation of islet xenograft survival by cryopreservation.

Our attempt to reduce islet immunogenicity by slow cooling to -40 degrees C, storage at -196 degrees C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (greater than or equal to 100 micron) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.

Animals↗