The effects of immunosuppression on islet transplant function in the dog.
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Animals↗
Biomedical subjects
N M Kneteman
Publications and source records attributed to N M Kneteman.
Long-term normoglycemia in pancreatectomized dogs transplanted with frozen/thawed pancreatic islets.
Storage of pancreatic islets by cryopreservation would greatly facilitate a large scale program of clinical islet transplantation. We report success on long-term follow-up with autotransplantation of frozen/thawed canine pancreatic fragments. Total pancreatectomy and islet isolation by collagenase ductal perfusion and mechanical disruption preceded either acute autotransplantation or cryogenic preservation prior to autotransplantation. Cryopreservation was by dimethylsulfoxide equilibration, cooling at 0.25 degrees C/min to -75 degrees C, storage in liquid N2 and thawing at 3.5 degrees C/min. Four of five acutely autotransplanted dogs remained normoglycemic for 20 months, with three of four maintaining normal K values on intravenous glucose tolerance test (IVGTT) and nondiabetic values on oral GTT. Four of four dogs transplanted with frozen/thawed islets remained normoglycemic for 15 months with three of four maintaining nondiabetic IV GTT K values and normal oral GTTs for 15 months. Both acutely transplanted and frozen/thawed islets are capable of maintaining long-term metabolic control. Cryopreservation preserved viability of sufficient canine pancreatic islets to reverse diabetes with autotransplantation. Function of the frozen-thawed islets showed minimal deterioration during a follow-up of 15 to 18 months.
Animals↗
Canine pancreatic fragment allotransplantation with cyclosporine A.
Allograft rejection is the major obstacle to clinical pancreatic islet transplantation. We attempted Cyclosporine A (CsA) immunosuppression of intrasplenic pancreatic dispersed fragment allografts in unrelated, unmatched dogs. Animals underwent (1) pancreatectomy only, (2) autotransplantation, or allotransplantation with (3) no immunosuppression (NIL), (4) azathioprine (AZA 3 mg/kg/day) and prednisone (PRED 2 mg/kg/day), or (5) CsA (25 mg/kg/day). Graft preparation was by collagenase ductal perfusion and mechanical disruption and transplantation was by splenic venous reflux. Rejection was defined by permanent hyperglycemia (greater than 150 mg/dl). Apancreatic dogs survived 6.0 +/- 0.5 days; autotransplanted dogs remained normoglycemic at 6 months. Rejection occurred at NIL, 5.0 +/- 0.6 days; AZA/PRED, 1.8 +/- 0.3 days; and CsA, 19.3 +/- 5.6 days. Survival was NIL, 16.0 +/- 3.4 days; AZA/PRED, 13.4 +/- 1.4 days; and CsA, 33.3 +/- 4.6 days. CsA showed significant advantage over both groups in both time to rejection (P less than 0.05) and survival (P less than 0.01). CsA toxicity was minimal. CsA achieved significant delay in rejection and improved survival in unmatched, unrelated dogs after intrasplenic allotransplantation with pancreatic dispersed fragments.
Animals↗
Isolation and cryopreservation of human pancreatic islets.
The outlined methods were able to isolate relatively large number of intact pancreatic islets from human cadaver pancreata. Confirmation of the nature of the endocrine and exocrine tissue was provided by electron microscopy. The islets were able to tolerate a 14 day period of storage in liquid N2 when frozen and thawed by the protocol given. Viability of the islets both before and after the freeze-thaw sequence was established by secretion of insulin in response to stimulation with both theophylline and high glucose in a dynamic perifusion system. The ability of the islets to survive a 24 hr. period of tissue culture after the freeze/thaw sequence and again respond with insulin secretion in response to hyperglycemia or theophylline stimulation confirms their viability and metabolic activity.
Freezing↗
Islet isolation assessment in man and large animals.
Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity and in vitro and in vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 mu diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason, in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.
Animals↗
Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis.
Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis. Can J Gastroenterol 2000;14(7):637-640. Acute graft-versus-host disease (GVHD) is a common complication after bone marrow transplantation, with characteristic rash and diarrhea being the most common features. After liver transplantation, however, this phenomenon is very rare. Most transplant patients are on a variety of medications, including immunosuppressants; therefore, the differential diagnosis of skin rash or diarrhea is broad. A 37-year-old man who underwent liver transplantation for primary biliary cirrhosis, and developed a rash and watery diarrhea, is presented. Skin and colonic biopsies confirmed acute GVHD. A pulse of intravenous steroids was given. The skin rash improved, but he developed pancytopenia. His course was complicated by central line infection, jugular and subclavian vein thrombosis, pseudomembranous colitis, recurrent bacteremia, cholestasis on total parenteral nutrition and cytomegalovirus infection. After the onset of pleuritic chest pain and clinical sepsis, spiral computed tomography scan of his chest and abdomen revealed septic infarcts in multiple organs. Despite empirical treatment with amphotericin B, he died of multiorgan dysfunction syndrome within 72 h. Autopsy revealed systemic aspergillosis with pancarditis, endocardial vegetations, and septic pulmonary, splenic, hepatic and renal infarcts. The pathogenesis and experience with this rare, but often fatal, complication of liver transplantation are reviewed. In contrast to GVHD after bone marrow transplantation, pancytopenia is common and liver dysfunction is rare. One should have a high level of suspicion in the liver transplant recipient presenting with rash and/or diarrhea.
Adult↗
High yield of rodent islets with intraductal collagenase and stationary digestion--a comparison with standard technique.
Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.
Animals↗
Effect of cryopreservation on the survival and function of murine islet isografts and allografts.
We compared the efficacy of fresh and frozen/thawed islets by determining the minimum number required to consistently reverse diabetes in mice. Defined numbers of islets, isolated from Balb/c (H-2d) and CBA/J (H-2k) mice, were transplanted into streptozotocin-induced diabetic Balb/c mice. Frozen/thawed grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. At 100 days after transplantation, isografts were recovered for measurement of insulin content. Mean (+/-SD) recovery of cryopreserved islets after thawing was 80 +/- 3% (range 67-89%). For both fresh and frozen/thawed isografts and allografts, 200 islets were required to establish normoglycemia. The degree of metabolic function provided by equivalent quantities of fresh and frozen/thawed grafts was similar; and all normoglycemic isograft recipients remained so until graft nephrectomy. The insulin content of fresh and frozen/thawed isografts containing 200 and 300 islets were 151 +/- 25 and 126 +/- 8 mU and 259 +/- 36 and 278 +/- 20 mU, respectively. Among allograft recipients, median survival ranged from 15 to 20 days, and was not influenced by cryopreservation or graft size. The results of this study demonstrate a high rate of recovery of viable islets following cryopreservation. The function of equivalent quantities of fresh and cryopreserved islet isografts and allografts in nonimmunosuppressed recipients is similar.
Animals↗
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↗