Prolongation of porcine islet allograft survival.
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Animals↗
Biomedical subjects
B J Hering
Publications and source records attributed to B J Hering.
Clinical islet transplantation--registry report, accomplishments in the past and future research needs.
This review provides the results of a recent analysis of the Islet Transplant Registry on clinical adult islet transplants performed worldwide through June 30, 1992. Between December 12, 1893 and June 30, 1992, 167 adult islet transplants were performed at 25 institutions worldwide, including 104 at 9 institutions in North America, 62 at 15 institutions in Europe, and 1 elsewhere. The total number of diabetic patients reported to be insulin independent after adult islet allotransplantation through June 30, 1992, was 19. In an analysis by era, the percentage of patients that showed positive basal C-peptide levels (i.e., > or = 1 ng/mL at > or = 1 mo) posttransplant, and that became insulin independent (> 1 wk) in the 1985-1989 era (n = 35 cases) were 20% and 6%, and in the 1990-1992 era (n = 69 cases) were 64% and 20%, respectively, and thus have improved significantly (p < 0.001 and p < 0.05). For the 1990-1992 period, the percentage of patients who showed positive basal C-peptide levels post-transplant, and who became insulin independent in the single donor pancreas group (n = 31 cases) were 52% and 13%, and in the multiple donor pancreata group (n = 36 cases) were 75% and 28%, respectively. Islet graft function rates were nearly identical for grafts prepared from pancreata stored < or = 6 h (n = 27) and > 6 < or = 12 h (n = 29), so that 67% and 72% showed positive basal C-peptide levels, and 30% and 21% of the recipients became insulin independent, respectively. No single patient showed islet graft function sufficient to allow withdrawal from insulin, if the pancreata have been stored for more than 12 h. In regard to recipient category for the six groups, namely IAK (islet after kidney), SIK (simultaneous islet kidney transplantation), SIL (simultaneous islet liver transplantation), SIL(C) (simultaneous islet liver transplantation after cluster operation), SIKL (simultaneous islet kidney liver transplantation), and SIH-L (simultaneous islet heart-lung transplantation), the number of patients who showed positive basal C-peptide levels post-transplant was 11 (58%), 17 (57%), 5 (83%), 8 (80%), 1 (50%), and 0 (0%), and the number of insulin independent patients was 4 (21%), 4 (13%), 0 (0%), 6 (60%), 0 (0%), and 0 (0%), respectively. Comparing the two largest recipient categories, namely IAK and SIK, no difference in the outcome of these transplants was apparent.(ABSTRACT TRUNCATED AT 400 WORDS)
Adult↗
Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.
Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.
Animals↗
Development of an automated computer-controlled islet isolation system.
Before clinical islet transplantation can become an effective and reliable treatment for type 1 diabetic patients, there must be significant improvements in the methods employed for the isolation of islets of Langerhans. We have developed an automated cell extraction system (ACES), which allows computer control of the isolation process. As well, it incorporates a novel method of recombining dissociated pancreatic tissue. Following initial system design and testing to determine the optimal system configuration, a series of 12 consecutive canine islet isolations were performed. Pancreases were perfused with collagenase via the duct and dissociated and recombined using either the standard Ricordi-based protocol (group 1, n = 6) or dissociated and recombined using the ACES system (group 2, n = 6). A total of 90.8 +/- 21 x 10(3) islet equivalents (IE) (mean +/- SEM) were recovered in group 1 vs. 99 +/- 14 x 10(3) IE in group 2 (p = NS, student unpaired t-test). Following Ficoll purification the recovery was 56.2 +/- 14 x 10(3) IE for group 1 vs. 54.7 +/- 11 x 10(3) IE for group 2 (p = NS). Viability was equivalent with an 8.6-fold increase in insulin secretion for group 1 and an 8.8-fold increase for group 2 when the islets were exposed to high glucose solution supplemented with IBMX (3-isobutyl-1-methylxanthine) during static incubation. In vivo function was equivalent following transplantation of 2000 IE under the kidney capsule of alloxan-induced diabetic nude mice with five of six and five of seven mice surviving long-term (> 50 days posttransplant) (groups 1 and 2, respectively). This data shows that an entirely automated pancreatic islet extraction system can result in effective canine islet recovery without compromising islet yields and viability. The ACES system has several advantages over the standard isolation protocol. These include: 1) computer control and monitoring over all phases of the isolation, 2) a single-use sterile disposable tubing set, and 3) a novel method of tissue recombination.
Animals↗
Assessment of intracellular insulin content during all steps of human islet isolation procedure.
This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.
Cell Separation↗