Glucose-dependent insulin release from genetically engineered K cells.

Genetic engineering of non-beta cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes. A tumor-derived K-cell line was induced to produce human insulin by providing the cells with the human insulin gene linked to the 5'-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP). Mice expressing this transgene produced human insulin specifically in gut K cells. This insulin protected the mice from developing diabetes and maintained glucose tolerance after destruction of the native insulin-producing beta cells.

Large scale isolation, growth, and function of porcine neonatal islet cells.

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice.

Treatment of the enhanced intestinal uptake of glucose in diabetic rats with a polyunsaturated fatty acid diet.

Intestinal absorption of most nutrients is enhanced in diabetic rats. We wished to test the hypothesis that manipulation of dietary fatty acids will modify enhanced uptake of glucose in rats with established streptozotocin-diabetes. Chow-fed control rats or animals with one week of streptozotocin-diabetes were continued on chow or were fed ad libitum for three weeks with semisynthetic isocaloric diets containing a high content of either essential polyunsaturated or non-essential saturated fatty acids. The jejunal and ileal in vitro uptake of varying concentrations of glucose was much higher in diabetic than control rats fed chow or the saturated fatty acid diet. In contrast, the enhanced uptake of this sugar was reduced or normalized in diabetic rats fed the polyunsaturated fatty acid diet. Feeding the polyunsaturated fatty acid diet was associated with increased brush-border membrane activity of alkaline phosphatase in diabetic jejunum and ileum, but neither the saturated fatty acid diet nor the polyunsaturated fatty acid diet altered brush-border membrane cholesterol or phospholipids in control or in diabetic rats. Mucosal surface area was similar in diabetic rats fed the saturated fatty acid diet or the polyunsaturated fatty acid diet. Thus, (1) feeding the polyunsaturated fatty acid diet diminishes the enhanced jejunal and ileal uptake of glucose in diabetic rats, and (2) the influence of the polyunsaturated fatty acid diet on uptake in diabetic rats was not explained by alterations in intestinal morphology or brush-border membrane content of cholesterol or phospholipids. This study suggests that manipulation of dietary lipids may play a role in the normalization of the enhanced intestinal glucose uptake in rats with established diabetes.

Polyunsaturated fat in the diet may improve intestinal function in patients with Crohn's disease.

To investigate the effect of increasing dietary polyunsaturated fat intake on fat absorption in Crohn's patients, normal subjects and subjects with inactive Crohn's disease consumed a high polyunsaturated to saturated fat ratio diet. Subjects participated in breath tests before and after six months of a high polyunsaturated to saturated (P/S) fat ratio diet to measure their response to [1-13C] 10:0 and [1-13C] 16:0 ingested with a test meal. Whole body absorption-oxidation of C10:0 was not affected by the diet treatment. Before diet treatment, whole body absorption-oxidation of C16:0 in Crohn's patients was 80% of that observed for control subjects. After consuming a high polyunsaturated to saturated fatty acid ratio diet, subjects increased oxidation of C16:0 by 85% compared to before the diet treatment period. It is concluded that (1) absorption of labelled C16:0 from a test meal is reduced in Crohn's patients, and (2) consumption of a high polyunsaturated to saturated fatty acid ratio diet improves the utilization of dietary C16:0 by Crohn's patients.

Cloning of human and rat p69 cDNA, a candidate autoimmune target in type 1 diabetes.

Triggering of autoimmunity in insulin-dependent diabetes was linked to dietary bovine serum albumin (BSA). Anti-BSA antibodies from diabetes-prone rats precipitate a protein, p69, from islet cell lysates. We have used these antibodies to identify rat p69 cDNAs. Human p69 cDNA was identified by crosshybridization. The p69 coding regions show 87% nucleotide and 89% amino acid homology. Recombinant p69 is recognized by autoantibody and T cells from diabetic children.

Critical mass of purified islets that induce normoglycemia after implantation into dogs.

Twenty grafts of highly purified islets of Langerhans (mean +/- SE vol 0.98 +/- 0.3 ml, islet diam 122 +/- 5 micron) were autoimplanted into the spleen or liver of totally pancreatectomized dogs. Portal venous pressure did not change significantly. Incremental doses of islets of 1000-3000 (n = 3), 3000-4000 (n = 6), 4000-5000 (n = 5), 5000-6000 (n = 3), and 6000-8000 (n = 3) per kilogram body weight resulted in corresponding fasting plasma glucose (PG) of 258 +/- 18, 163 +/- 13, 158 +/- 17, 138 +/- 15, and 108 +/- 6 mg/dl. In 3 apancreatic control dogs, PG was 338 +/- 9 mg/dl. One (normoglycemic) dog died of wound complications, and follow-up PG at 1 mo was 89 +/- 5 mg/dl in 6 of 10 dogs that received 3000-5000 islets/kg and 91 +/- 6 mg/dl in all 6 that received greater than 5000 islets/kg. K values 1 mo after surgery during glucose tolerance tests were 1.8 +/- 0.3 for 6 spleen dogs and 1.6 +/- 0.3 for 6 liver dogs. Six months after splenic implantation, PG was 75 +/- 4 mg/dl and rose to greater than 350 mg/dl after splenectomy. These data define the critical number of purified dog islets of known size that is necessary to induce prolonged normoglycemia. Sufficient pure islets can be collected from 1 dog pancreas to correct diabetes after autoimplantation into the liver or spleen.

Insulin sensitivity and glucose effectiveness in long-term islet-autotransplanted dogs.

Parameters determining glucose tolerance were quantified with Bergman's minimal-model method applied to frequently sampled intravenous glucose tolerance tests in five normal and five islet-autotransplanted mongrel dogs 8-10 mo posttransplantation. Despite normal fasting glucose and insulin levels, glucose tolerance was reduced in the islet-transplanted dogs (1.5 +/- 0.4 vs. 4.2 +/- 0.4%/min in normal controls, P less than .002). The reduction in glucose tolerance was due to a reduced insulin response to glucose injection (4 +/- 1 vs. 32 +/- 5 microU/ml, P less than .001) and reduced glucose effectiveness (1.7 +/- 0.5 vs. 5.3 +/- 0.6 x 10(2) min-1, P less than .005) but not to a reduction in insulin sensitivity (8.4 +/- 0.6 vs. 7.8 +/- 0.7 x 10(4) min-1.microU-1.ml-1, P greater than .5). Our results suggest that reduced insulin secretory response as a result of islet transplantation may result in a defect in glucose's ability to promote its own disposal but not necessarily in a defect in insulin sensitivity.

Prevention of recurrence of IDDM in islet-transplanted diabetic NOD mice by adjuvant immunotherapy.

Insulin-dependent diabetes mellitus (IDDM) involves the destruction of the insulin-producing cells in the islets of Langerhans. One possible cure is by transplanting the islet cells; however, transplanted islets, even between identical twins, are subject to autoimmune destruction by the disease process, resulting in diabetes recurrence. We recently reported that complete Freund's adjuvant (CFA), an immunomodulating agent, prevented development of autoimmune diabetes in the NOD mouse. In this study, we evaluated adjuvant therapy in prevention of autoimmune destruction and rejection of transplanted islets in diabetic NOD mice. After transplantation, untreated syngeneic islet recipients (n = 16) initially became normoglycemic and then hyperglycemic, with a median survival time (MST) of the graft of 17 days. When CFA was administered at the time of transplantation, 11 of 13 CFA-treated syngeneic islet recipients remained normoglycemic long term (greater than 100 days) with an MST greater than 107 days. Ten of 11 mice maintained indefinite normoglycemia until the conclusion of follow-up (101 to 172 days). When adjuvant therapy was used in conjunction with allogeneic islet transplantation, graft survival was not extended, with MST being similar to the untreated allogeneic islet recipients (12 [n = 5] and 13 [n = 5] days, respectively). The extended acceptance of second syngeneic islet grafts by CFA-treated mice indicates that the persistent autoimmunity against the transplanted islets can be reversed in the diabetic NOD mice after CFA treatment.

Prolongation of islet xenograft survival by cryopreservation.

Our attempt to reduce islet immunogenicity by slow cooling to -40 degrees C, storage at -196 degrees C, and rapid thawing is based on the differential susceptibility of various cell types to a freeze-thaw process. Five hundred rat islets (greater than or equal to 100 micron) were immediately implanted or cryopreserved and then implanted beneath the renal capsule of streptozocin-induced diabetic mice with or without an injection of anti-lymphocyte serum at the time of transplantation. Thirteen days after transplantation, all fresh xenografts had rejected, whereas 37.5% of cryopreserved grafts were still functioning. In immunosuppressed mice, 6.2% of fresh xenografts and 54.5% of cryopreserved grafts were functioning 19 days after transplantation. These results show that cryopreservation can extend xenograft survival.

Both CD4+ and CD8+ T-cells in syngeneic islet grafts in NOD mice produce interferon-gamma during beta-cell destruction.

Syngeneic pancreatic islet grafts in diabetic NOD mice are infiltrated by mononuclear leukocytes, beta-cells are selectively destroyed, and autoimmune diabetes recurs. This model was used to identify islet graft-infiltrating mononuclear leukocytes associated with beta-cell destruction and diabetes recurrence. We compared cell surface antigen and cytokine-producing phenotypes of mononuclear leukocytes in islet grafts from NOD mice that were protected from diabetes recurrence by complete Freund's adjuvant (CFA) administration (beta-cell nondestructive insulitis) and in islet grafts from control phosphate-buffered saline (PBS)-injected NOD mice (beta-cell destructive insulitis). Islet grafts from CFA-injected mice contained fewer CD4+ and CD8+ cells and more B cells; also fewer interferon gamma (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha)-positive cells and more IL-4 and IL-10 positive cells. By performing two-color immunostaining of cell surface antigens and intracellular IFN-gamma, we found that IFN-gamma positive cells in islet grafts from CFA- and PBS-injected mice were approximately equally divided between CD4+ and CD8+ T-cell subsets. Also, the frequencies of both CD4+ IFN-gamma + and CD8+ IFN-gamma + cells were decreased in islet grafts from CFA-injected mice. These findings suggest that destruction of beta-cells in syngeneic islets transplanted into NOD mice is promoted by cells producing Th1-type cytokines (IFN-gamma, IL-2, and TNF-alpha) and prevented by cells producing TH2-type cytokines (IL-4 and IL-10). Furthermore, both CD4+ and CD8+ IFN-gamma-producing T-cells in the islet grafts appear to be involved in beta-cell destruction and diabetes recurrence.

Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression.

We prepared single-cell suspensions of Lewis rat ¿RT1(1/l)¿ testicular cells and cultured these in vitro for 48 h under conditions that promoted the formation of cellular aggregates. In the absence of systemic immunosuppression, the transplantation of a sufficient quantity of these aggregates (containing 11 x 10(6) cells, (75% Sertoli cells), together with 2,000 purified Lewis rat islets, reversed the diabetic state for >95 days in 100% (5/5) of the chemically diabetic Wistar-Furth ¿RT1(u/u)¿ recipients. Similar grafts consisting of islets alone or islets plus 50% fewer testicular cell aggregates survived for only 10 days. Functioning composite allografts harvested from normoglycemic animals at approximately 100 days showed healthy beta-cells in close association with Fas ligand-expressing Sertoli cells. Because no gene therapy protocol is required, the transplantation of composite grafts consisting of purified human allogeneic islets plus human allogeneic testicular cell aggregates can be applied in clinical islet transplantation as soon as it has been proven in a large animal model.

Normoglycemia after reflux of islet-containing pancreatic fragments into the splenic vascular bed in dogs.

Canine pancreata obtained at total pancreatectomy were cannulated via the ducts and perfused with collagenase to prepare a tissue suspension that was isografted into the spleen (preparation congruent to 2 h, mean graft vol = 10 +/- 1 ml containing 24% of the B-cell mass/pancreas). In 13 dogs the tissue was implanted by reflux into terminal splenic veins: two died postoperatively, and in two the intrasplenic vein wall was inadvertently punctured during cannulation. In the remaining nine, mean fasting blood glucose (BG) was less than or equal to 150 mg/dl initially; one was killed at 2 wk (distemper) and one at 6 wk (sepsis, diabetes), and one died at 9 wk (intestinal obstruction). Mean BG was 94 +/- 4 mg/dl at 1 mo and remained in this range until the dogs were killed at 5 mo (91 +/- 13 mg/dl). During glucose-tolerance testing 1 wk preimplantation and 1 mo and 2-3 mo postimplant, mean values were: K (decline in glucose concentration, %/min), 3.4 +/- 0.2, 1.4 +/- 0.1, and 1.5 +/- 0.1; peak insulin (microU/ml), 50 +/- 5, 12 +/- 1, and 11 +/- 2; fasting serum glucagon (pg/ml), 33 +/- 3, 59 +/- 12, and 53 +/- 9, with no change in the glucagon response. Histologically, the spleens contained prominent islets. In five other dogs, the tissue was injected into the splenic pulp: mean BG rose to greater than or equal to 250 mg/dl at 2 wk (compared with initial series, P less than 0.001) and remained elevated until death at 6 wk, when histologic examination of the spleens showed severe fibrosis and no islets. Apancreatic controls (N = 4) survived 10 +/- 3 days; BG was 343 +/- 11 mg/dl terminally. We conclude that this modified method for collagenase perfusion of a single large-mammal pancreas via the ducts provides sufficient viable islets to induce prolonged normoglycemia (5 mo) and preserve the response to glucose challenge. Reflux of pancreatic fragments into splenic veins appears more efficient than intrapulp implantation.

Expression of Gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis.

Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.

Viable purified islets of Langerhans from collagenase-perfused human pancreas.

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 x 10(3), distributed as 76 x 10(3) large (greater than 100-micron) and 85 x 10(3) small (less than 100-micron) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 microliter, with purity of 70-90% (large islets) and 20-40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.

Markedly reduced beta-cell function does not result in insulin resistance in islet autografted dogs.

Autotransplantation of islets of Langerhans has resulted in long-term normoglycemia in pancreatectomized dogs. This canine model is useful in evaluating both the progress of islet transplantation and the effect of a reduced islet mass upon the determinants of glucose tolerance: i.e., insulin secretion, insulin sensitivity, and glucose effectiveness. To determine the effect of a reduced islet mass on these factors, we measured the acute insulin response to arginine (AIRa) and glucose (AIRg), the slope of glycemic potentiation of AIRa (SP), insulin sensitivity (Sl), and glucose effectiveness (SG) in control (CN), diabetic (DM), and pancreatectomized dogs rendered normoglycemic with transplanted autografts of islets of Langerhans (TX). Normal fasting plasma glucose (FPG) (TX 4.7 +/- 0.2 mM; CN 4.9 +/- 0.1 mM; P greater than 0.05) was maintained despite a markedly reduced insulin secretion in TX (AIRa 24%, AIRg 15%, and SP 11% of CN). All measures of insulin secretion were significantly correlated (SP vs. AIRg, r = 0.80, P less than 0.0001; AIRa vs. AIRg, r = 0.92, P less than 0.0001) across all animals, but none of the measures of secretion were significantly correlated with either the number of islets transplanted or time posttransplant (P greater than 0.10). Insulin sensitivity was normal in islet autografted dogs (TX: 136 +/- 12 min-1/(nmol/ml); CN: 101 +/- 11 min-1/(nmol/ml), P greater than 0.05) but SG was reduced (TX: 1.93 +/- 0.28 x 100 min-1; CN: 3.53 +/- 0.35 x 100 min-1, P less than 0.05), as determined by the minimal-model method. In diabetic animals (FPG = 16.1 +/- 1.3 mM), insulin secretion was negligible by all measures (P greater than 0.05), and was associated with insulin resistance (Sl = 28 +/- 8 min-1/(nmol/ml)) and reduced SG (1.72 +/- 0.11 x 100 min-1). These studies indicate that across a range of insulin secretion in dogs, the secretagogues arginine and glucose provide similar estimates of beta-cell function. This markedly reduced beta-cell function does not result in insulin resistance when fasting normoglycemia is maintained, but is associated with a decrease in glucose action at basal insulin.

Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen.

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Testicular sertoli cells protect islet beta-cells from autoimmune destruction in NOD mice by a transforming growth factor-beta1-dependent mechanism.

Testicular Sertoli cells protect pancreatic islet grafts from allo- and autoimmune destruction; however, the mechanism(s) of protection is unclear. The aim of this study was to determine whether Fas ligand (FasL) and/or transforming growth factor (TGF)-beta, immunoregulatory proteins produced by Sertoli cells, might mediate the protective effects of these cells against autoimmune destruction of islet beta-cells. Sertoli cells were purified from testes of NOD mice and implanted under the right renal capsule of diabetic NOD mice, whereas NOD islets were implanted under the left renal capsule. Of the mice that received islet and Sertoli cells grafts, 64% (9 of 14) remained normoglycemic at 60 days posttransplantation compared with 0% (0 of 6) of the mice that received islet grafts alone. Immunohistochemical examination of Sertoli cell grafts in normoglycemic mice revealed that TGF-beta1 expression by Sertoli cells remained high, whereas FasL expression by Sertoli cells decreased progressively posttransplantation. Also, plasma levels of TGF-beta1 were significantly elevated in mice that received Sertoli cells and islet grafts, and anti-TGF-beta1 antibody administration completely abrogated the protective effect of Sertoli cells on islet graft survival, whereas anti-FasL antibody did not. Islet graft destruction in anti-TGF-beta1-treated mice was associated with increases in interferon (IFN)-gamma-producing cells and decreases in interleukin (IL)-4-producing cells in the islet grafts. We conclude that 1) Sertoli cell production of TGF-beta1, not FasL, protects islet beta-cells from autoimmune destruction and 2) TGF-beta1 diverts islet-infiltrating cells from a beta-cell-destructive (IFN-gamma+) phenotype to a nondestructive (IL-4+) phenotype.

Beta-cell destruction in NOD mice correlates with Fas (CD95) expression on beta-cells and proinflammatory cytokine expression in islets.

A mechanism of autoimmune destruction of islet beta-cells in type 1 diabetes has been proposed to be the binding of Fas ligand (FasL) on T-cells to Fas receptors on beta-cells. We investigated this proposal by examining the expression of FasL and Fas on islet-infiltrating T-cells and beta-cells in relation to beta-cell destruction in a syngeneic islet transplant model in NOD mice. Diabetic NOD mice were transplanted with syngeneic islets and injected with complete Freund's adjuvant, which prevented diabetes recurrence (nondestructive insulitis), and with phosphate-buffered saline, which did not (beta-cell destructive insulitis). Two-color immunohistochemical assays revealed that FasL was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from both diabetic and normoglycemic mice, and the percentage of each type of cell that expressed FasL was greater in islet grafts from normoglycemic compared with diabetic mice. In contrast, Fas was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from diabetic mice, but it was nearly or totally absent on these cells in islet grafts from normoglycemic mice. Similarly, polymerase chain reaction analysis of islet grafts revealed that Fas mRNA expression was significantly lower in islet grafts from normoglycemic compared with diabetic mice. Also, mRNA levels of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were significantly lower in islet grafts from normoglycemic mice. Finally, Fas was induced on NOD islet cells by incubation with IL-1beta, IFN-gamma, and the combination of IL-1beta, TNF-alpha, and IFN-gamma. These findings support the concept that cytokine-induced Fas receptor expression on islet beta-cells is a mechanism for their destruction by FasL-expressing CD4+ and CD8+ T-cells and, possibly, by FasL-expressing beta-cells themselves.

Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction.

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-3% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-3% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.