Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts.

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

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.

Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function.

The aim of the present study was to clarify whether prolonged in vitro exposure of human pancreatic islets to high glucose concentrations impairs the function of these cells. For this purpose, islets isolated from adult cadaveric organ donors were cultured for seven days in RPMI 1640 medium supplemented with 10% fetal calf serum and containing either 5.6, 11, or 28 mM glucose. There was no glucose-induced decrease in islet DNA content or signs of morphological damage. However, islets cultured at 11 or 28 mM glucose showed a 45 or 60% decrease in insulin content, as compared to islets cultured at 5.6 mM glucose. Moreover, when such islets were submitted to a 60-min stimulation with a low (1.7 mM) followed by a high (16.7 mM) concentration of glucose, the islets cultured at 5.6 mM glucose showed a higher insulin response to glucose than those of the two other groups. Islets cultured at the two higher glucose concentrations showed increased rates of insulin release in the presence of low glucose, and a failure to enhance further the release in response to an elevated glucose level. Islets cultured at 28 mM glucose showed an absolute decrease in insulin release after stimulation with 16.7 mM glucose, as compared to islets cultured at 5.6 mM glucose. The rates of glucose oxidation, proinsulin biosynthesis, and total protein biosynthesis were similar in islets cultured at 5.6 or 11 mM glucose, but they were decreased in islets cultured at 28 mM glucose. These combined results suggest that lasting exposure to high glucose concentrations impairs the function of human pancreatic islets.

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.

Cold storage of rat pancreas before purification of islet beta-cells.

Allotransplantations in rats have demonstrated that aggregates of purified islet endocrine cells are less immunogenic than isolated islets. If purified islet cells are to be tested in humans, their preparation should be feasible from cold-preserved organs. We compared the yield in purified beta-cells from freshly harvested rat pancreases with that from cold-stored organs. After 24 h of preservation in Collins' solution or in University of Wisconsin (UW) solution, the number of purified beta-cells per pancreas was 40% lower (P less than 0.05) than that obtained from nonpreserved controls. Addition of 5 mM benzamidine/4% bovine serum albumin to Collins' solution resulted in similar recoveries as with freshly harvested organs; this addition did not increase the yield from UW solution-stored organs. When compared to preparations from fresh pancreases, islets and islet cells isolated from pancreases preserved in Collins' solution-bovine serum albumin-benzamidine were comparable in structural integrity, viability in culture, secretory responsiveness in vitro, and capability of correcting hyperglycemia in streptozocin-induced diabetic rats. We conclude that addition of benzamidine to Collins' preservation solution allows purification of islet beta-cells from 24-h-preserved rat pancreases in the same yield and quality as from freshly harvested organs. These results indicate that cold-preserved pancreases can be used for the preparation of purified islet cell grafts.

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.

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.

Neonatal porcine islet cells induce human CD4+, but not CD8+, lymphocyte proliferation and resist cell-mediated cytolytic injury in vitro.

Xenotransplantation of porcine tissue to human recipients promises to alleviate the organ shortage. Human antibody-mediated and cell-mediated immune responses against porcine grafts, however, represent barriers to successful xenotransplantation. We compared neonatal porcine islet cells (NPICs) and neonatal porcine splenocytes for the ability to stimulate proliferation of human peripheral blood lymphocytes (PBLs), and for their susceptibility to human natural killer (NK) and cytotoxic T-lymphocyte (CTL)-mediated lysis. Human peripheral blood CD4+ lymphocytes showed strong proliferation in response to NPICs, likely because of occasional swine leukocyte antigen (SLA) class II+ cells in the NPIC preparations. In contrast, human peripheral blood CD8+ lymphocytes did not proliferate in response to NPICs, although they showed clear responses to both porcine splenocytes and endothelial cells. Both human CTL-raised-against-porcine splenocytes and endogenous NK cells lysed porcine splenocytes, but the same cells showed little or no lytic activity against NPICs. Lysis of porcine splenocyte targets was completely abrogated by pretreatment of the human NK or CTL populations with concana-mycin A, suggesting a perforin-dependent effector mechanism. Pretreatment of the NPIC targets with proinflammatory porcine cytokines to upregulate SLA class I expression failed to enhance human CTL-mediated lysis. However, lysis of NPICs by human CTLs could be elicited when a lectin was added to form stable effector:target cell conjugates. It appears that NPICs do not express sufficiently high levels of co-stimulatory and/or adhesion molecules to either activate human CD8+ T-cells or to be effective targets for activated human CTLs. These data suggest that NPICs may not be destroyed by NK- or CTL-mediated lytic mechanisms after transplantation into humans.

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.

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.

Rat pancreas preparation for cold storage and subsequent islet cell isolation.

Pancreatic islets can be isolated from cold-preserved organs. They contain 60% more beta cells when prepared from organs that were stored for 24 hr in Collins solution supplemented with albumin and benzamidine (CAB) instead of University of Wisconsin (UW) solution. Recovery from CAB-stored organs was similar when CAB was perfused in situ before organ removal or ex vivo after organ harvesting in UW. In situ flush with cold Ringers before ex vivo replacement by CAB resulted in 25% lower recovery of islet beta cells and in higher contamination with nonendocrine and damaged cells. Recovery of beta cells was 50% reduced when cold storage solution was not chased before collagenase digestion of the organ. It is concluded that the isolation of rat islets from cold-preserved organs can be improved by using UW or CAB instead of Ringers for situ perfusion, by cold storage in CAB, and by adequate chase of cold storage solution with physiologic medium before collagenase digestion. These conditions can be tested in current human islet isolation protocols.

Microencapsulation of neonatal porcine islets: protection from human antibody/complement-mediated cytolysis in vitro and long-term reversal of diabetes in nude mice.

BACKGROUND: Recently, we have developed a simple and reliable method to efficiently isolate large numbers of neonatal porcine islets (NPI). We and others have shown that NPI are susceptible to cytolysis by the activation of human complement in vitro. Microencapsulation of islets may be one strategy to protect NPI from this form of rejection. We examined whether microencapsulation can prevent lysis of NPI induced by human antibody and complement in vitro and also assessed their ability to reverse hyperglycemia in diabetic nude mice. METHODS: NPI were microencapsulated with purified alginate, cultured for 2 days, then tested for sensitivity to fresh human serum using an established in vitro cytotoxicity assay or transplanted into alloxan-induced diabetic nude mice. RESULTS: Incubation of nonencapsulated NPI for 24 hr in the presence of fresh human serum resulted in a 53% loss of cellular insulin content, a 51% reduction in recoverable DNA content, and a marked reduction of insulin secretory responsiveness when compared with controls cultured in heat-inactivated human serum. In contrast, exposure of encapsulated islets to fresh human serum had no cytotoxic effect on the islets. Transplantation of 2000 encapsulated NPI i.p. into diabetic nude mice (n=16) corrected hyperglycemia in all mice within 8 weeks. Similar results were obtained when 2000 nonencapsulated NPI were implanted under the kidney capsule (n=10); however recipients of nonencapsulated NPI placed i.p. failed to obtain euglycemia and survived for only 3 weeks posttransplantation. CONCLUSION: Microencapsulation protects NPI from the cytotoxic effects of human antibody and complement and allows for long-term reversal of diabetes in nude mice.

Cryopreservation of rat pancreatic islets: effect of ethylene glycol on islet function and cellular composition.

BACKGROUND: Inasmuch as cryopreservation can facilitate clinical islet transplantation by providing a means of storing supplemental islets in order to augment marginally adequate grafts, protocols are needed to allow for a minimal loss in viable beta cells. By replacing the cryoprotectant dimethyl sulfoxide (DMSO) with ethylene glycol (EG), a more simplified cryopreservation protocol was developed, which resulted in improved survival and function of rat pancreatic islets. METHODS: Nonfrozen islets, islets cryopreserved in DMSO, and EG-cryopreserved islets were compared for percent recovery, cellular composition, in vitro viability, and metabolic function after transplantation. RESULTS: After cryopreservation in DMSO or EG, islet yield was similar to that of nonfrozen controls; however, islets cryopreserved in DMSO exhibited lower cellular DNA, insulin, and glucagon content, as well as an impaired insulin secretory capacity in vitro than the nonfrozen controls. When compared with controls, islets cryopreserved in DMSO contained a higher proportion of beta cells but a lower number of glucagon-positive cells, whereas cryopreservation with EG resulted in similar DNA/hormone contents, in vitro viability, and cellular composition. Transplantation of islet grafts composed of comparable numbers of beta cells (2.1-2.3 million) corrected diabetes in 100% (6/6; nonfrozen controls), 92% (10/11; DMSO), and 100% (14/14; EG) of the recipients; however, those who received DMSO-treated islets took longer to achieve euglycemia and remained glucose-intolerant. CONCLUSIONS: These results demonstrate that EG allows for the successful cryopreservation of rat islet beta and a cells with the same yield and quality as nonfrozen islets. The observation that alpha-cell survival was better after cryopreservation with EG may explain the improved functional viability of these grafts. Further studies are needed to assess whether this protocol provides any advantage for cryopreserving large numbers of human islets.

Single injection of insulin delays the recurrence of diabetes in syngeneic islet-transplanted diabetic NOD mice.

BACKGROUND: Insulin has been implicated in the pathogenesis of type 1 diabetes and oral administration of insulin has been shown to delay the onset of diabetes in NOD mice. In this study we determined whether a single footpad injection of insulin will protect syngeneic islet grafts from autoimmune destruction when placed under the kidney capsule of diabetic NOD mice. METHODS: Five hundred islets were transplanted under the kidney capsule of diabetic female NOD mice in conjunction with a single footpad injection of either pork insulin in saline or mixed with incomplete Freund's adjuvant (IFA). Control groups received either IFA or saline alone. RESULTS: Seven of 11 animals (63.6%) given insulin in IFA exhibit long-term graft survival (>75 days; mean +/- SEM >85.4+/-16.1) whereas only 3 of 12 animals (25.0%) in the IFA group had graft survival longer than 75 days (mean +/- SEM >41.9+/-12.8 days). In contrast, none of the animals that received insulin in saline (17.3+/-2.5 days) and saline only (16.1+2.0 days) exhibit prolonged graft survival. CONCLUSION: These results suggest that a single footpad injection of insulin can protect the islet graft from immune attack in NOD mice.

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival.

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.

Successful biolistic transformation of mouse pancreatic islets while preserving cellular function.

To utilize gene therapy, we required an efficient method to transfect intact islets before their use in transplantation. The biolistic method transforms cells by bombarding them with microprojectiles coated with DNA. Once internalized, the DNA is solubilized and expressed. We used the firefly luciferase gene driven by the human cytomegalovirus immediate early promoter as a reporter construct in freshly isolated BALB/c mouse islets to compare the transfection efficiency using either the biolistic method, lipofection, or recombinant adenoviral infection (n=4 in each case). The biolistic method achieved, on average, a 35-fold higher level of luciferase activity than the lipofection method (mean +/- SEM: 42.6 +/- 14.2 vs. 1.1 +/- 0.2 relative light units (RLU)/islet). Adenoviral infection achieved, on average, a further 25-fold higher level of luciferase activity than the biolistic method (1136.0 +/- 542.0 RLU/islet). The average proportion of islets recovered 48 hr after the biolistic blast was 53% (n=20). The average number of dissociated cells found to express the foreign gene product using beta-galactosidase as a reporter construct was 3% (n=6). Furthermore, nontransformed and biolistically transformed islets responded similarly to an in vitro glucose challenge (stimulation index of insulin release at 20.0 mM glucose/insulin release at 2.8 mM glucose = 2.8 and 3.0, respectively, P=0.9). Syngeneic, biolistically transfected islets functioned to reverse the diabetic state when transplanted (500 islets) beneath the renal capsule of alloxan-induced diabetic BALB/c recipients (n=7). This methodology can achieve efficient transfection of pancreatic islets while preserving their function and thus holds promise for ex vivo gene therapy of isolated islets prior to transplantation.

Interleukin-4 or interleukin-10 expressed from adenovirus-transduced syngeneic islet grafts fails to prevent beta cell destruction in diabetic NOD mice.

BACKGROUND: We performed ex vivo adenoviral gene transfer in a mouse pancreatic islet transplant model to test the efficacy of this expression system. We then determined whether adenoviral-mediated expression of mouse interleukin (IL) 4 or IL-10 from transduced syngeneic islet grafts could prevent disease recurrence in diabetic nonobese diabetic (NOD) mice. METHODS: An adenoviral vector expressing beta-galactosidase (AdCMV betaGal) was used to transduce BALB/c islets (2.5 x 10(3) plaque-forming units/islet), which were analyzed for glucose responsiveness, islet cell recovery, and efficiency of gene transfer. In vivo function and reporter gene expression were examined with AdCMV betaGal-transduced islet grafts in alloxan-induced diabetic syngeneic recipients. Adenoviruses expressing either IL-4 or IL-10 were used in a similar fashion to infect NOD islets, which were characterized in vitro, as well as transplanted into diabetic syngeneic recipients. RESULTS: In vitro functional studies showed no significant difference between control or transduced islets, with 50+/-4% of AdCMV betaGal-infected islet cells staining positive for beta-galactosidase. Transplant recipients became nomoglycemic within 48 hr after transplant, and, although beta-galactosidase expression decreased over time, it was detectable in the graft for up to 8 weeks. Despite the nanogram quantities of IL-4 or IL-10 produced/day from each graft equivalent in vitro, transduced and transplanted NOD islets failed to prevent disease recurrence. CONCLUSIONS: These results suggest that adenoviruses are efficient for at least medium term gene expression from islets in vivo, but neither IL-4 nor IL-10 alone can prevent autoimmune disease recurrence in NOD mice.

Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia.

BACKGROUND: Islet isolation from the pancreatic tissue matrix remains highly variable. Recent evidence suggests that intrinsic human pancreatic proteases, including trypsin, may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. In this study we have hypothesized that serine protease inhibition applied during pancreatic digestion, could improve yield and/or functional viability of islets isolated from human pancreases. METHODS: Twelve organ donor pancreases with 12.9+/-0.6 hr cold storage (mean+/-SEM) were perfused via their ducts with Liberase-HI enzyme in the presence (n=6) or absence (n=6) of 0.4 mM Pefabloc. All were then gently dissociated and their purified islets separated with Ficoll density gradient centrifugation. RESULTS: Donor-related factors (age, gender, cold storage time, body mass index, and pancreas weight) did not differ significantly between the two experimental groups. Pefabloc supplementation did not affect the digestion time, islets remaining trapped in exocrine tissue, or final islet purity. Islet recovery was increased in the Pefabloc-treated group (mean+/-SEM yield 323.8+/-80.8 x 10(3) islet equivalents vs. 130.8+/-13.6 x 10(3) islet equivalents, P<0.05). Cellular composition, DNA and insulin content, and insulin secretory activity of the isolated islets was similar. CONCLUSIONS: Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.