Rejection of islets versus immediately vascularized pancreatic allografts: a quantitative comparison

It has been suggested that free grafts of islets are rejected more vigorously than immediately vascularized intact organs grafts. However, the physiological manifestations of rejection depend, in part, upon the functional reserve of the transplanted tissue. If the number of islets transplanted is just adequate to maintain normoglycemia, the immune destruction of only a few islets will be manifested by hyperglycemia. Thus, differences in rejection time could be an artifact of the islet mass transplanted. We compared the onset of rejection of immediately vascularized segmental pancreatic grafts and of free grafts of islets under conditions in which the β cell mass transplanted, as determined by tissue insulin content, was equivalent. Lewis rats, made diabetic (plasma glucose > 400 mg/dl) by streptozotocin, received either free islet allografts by portal embolization or vascularized segmental pancreatic allografts derived from Fischer donors. Identical pancreatic segments that were not transplanted had a mean (± SE) total tissue insulin content of 33 ± 3 μg. The mean total insulin content of Fischer islets prepared by collagenase digestion in a quantity identical to that used for transplantation to single recipients was 35 ± 7 μg. Similar measurements were made in Fischer to Fischer and Lewis to Lewis isograft control groups. Recipients of both segmental pancreas and free islet grafts became normoglycemic after transplantation and this state was sustained indefinitely in recipients of syngeneic grafts. In rats receiving allografts, the day of rejection, defined as an elevation of plasma glucose to >200 mg/dl, occurred at a mean of 12.1 ± 0.3 days for recipients of pancreatic grafts (n = 17) and 5.2 ± 0.3 days in recipients of islet grafts (n = 17) (P < 0.001). The functional survival of free grafts of allogeneic islets is less than that of islets contained within immediately vascularized pancreatic grafts, even when the transplanted β cell mass is equivalent. However, this difference could still be due to nonimmunologic, quantitative factors that influenced the rate with which hyperglycemia occurred after initiation of the rejection process. The insulin content in the livers of islet isograft recipients showed that only 53 to 71% of the transplanted islets survived. Further experiments that compensate for this factor are needed to determine whether or not there are differences in susceptibility to rejection of the two types of grafts. Nevertheless, on the basis of the number of donors used per recipient, islet allotransplantation is less efficient than pancreas allotransplantation.     

同种异体胰岛及胰腺干细胞来源的胰岛样结构序贯移植治疗糖尿病

目的 观察同种异体大鼠胰岛及胰腺干细胞来源的胰岛样结构序贯移植在糖尿病治疗中的作用.方法 分离胰腺组织获得胰岛及胰腺导管上皮细胞,将具有干细胞潜能的胰腺导管上皮细胞在体外培养27d.将新鲜分离的胰岛(200±50)个及诱导分化2周的胰腺干细胞来源的胰岛样结构(2×106)个序贯移植到糖尿病大鼠的肾被膜下观察大鼠的血糖及生存情况.结果 将胰岛及胰腺干细胞来源的胰岛样结构序贯移植到同一糖尿病大鼠3周后血糖仍在5 mmol/L水平,对照组血糖无明显下降.结论 胰腺干细胞可诱导分化为分泌胰岛素的胰岛样结构,胰岛及胰腺干细胞来源的胰岛样结构序贯移植对大鼠糖尿病有治疗作用.     

Transplantation of porcine Langerhans islets for therapy of type I diabetes. The way to clinical application]

Transplantation of isolated pancreatic islets provides an interesting alternative to the present cure for diabetes: insulin injections and pumps. These are characterized by an insufficient glucose haemostasis and in the long run can induce kidney failure, blindness, heart failure, and amputations. Up to now more than 293 allogeneic islet transplantations have been performed in diabetics with chronical kidney failure. Despite some success, no real breakthrough has been yet achieved, though great efforts are being made to improve the various methodological steps on the way to clinical transplantation. The use of animal (xenogeneic) organs could be a solution to overcome the shortage of allogeneic donors. The current experimental and clinical research focuses on the use of pigs as organ donors, which have a number of advantages over the immunologically more compatible primates. This article reports on success and open questions concerning the efforts to isolate porcine islets for future clinical transplantation: the search for a suitable pig breed, the various isolation steps, purification and in vitro culture, transplantation models using-small and large animals, first clinical trials, and immunological reactions against the xenogeneic tissue. In addition, strategies to circumvent tissue rejection and future perspectives are discussed.     

Transplantation of macroencapsulated human islets within the bioartificial pancreas βAir to patients with type 1 diabetes mellitus

Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell–derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1‐2 βAir devices, each containing 155 000‐180 000 islet equivalents (ie, 1800‐4600 islet equivalents per kg body weight), and monitored for 3‐6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C‐peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose‐stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).     

Pulsatile insulin release from islets isolated from three subjects with type 2 diabetes

Plasma insulin in healthy subjects shows regular oscillations, which are important for the hypoglycemic action of the hormone. In individuals with type 2 diabetes, these regular variations are altered, which has been implicated in the development of insulin resistance and hyperglycemia. The origin of the change is unknown, but derangement of the islet secretory pattern has been suggested as a contributing cause. In the present study, we show the dynamics of insulin release from individually perifused islets isolated from three subjects with type 2 diabetes. Insulin release at 3 mmol/l glucose was 10.5 +/- 4.5 pmol.g(-1).s(-1) and pulsatile (0.26 +/- 0.05 min(-1)). In islets from one subject, 11 mmol/l glucose transiently increased insulin release by augmentation of the insulin pulses without affecting the frequency. Addition of 1 mmol/l tolbutamide did not increase insulin release. In islets from the remaining subjects, insulin release was not affected by 11 mmol/l glucose. Tolbutamide transiently increased insulin release in islets from one subject. Insulin release from four normal subjects at 3 mmol/l glucose was 4.3 +/- 0.8 pmol.g(-1).s(-1) and pulsatile (0.23 +/- 0.03 min(-1)). At 11 mmol/l glucose, insulin release increased in islets from all subjects. Tolbutamide further increased insulin release in islets from two subjects. It is concluded that islets from the three individuals with type 2 diabetes release insulin in pulses. The impaired secretory response to glucose may be related to impaired metabolism before mitochondrial degradation of the sugar.     

Functional and molecular defects of pancreatic islets in human type 2 diabetes

To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.     

Transplantation of cultured pancreatic islets to BB rats

Pancreatic islets held in tissue culture before transplantation into artificially induced diabetics are not rejected. In animals and human identical twin transplants, the autoimmunity of naturally occurring diabetes may destroy islets, even if rejection is avoided. Therefore we studied whether autoimmune damage of islets can be avoided by pretransplant culture. Recipients were BB rats, which spontaneously developed diabetes. Donors were either Wistar Furth (WF) (major histocompatibility [MHC] identical to BB rats) or Lewis (MHC nonidentical to BB rats). Islets were inoculated into the portal vein either immediately after isolation or after 14 days in tissue culture (95% air, 5% CO2, 24 degrees C). Recipients of cultured islets received a single injection of 1 ml of antilymphocyte serum at the time of transplant. Recurrence of diabetes after transplantation of freshly isolated MHC incompatible Lewis islets occurred rapidly on the basis of rejection or autoimmune damage (or both). Precultured Lewis islets had prolonged or permanent survival. Freshly isolated MHC compatible WF islets were destroyed, and no improvement was seen with culture. We conclude that autoimmune destruction of transplanted islets can be avoided by tissue culture, as can rejection. This is important because this strategy is effective only if recipient and donor differ at the MHC locus. Islet donors may need to be selected on the basis of disparity of histocompatibility factors.     

Transplantation of pancreatic islets into cleared mammary fat pads

Hyperplastic islets of Langerhans from 129/J db3J/db3J mice were used to test the efficacy of the cleared mammary fat pad as an ectopic transplantation site. Eight to 10 islets were inoculated into a cleared mammary fat pad of syngeneic 129/J or allogeneic BALB/cByJ female mice following diabetes induction using streptozotocin. Islets transplanted into syngeneic hosts reversed the weight loss and hyperglycemia characteristic of nontransplanted streptozotocin-diabetic mice. In contrast to the degenerate islets found in the host pancreas, the ectopic islets were filled with well-granulated beta cells and they incorporated 3H-thymidine. Islets transplanted into allogeneic BALB/cByJ diabetic hosts produced weight gain and remission of hyperglycemia in four of six recipients. Extirpation at the 9th week of the cleared fat pad containing the islet graft in one of the cured mice returned the animal to a hyperglycemic state within 1 week. Although some of the grafted islets in allogeneic hosts appeared to be completely intact at 8 weeks, others appeared to be undergoing rejection by the host. Nevertheless, the maintenance of viable and functional islets in the cleared fat pads in H-2-histoincompatible recipients for 8 weeks without immunosuppression of the host or prior culture of the islets makes this model attractive for further study as a potentially immunoprivileged site.     

Transplantation of isolated hepatocytes into the pancreas

In previous research into hepatocyte transplantation (HTX) the spleen was the preferred acceptor organ for isolated donor hepatocytes. In this study the pancreas was tested as an acceptor organ for HTX. HTX into the pancreas or spleen was performed by injection of 10(7) isolated hepatocytes into the parenchyma of these organs. Intrapancreatic hepatocytes showed good viability 3 months after syngenic HTX as assessed by histological and immunocytochemical parameters. Definite proof of sustained metabolic activity of normal hepatocytes, 3 months after transplantation into the pancreas of congenitally jaundiced rats, was obtained by demonstration of bilirubin conjugates in bile of the recipients: 4.0% of total biliary bilirubin was conjugated. Intrasplenic HTX, however, was more effective and resulted in a conjugated fraction of 17.7% of total biliary bilirubin (p less than 0.001). Reduction of total plasma bilirubin was significant with both methods, but more pronounced in intra-splenic HTX. Bile drainage from the hepatocellular transplant via the pancreatic excretory system into the gut was not observed: conjugated bilirubins were not found in pancreatic juice of HTX-treated jaundiced rats. Intrapancreatic HTX did not adversely affect the host rat; evidence of pancreatitis or diabetes was not found. It is concluded that the pancreas is a suitable acceptor organ for HTX. However, intrapancreatic HTX appears to be less effective than intrasplenic HTX in the treatment of enzyme deficiency disease.     

Simultaneous cadaver renal and pancreas transplantation in type I diabetes

Between February 1985 and April 1986, we performed 11 simultaneous cadaver kidney and segmental pancreatic transplants in patients with type I diabetes. There were nine men and two women ranging in age from 25 to 47 years (mean, 38.5 years). All pancreatic grafts were extraperitoneal, and the pancreatic duct was managed by pancreaticocystostomy utilizing an internal stent. Three patients died from two to six weeks postoperatively of septic complications. Four pancreatic grafts were functioning at 2, 5, 11, and 14 months after operation, and eight patients had had functioning renal allografts from two to 14 months (mean, 6.8 months) with a mean serum creatinine level of 2.4 mg/dL (210 mumol/L). Graft failure occurred in the other four patients from vascular thrombosis (three patients) or hemorrhagic pancreatitis (one patient). Significant morbidity included an infected arterial anastomosis (two patients), pancreatic fistulas (four patients), and bladder leak (four patients). In conclusion, this procedure is an effective option for selective diabetics with end-stage renal disease. Although technical complications were frequent, no adverse effect on renal allograft function was evident. With technical refinements, this procedure should be applicable to most type I diabetics with renal failure.     

Growth of a nation part I: impact of organ donor obesity on whole-organ pancreas transplantation

Obesity has reached epidemic proportions in the USA. Consequently, there are an increasing number of potential organ donors that are obese, but would otherwise be appropriate donors for pancreas transplantation (PTx). This is a retrospective study of all PTx performed at Indiana University between 2003 and 2009 (n = 308) comparing donors with body mass index (BMI) <25, 25-29.9, and ≥30 kg/m(2) . Data included recipient and donor demographics, seven and 90-d graft loss, one-yr pancreas, kidney (for simultaneous pancreas and kidney transplant only) and patient survival, causes of graft loss and death, peak amylase and lipase, length of stay, readmissions, complications, HbA1C, and c-peptide. Of the 308 donors, 84 (27%) were overweight and 43 (14%) were obese. The overweight donors were significantly older, and the obese donors had hypertension significantly more frequently than the other two groups. There were no significant differences in recipient transplant demographics. There was no significant difference in length of stay or 90-d readmissions, seven or 90-d pancreas graft loss, one-yr graft or patient survival, peak serum amylase or lipase, HbA1C, or c-peptide. The incidence of post-transplant technical, immunological, and infectious complications were similar. Although technically challenging, PTx of allografts from obese donors can be accomplished with similar results compared to normal BMI donors.     

Transplantation of cryopreserved human pancreatic islets into diabetic nude mice

If pancreatic islet transplantation becomes clinically applicable, cryogenic storage of human islet preparations could solve many problems related to transportation and banking of islets, HLA matching, recipient pretreatment, or the possible use of multiple donors. The diabetic nude mouse could offer a simple model to test in vivo the function of cryopreserved human islet preparations before transplantation into diabetic patients. In this study, 6 nude mice made diabetic by an intravenous injection of streptozocin were transplanted with 400–600 cryopreserved human islets beneath the renal capsule. All the mice became normoglycemic within 3 weeks from transplantation. Nephrectomy of the kidneys bearing the grafts 45 days after transplantation resulted in an immediate return to the diabetic state, demonstrating that the only functional tissue was located in the excised kidneys. Histologic study of the renal subcapsular grafts demonstrated morphologic integrity of the islets with a normal degree of beta granulation. The results demonstrated that cryopreserved human islets will function in diabetic nude mice. This model to test human islet preparations in vivo may be of assistance in the application of cryopreserved islet transplantation in diabetic patients.

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Resumen Si el trasplante de islotes pancreáticos se hace clínicamente aplicable, la criopreservación de preparaciones de islotes pancreáticos humanos podría resolver muchos de los problemas relacionados con el transporte y almacenamiento de tales islotes, con la compatibilización HLA, con el pretratamiento del receptor, y con el posible uso de multiples donantes. El ratón atímico diabético puede representar un modelo sencillo para probarin vivo la función de preparaciones criopreservadas de islotes humanos antes de ser trasplantadas a pacientes diabéticos.En el presente estudio 6 ratones atímicos hechos diabéticos mediante la inyección intravenosa de estreptozotocina, fueron trasplantados con 400–600 islotes humanos criopreservados al tejido subcapsular renal a través de una incisión en la cápsula del riñón. Todos los ratones convirtieron a normoglicemia dentro de las primeras 3 semanas después del trasplante. La nefrectomía del riñón portador de los islotes, realizada a los 45 días del trasplante, resultó en el retorno inmediato al estado diabéticos, demostrando así que el único tejido funcional estaba representado por los islotes trasplantados al riñón. El estudio histológico de los trasplantes renales subcapsulares demostró morfología normal de los islotes con grado normal de granulación de tipo beta.Nuestros resultados demuestran que los islotes criopreservados funcionan en trasplantes al ratón atímico. Este modelo escogido para probar el funcionamiento de preparaciones de islotesin vivo puede ser de utilidad en la aplicación práctica del trasplante de islotes pancreáticos criopreservados en pacientes diabéticos.

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Résumé Si la transplantation des ilôts pancréatiques devient un jour applicable cliniquement, le stockage cryogénique des préparations d'ilôts humains pourrait résoudre bien des problèmes concernant le transport et la banque d'ilôts, l'appariement HLA, le prétraitaient du receveur ou la possibilité d'utiliser plusieurs donneurs. La souri nude diabétique est un modèlein vivo simple pour tester la fonction des préparation des ilôts humaines avant de pratiquer la transplantation chez le diabétique.Dans cette étude, on a pratiqué la transplantation de 400–600 ilôts humains conservés au froid sous la capsule rénale chez 6 souris nude, rendues diabétiques par injection intraveineuse de Streptozotocine. Toutes les souris sont redevenues normoglycémiques en moins de 3 semaines. La néphrectomie des reins portant les greffons 45 jours après a provoqué immédiatement le retour à l'état diabétique, démontrant la qualité fonctionnelle des seuls ilôts transplantés. L'étude histologique des greffons sous capsulaires a montré l'intégrité morphologique des ilôts avec un taux normal de granulation bétâ.Ces résultats prouvent que les ilôts humains conservés fonctionnent bien chez la souri-nude diabétique. Ce modèle pour tester les préparationsin vivo peut servir d'aide dans l'application de la transplantation d'ilôts conservés au froid chez le diabétique.

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Supported in part by NIH Training Program Grant No. 5T32A107163; Brown and Williamson Tobacco Corporation, Philip Morris Incorporated, R.J. Reynolds Tobacco Company and United States Tobacco Company; Project Transplant; The Humana Foundation; and the PJD Fund.