Philip C. Watt

Human islet isolation in 104 consecutive cases : factors affecting isolation success

One of the major steps toward successful islet transplantation for the treatment of type diabetes is to obtain islets of sufficient number and viability. Using a standardized method of isolating islets, the goal of this study was to analyze the factors influencing the outcome of islet isolation. A total of 104 cadaveric human pancreata were processed for islets by the same team. Data from the islet-processing charts were reviewed retrospectively. The two endpoints were the recovery of islets, viable after 2 days of culture (group V = viable, group NV = nonviable) and the islet yield. Viable islets were recovered in 61% of cases (n = 63). Minimal blood glucose recorded during hospitalization was very significantly lower in group V (124 +/- 5 vs. 148 +/- 9, P = 0.01). Lack of significant medical history in the donor was associated with better viability as compared with various donor predispositions (chi-2 4.21, P = 0.04). Cold ischemia time (8.1 +/- 0.5 hr in group V vs. 9.8 +/- 0.9 hr in group NV, P = 0.07) and collagenase lot (5 lots tested, chi-2 13.1, P = 0.01) also affected the recovery of viable islets. Hospital time was shorter in group V (65.3 +/- 6.8 vs. 80.9 +/- 17.9 hr, P = 0.35). Multivariate logistic regression analyses of viable islet recovery identified minimal blood glucose (P = 0.03) and collagenase lot (P = 0.06) as the most significant risk factors. However, the best multivariate predictive model--which includes blood glucose, collagenase lot, donor age and surgical procurement team--correctly predicted 66.2% of cases only. Multivariate analysis of final islet yield designed hospitalization length, cardiorespiratory arrest, surgical procurement team, and collagenase lot as the best predictors. These data obtained in a large series of pancreata emphasized several donor and technical factors that should target the attention of islet transplant researchers in order to improve islet yield and viability.

Immunoneutralization of somatostatin, insulin, and glucagon causes alterations in islet cell secretion in the isolated perfused human pancreas.

Introduction In this study, immunoneutralization of endogenous insulin, glucagon, and somatostatin with specific antibodies was used in an isolated perfused human pancreas (IPHP) model. Aims To study intrapancreatic cellular interactions and pancreatic hormonal secretion. Methodology Randomized, sequential 10-minute test intervals of single-pass perfusion with each antibody were performed at 3.9 m M or 11.5 m M steady-state glucose concentrations. Somatostatin, insulin, and glucagon levels were measured in the effluent during basal and immunoneutralization intervals. Results At 3.9 m M glucose concentration, somatostatin antibody (SS-Ab) stimulated insulin and glucagon secretion, insulin antibody (IN-Ab) inhibited glucagon secretion, and glucagon antibody (GN-Ab) stimulated insulin secretion. At 11.5 m M glucose concentration, SS-Ab stimulated insulin secretion, IN-Ab stimulated glucagon and inhibited somatostatin secretion, and GN-Ab stimulated insulin secretion. Conclusion The variation in hormonal responses to immunoneutralization during stimulated and nonstimulated glucose conditions suggests that a dynamic association exists between the pancreatic cells.

Regulatory role of intraislet somatostatin on insulin secretion in the isolated perfused human pancreas

This study was undertaken to determine whether intraislet somatostatin inhibits insulin secretion in the human islet. A high-affinity monoclonal somatostatin antibody was used to immunoneutralize somatostatin in the isolated, perfused human pancreas. Single pass perfusion was performed in pancreata obtained from cadaveric organ donors using a modified Krebs medium with either 3.9 or 12.9 mM glucose. Sequential test periods separated by basal periods were performed with either somatostatin-14 (SS-14), somatostatin monoclonal antibody (CURE.S6), or a combined infusion. Infusion of SS-14 resulted in inhibition of insulin secretion under both low glucose (δ = −712 ± 212 pM) p < 0.05) and high glucose (δ = −21,913 * 10,003 pM) (p = 0.06) conditions. Immunoneutralization of intraislet somatostatin with CURE.S6 resulted in a significant increase in insulin secretion under both low glucose (454 ±162 pM) (p < 0.05) and high glucose (2,177 ± 829 pM) (p < 0.05) conditions. Combined infusion of SS-14 and CURE.S6 resulted in a reversal of the inhibitory effect of exogenous SS-14. The data suggest that intraislet somatostatin has an inhibitory role in the regulation of insulin secretion in the human islet.

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

BACKGROUNDnThe 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.nnnMETHODSnFrom 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.nnnRESULTSnAlthough 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.nnnCONCLUSIONSnIslet 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.

Biologic behavior of sporadic gastrinoma located to the right and left of the superior mesenteric artery

Among a series of 107 closely followed patients with gastrinoma, 60 patients with sporadic type tumors were identified and evaluated. There were 44 patients (73%) with tumors to the right of the superior mesenteric artery (SMA). Of these, 16 (36%) had extrapancreatic tumors, 28 (64%) had tumor within lymph nodes, and 9 (20%) had multiple tumors. In this group of patients, there were 19 (43%) cures, and only 9 (20%) patients had hepatic metastases. In contrast, in 16 patients (27%) with tumors to the left of the SMA, there were no extrapancreatic tumors, only 3 patients (19%) had tumor within lymph nodes, and 7 (44%) had multiple tumors. In this group, there was only one cure (6%), and nine (56%) patients had hepatic metastases. These findings suggest two distinct populations of sporadic gastrinoma, one to the right (gastrinoma triangle) and the other to the left (outside triangle) of the SMA, which appear to have different biologic behaviors. These differences may reflect divergent etiologies for these two groups of tumors.

A New Culture Method for Human Pancreatic Islets Using a Biopore Membrane Insert

The availability of highly purified human islets has increased with the progressive improvement of isolation methods. This has provided opportunities to perform various in vitro studies on human islets. However, when islets are maintained in culture, the overgrowth of fibroblasts results in a reduced islet purity and often has an adverse effect on islet function. To reduce fibroblast growth and to maintain normal islet function, we have investigated a new three-dimensional culture technique using a noncoated transparent Biopore membrane insert (Millicell CM, Millipore). Islets were isolated from seven human pancreata and cultured for 2 months using this membrane insert. At various time intervals, the functional viability of is lets was assessed by measurements of insulin released into the culture medium, static incubation assays of basal and stimulated insulin release, islet insulin contents, and insulin biosynthesis. Results were compared to those of islets cultured in hydrophobic plastic petri dishes, our standard procedure. We found that the non-coated membrane does not allow islet attachment to the surface and prevents fibroblast growth, so that islets maintain a three-dimensional structure and remain in a free-floating form. Islets cultured in a membrane insert showed a function similar to or better than that of islets cultured in plastic petri dishes.

A review of gene transfer techniques

The ability to transfer genetic material from one cell to another provides a novel approach for the investigation and potential treatment of a variety of diseases. Several gene transfer techniques have been developed. Of these, the use of retroviruses as vectors has allowed highly efficient and stable introduction of foreign genes into target mammalian cells, thus making gene therapy possible. The current prospects are that some unusual lethal diseases can be corrected and that various forms of cancer will be treated in this manner.