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. ≥ 1 ng/mL at ≥ 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 ≤6 h (n = 27) and >6 ≤ 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. The only sites of transplantation in the period between 1990-1992 were the liver, epiploic flap, and spleen, with the total number of recipients being 60 (90%), 4 (6%), and 3 (4%), respectively. For these three groups, the number of patients who showed positive basal C-peptide levels was 38 (63%), 2 (50%), and 1 (33%), and the number of insulin independent patients was 13 (22%), 0 (0%) and 0 (0%), respectively. In the overwhelming majority of cases, recipient selection was not based on prospective HLA donor/recipient matching. In different categories of HLA mismatching (i.e., AB-, DR-, BDR-, and ABDR-mismatch) no obvious tendency or difference in outcome could be demonstrated. In the 1990-1992 period, 16 patients (24%) received OKT3, 26 patients (39%) received ALS, ALG, or ATG, and 25 patients (37%) received neither monoclonal nor polyclonal T-cell antibodies for induction immunosuppression. For the three groups mentioned, the number of patients who showed positive basal C-peptide levels was 12 (86%), 16 (62%), and 15 (60%), and the number of insulin independent patients was 2 (14%), 6 (23%), and 6 (24%), respectively. A synopsis of all pretransplant C-peptide-negative Type I diabetic patients who succeeded in insulin independence revealed certain common characteristics, such as transplantations of ≥8000 islet equivalents per kilogram body weight, a purity of transplanted islets ≥ 50% (in all but one case), the liver as implantation site, and OKT3 or ALG/ ALS/ATG for induction immunosuppression (“state of the art” cases). Because it has been unequivocally proven that islet transplantation can be performed successfully in association with other organ transplants, more attention should be drawn to the nonuremic, nonkidney Type I diabetic patients, who have not been reported in a single case since 1990. This is the real target group, that could benefit most from islet replacement. However, islet transplantation in this recipient category will only have an undisputable indication, if low-risk, but effective methods other than life-long immunosuppression to protect the islet graft from rejection can be developed.

Improved glucose counterregulation and autonomic symptoms after intraportal islet transplants alone in patients with long-standing type I diabetes mellitus.

BACKGROUNDnDefective glucose counterregulation and hypoglycemia unawareness are both well-recognized risk factors for recurrent episodes of severe hypoglycemia in patients with type I diabetes. At present, no conventional therapy is available to routinely overcome these acquired impairments in long-standing diabetes.nnnMETHODSnTo test the hypothesis that successful intraportal islet transplantation could improve this syndrome, hormonal counterregulatory responses and symptoms were studied during stepped hypoglycemic clamp tests before and after intraportal islet transplantation in three patients with type I diabetes who were prone to severe hypoglycemia.nnnRESULTSnAs compared with matched nondiabetic control subjects, before islet transplantation, glucagon responses were absent while epinephrine and cortisol responses were either markedly decreased or absent in all diabetic subjects. One patient also had decreased norepinephrine and growth hormone responses. Autonomic warning symptoms were absent in all patients during hypoglycemia. One month after successful islet transplantation, there was no improvement in the glucagon response. However, glycemic thresholds and/or peak incremental responses of epinephrine, norepinephrine, and cortisol improved in all patients. Moreover, all patients had developed autonomic warning symptoms so that glycemic thresholds were detectable within the examined range.nnnCONCLUSIONnWe conclude that intraportal islet transplantation does not restore hypoglycemia-induced glucagon secretion, but it improves the responses of most counterregulatory hormones and hypoglycemic warning symptoms even in long-standing type I diabetes.

Leptin inhibition of insulin secretion from isolated human islets

Abstract Leptin is a hormone produced and secreted from the adipose tissue. Its physiological actions include the regulation of satiety, food intake and energy balance. The production of leptin is increased by high insulin levels. Here, we demonstrate that leptin acts as an inhibitor of glucose-induced (20 mM) insulin secretion from isolated human islets. No effect was observed in the presence of lower glucose levels (2.8 and 10 mM glucose). The pancreatic β-cell might represent a target of a direct physiological action of leptin. We suggest the presence of an “adipo-insular axis” in which leptin mediates negative feedback from the adipose tissue to the endocrine pancreas.

Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll

Endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. By the chromogenic Limulus amebocyte lysate assay we found rather high but variable endotoxin concentrations in the chemicals used for islet isolation, i.e. collagenase and Ficoll. Liberase, a special collagenase preparation from Boehringer, was nearly endotoxin-free. Correlating to the endotoxin content, collagenase and Ficoll had the capacity to induce interleukin-1β release from human peripheral blood mononuclear cells. Because collagenase and density gradient media are needed in most cell isolation procedures from solid organs, each lot of these chemicals should be tested for endotoxin contamination. In pancreatic islet transplantation, the use of endotoxin-free chemicals may diminish early local inflammatory reactions at the graft site and thereby reduce the number of islets needed for successful islet transplantation.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft [1]. With improved isolation methods based on the original procedure published by Ricordi et al. [2] yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation [3, 4]. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.

Large variability of the intracellular ATP content of human islets isolated from different donors.

Abstract Observations in experimental heart, liver, kidney and pancreas transplantation indicated that graft function and survival correlates significantly with ATP content of transplanted tissue. The ATP content of cells can be reduced by several factors i.e. the nutritional donor status, storage technique, warm ischemia and cold ischemia time. This study investigates the intracellular ATP content of isolated human islets for the first time.Quantified samples of freshly isolated (digestion-filtration, continuous ficoll gradient purification) and cultured (22°C, CMRL+10% FCS) islet equivalents (IEQ) of consecutively processed human pancreata from multiorgan donors (UW vascular flush) were shock frozen in liquid nitrogen and stored at –196°C until rapid thawing, sonification and subsequent luminometric determination of ATP (Luciferin-Luciferase-reaction) and assessment of islet protein (IP). The ATP content was analysed for freshly isolated and subsequently 5±1 days cultured islets (n=10).The ATP content of freshly isolated human islets was 130.4±53.4 pg/µg IP (mean ± SEM) corresponding to 20.7±6.3 pg/IEQ. After culture ATP content increased to 265.5±113.3 pg/µg IP (204.2±41.5%) corresponding to 43.7±15.3 pg/IEQ (216.1±34.9%; p<0.05). The coefficient of variation was 129.5%, 96.5% (fresh) and 135.0%, 111.0% (cultured) for ATP/µg IP and ATP/IEQ, respectively.The present data show that: (1) the ATP content of freshly isolated human islets varies enormously; (2) intraislet ATP levels increase significantly during 22°C culture suggesting that the capacity to produce ATP is maintained despite hypothermic environment. More data are necessary to clarify the relevance of intraislet ATP content for graft function and survival after islet transplantation.

Critical islet mass for successful porcine islet autotrasplantation

A major reason for the failure of clinical islet transplantations may be a limited islet mass. The aim of this study was to determine the critical islet mass necessary for normalization of glucose metabolism in a porcine model. Diabetes was induced by total pancreatectomy. The splenic lobe of the pancreas was intraductally distended with UW-solution containing 2.67–3.33 mg/ml collagenase, and the distended pancreas was digested in a continuous digestion filtration device. The islets were purified on a isoosmotic Ficoll-sodium-diatrizoate gradient. The survival period of the diabetic recipients in group 2 and 3 receiving, respectively, a low (2.14±0.39 µL/kg body weight) and a high (4.99±0.83 µL/kg body weight) islet mass was significantly prolonged compared to that of diabetic recipients in group 1 receiving no islet transplantation. However, the survival period of the recipients in group 2 was not significantly different to that in group 3. Three recipients of an islet mass of >5 µl/kg body weight became normoglycemic (fasting blood glucose <100 mg/dl) for more than two months. Furthermore, the glucose and insulin release reactions to the glucose challenge were comparable to that before pancreatectomy. Contrarily, another five diabetic recipients of an islet mass of <4 µL/kg body weight became a fasting blood glucose level of <200 mg/dl. The glucose and insulin release reactions to the glucose challenge were improved only, but not normalized compared to that before pancreatectomy. The data presented in this study demonstrate that metabolic normalization in pancreatectomized diabetic minipigs can be established by autotransplantation of an islet mass of >5 µl/kg body weight.

Glutamic acid decarboxylase antibodies are more frequent than islet cell antibodies in islet transplanted IDDM patients and persist or occur despite immunosuppression.

Pancreatic islet grafts transplanted into patients with autoimmune diabetes are potentially threatened by two immune responses, allograft rejection and the recurrence of autoimmune insulitis. In the present study we investigated the humoral autoimmune response directed to islet autoantigens by studying islet cell antibodies and glutamic acid decarboxylase (GAD 65) antibodies in twenty-one insulin-dependent diabetes-mellitus (IDDM) patients undergoing intraportal islet allotransplantation. Islet transplantation was performed according to the following recipient categories: Islet after kidney transplantation (n=10), simultaneous islet and kidney transplantation (n=6) and islet transplant alone (n=5). GAD 65 antibodies were detected in a radioligand GAD 65 antibody assay using recombinant, in vitro translated, human 35S-methionin labelled GAD 65 as tracer. Islet cell antibodies were determined by indirect immunofluorescence technique on human pancreas. In six out of twenty-one patients we observed GAD 65 antibodies before islet transplantation and the GAD 65 antibodies persisted despite immunosuppression. In contrast only two subjects were concordantly islet cell antibody positive and the titre decreased post transplantation. In addition we observed occurrence of GAD 65 antibodies in five subjects that were shown to be antibody negative before islet transplantation with three of them subsequently becoming positive for islet cell antibodies. The remaining ten patients were GAD 65 antibody and islet cell antibody negative before islet transplantation and remained negative thereafter. Interestingly none of the patients was exclusively positive for islet cell antibodies without being positive for GAD 65 antibodies. In summary we have demonstrated in twenty-one islet grafted individuals that humoral autoimmunity to islet antigens can persist or occur despite immunosuppression. Islet cell antibodies appear to be less frequent (5 out of 21, 23%) compared to GAD 65 antibodies (11 out of 21, 52%) suggesting that they are more affected by immunosuppressive therapy. We conclude that GAD 65 antibodies are a useful tool to further evaluate a possible link between persistent autoimmunity and early or late graft failure after islet transplantation.

Viability and recovery of frozen-thawed human islets and in vivo quality control by xenotransplantation

Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3±8.0% for vitrified and 51.8±3.0% for conventionally cryopreserved islets; the recovery rate was 84.8±12.2% and 92.8±12.4%, respectively. Insulin recovery after transplantation was 25.6±7.3% for fresh and 24.1±7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.

Enterostatin inhibits insulin secretion from isolated perifused rat islets

The effect of enterostatin on glucose-induced insulin secretion was examined in isolated, perifused rat islets. In the presence of 16.67 mM glucose, there was significant inhibition of insulin secretion at concentrations of 200 nM, 2, 20 and 40 μM enterostatin. In particular, the second phase of insulin secretion was inhibited. With a low concentration of glucose (2.78 mM), there was no significant effect on insulin secretion by enterostatin. The inhibition of insulin secretion exerted by enterostatin may be an important effect in the prevention of insulin resistance.