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Z Ao

Publications and source records attributed to Z Ao.

49 records · Page 3Linked to original sources
PubMed1993

Survival and function of purified islets in the omental pouch site of outbred dogs.

The aims of this study were to devise an omental pouch site for islet implantation in a preclinical large animal and to compare the function of islets engrafted to this site with islets implanted into the spleen. Highly purified islets were isolated from outbred mongrel dogs, then grafted into totally pancreatectomized outbred recipients. Autografts of islets were implanted into a greater omental pouch (group [gp] 1, n = 12) or into the spleen by venous reflux (gp 2, n = 12). Allografts of single donor islets were implanted into the omental pouch (gp 3) of dogs that received CsA (n = 9) or untreated controls (n = 3). The threshold islet mass that consistently reversed diabetes in gp 1 was 10 microliters/kg, which exceeded by 2.5-fold that required in gp 2. Normoglycemia was induced and maintained for 2 months in 6 gp 1 and 8 gp 2 dogs. At IVGTT, the K value (decline in glucose, %/min) was 1.3 +/- 0.4 in gp 1 versus 1.5 +/- 0.2 in gp 2 (P > 0.2). Peripheral venous insulin levels were significantly lower (P < 0.01) in gp 1. Omentectomy (gp 1) or splenectomy (gp 2) induced prompt hyperglycemia. All gp 3 dogs (received > 10 microliters/kg) were initially normoglycemic: grafts of untreated controls failed at 4.2 +/- 1.8 days. In 1 CsA-treated dog the graft failed for technical reasons; normoglycemia persisted in the other 8 for 10, 15, and 21 days, and in 5 instances for > 30 days. When CsA therapy was stopped at 30 days, normoglycemia persisted for 34 +/- 9.5 days. We conclude that purified islets restore normoglycemia after implantation into the omental pouch of diabetic dogs. Compared with intrasplenic islet implantation, an increased graft volume is required and insulin levels are lower.

Animals↗
PubMed1993

Prolongation of canine pancreatic islet allograft survival with combined rapamycin and cyclosporine therapy at low doses. Rapamycin efficacy is blood level related.

We studied the survival of 5 groups of apancreatic mongrel dogs that received 30 days of treatment with CsA adjusted to 300 micrograms/L, rapamycin (0.05 mg/kg/day), both, or no immunosuppression after intrasplenic allotransplantation with purified pancreatic islets. Autografts survived indefinitely. Neither CsA nor rapamycin alone at low doses showed significant increase in islet allograft survival: 6.2 +/- 1.7 and 5.0 +/- 1.1, respectively, versus 3.4 +/- 1.0 days in controls. Dogs treated with low doses of both CsA and rapamycin demonstrated prolongation of graft function to 23.6 +/- 13.2 days (P < 0.05). These findings support synergism between these 2 agents, especially as CsA was not shown to increase trough rapamycin blood concentration when given together. In the combined treatment group, a significant (r = 0.90, P < 0.001) relationship was found between rapamycin blood levels and graft survival. Animals having trough rapamycin concentrations > 10 micrograms/L had significantly (P < 0.05) prolonged graft survival, which suggests that dosing of rapamycin according to blood levels may optimize the effectiveness of the drug. Given at these low doses, combination CsA and rapamycin gave no evidence of adverse effects as measured by hepatic and renal function tests, histology, or electron microscopy.

Animals↗
PubMedDate not supplied

Bulk cryopreservation of isolated islets of Langerhans.

Current methods to isolate human islets of Langerhans are limited and multiple donors are required for successful reversal of longstanding Type 1 diabetes mellitus. Cryopreservation of isolated islets is an effective method of storing and pooling islets. Current cryopreservation protocols are cumbersome due to current practices of placing small aliquots of islets per individual freezer tube. In the present study, we examined the application of a blood freezer bag for the cryopreservation of isolated islets by slow cooling and rapid thawing. Freezing and thawing profiles generated using thermocouples placed inside a 500 mL Cryocyte (Baxter) blood freezer bag showed that a longer equilibration period at -7.4 degrees C was necessary to consistently achieve nucleation and cooling profiles similar to those observed in glass tubes. When known numbers of rat islets were placed in the freezer bag and the cryoprotectant dimethyl sulfoxide (DMSO) was added in a stepwise fashion and removed using a sucrose dilution, the islet recovery compared with glass tubes was 92 +/- 4.8 vs. 90 +/- 2.3% (n = 4, p = ns, Mann-Whitney U-test). When purified canine islets were cryopreserved in a single freezer bag or in multiple glass tubes, the recovery was similar (78.8 +/- 12.5% recovery for freezer bag vs. 82.3 +/- 5.3% for glass tubes; n = 6, p = ns). In vitro function was equivalent for both groups. The stimulation index of insulin release during glucose perifusion (stimulated over basal insulin secretion) for canine islets cryopreserved in a freezer bag vs. glass tubes was 3.2 +/- 1.0 and 2.3 +/- 1.3, respectively (n = 6, p = ns). These values were significantly lower than the nonfrozen control islets (6.9 +/- 2.4, p < 0.05). When 2000 canine islets cryopreserved in either a freezer bag, or glass tubes were transplanted into diabetic nude mice, the animals became and remained normoglycemic posttransplant. We conclude that the survival of freshly isolated canine islets cryopreserved in a single freezer bag is equivalent to the glass tube method. Bulk cryopreservation of islets in a single freezer bag will facilitate effective low temperature tissue banking to support ongoing clinical trials of islet transplantation.

Animals↗
PubMedDate not supplied

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↗
PubMedDate not supplied

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Adenosine↗