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Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway-sparing immunomodulatory strategies in a pig-to-nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ≈ 50 000 islet equivalents/kg wild-type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA-1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA-1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin-independent normoglycemia (median rejection-free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway-sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

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Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

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The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig-to-human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long-term survival of porcine islets has been achieved using CD154-specific antibodies to interrupt the CD40/CD154 costimulation pathway; however, CD154-specific antibodies seem unlikely candidates for clinical translation. An alternative strategy for CD40/CD154 pathway interruption is use of CD40-specific antibodies. Herein, we evaluate the ability of a chimeric CD40-specific monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (~50,000 IEQ/kg) were transplanted intraportally into pancreatectomized diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and the IL-2 receptor-specific antibody basiliximab, and maintenance therapy with sirolimus and the B7-specific fusion protein belatacept. Chi220 effectively promoted xenoislet engraftment and survival, with five of six treated recipients achieving insulin-independent normoglycemia (median rejection-free survival 59 days; mean 90.8 days, maximum 203 days). No thromboembolic phenomena were observed. CD40 represents a promising alternative to CD154 as a therapeutic target, and the efficacy of CD40-specific antibodies in islet xenotransplantation warrants further investigation.

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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.

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OBJECTIVES: Islet-like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin-induced diabetic immuno-incompetent mice. MATERIALS AND METHODS: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). RESULTS: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C-peptide and glucagon, and for the ductal epithelial marker cytokeratin-19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas-specific cell markers was maintained for 70 days post-transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C-peptide in response to an oral bolus of glucose. CONCLUSIONS: hESC-derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C-peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin-producing cells for transplantation into patients with type 1 diabetes.

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Surgical correction in severe cases of AIS is often hampered by insufficient autograft bone to facilitate the fusion. The development of other sources of bone generating cells would greatly enhance the surgical. Bone marrow derived stem cells were harvested from femoral reaming during total hip arthroplasty for the purpose of differentiating into osteoblasts. Stem cells were isolated from the marrow and successfully differentiated into three cell lines (osteoblasts, chondrocytes and adipocytes) to confirm multilineage potential. Osteoblasts were developed from the stem cells and demonstrated the ability to be cultured to possibly provide a source of bone generating cells to augment surgical fusions. It is anticipated that the addition of osteoblasts created from stem cells (combined with appropriate matrix) will have significant influence on the success of AIS surgery through improvement of bone fusion.

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AIMS/HYPOTHESIS: The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue. METHODS: HDCs (n=4), NPIs (n=9) and human islets (n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days. RESULTS: HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control (p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups. CONCLUSIONS/INTERPRETATION: Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance.

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AIMS/HYPOTHESIS: Neurogenin 3 (NEUROG3), a basic helix-loop-helix transcription factor that is needed for endocrine cell development in the embryonic pancreas, has been shown to induce transdifferentiation of duct cells from adult pancreas towards a neuro-endocrine phenotype. Our study explored the endocrine transdifferentiation potential of NEUROG3 in neonatal pancreatic precursor cells. MATERIALS AND METHODS: A replication-deficient adenovirus expressing Neurog3 and green fluorescent protein (GFP) (Ad-NEUROG3) was used to infect neonatal pig pancreatic cell preparations enriched for endocrine islet and cytokeratin-positive precursor cells. GFP-positive cells were sorted using flow cytometry on days 3 and 8 after infection and characterised at the transcript and protein level. For in vivo experiments, the total population of Ad-NEUROG3-infected pancreatic cells was transplanted, then later removed for determination of graft hormone content and immunohistochemistry. RESULTS: Among the GFP-positive cells, the fraction of precursor cells decreased by more than 85% at day 8 after infection, while the fraction of glucagon-positive cells increased 2.5-fold and the beta cell number remained the same. Transplantation of the Ad-NEUROG3-infected pancreatic cell preparation failed to reverse streptozotocin-induced hyperglycaemia, while non-infected cells and a control cell preparation infected with replication-deficient adenovirus expressing only GFP were able to do so. At day 109 after transplantation, kidneys grafted with Ad-NEUROG3-infected pancreatic cells contained significantly decreased insulin and increased glucagon levels. Abundant glucagon-immunopositive cells were seen in Ad-NEUROG3-infected grafts, which were virtually devoid of proliferating insulin-positive cells. CONCLUSIONS/INTERPRETATION: In summary, adenoviral delivery of NEUROG3 to pancreatic precursor cells from neonatal pig pancreas promotes alpha cell differentiation in vitro and in vivo.

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AIMS/HYPOTHESIS: The aim of this study was to determine whether a simple alginate capsule can prolong islet survival and function during long-term tissue culture. We also wanted to observe the ability of these encapsulated islets to restore glucose responsiveness to diabetic recipients, along with the quantity of islets required to do so. METHODS: We compared the recovery and metabolic function of encapsulated canine islets with that of non-encapsulated canine islets following 1, 2 or 3 weeks of tissue culture. These culture preparations were also transplanted into diabetic nude mice and compared for their ability to reverse diabetes. Furthermore, short-term cultured encapsulated and non-encapsulated islets were transplanted in varying numbers to determine the minimum dose required to normalise blood glucose and prolong recipient survival. RESULTS: Islet recovery following 1, 2 and 3 weeks of tissue culture was significantly higher when islets were encapsulated. When these islets were recovered at 1, 2 and 3 weeks and transplanted into diabetic nude mice, survival at 100 days was 100% for all encapsulated groups, versus 66%, 33% and 33% respectively for the non-encapsulated islets. Additionally, substantially fewer short-term cultured islets were required to normalise blood glucose when the islets were encapsulated. Recipients of encapsulated islets also had significantly longer survival times than recipients of non-encapsulated preparations. CONCLUSIONS/INTERPRETATION: This study demonstrates that encapsulation of islets with purified alginate improves islet survival and function in vitro and in vivo.

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The discovery of a pancreatic adult stem cell would have significant implications for cell-based replacement therapies for type 1 diabetes mellitus. Nestin, a marker for neural precursor cells, has been suggested as a possible marker for islet progenitor cells. We have characterized the expression and localization of nestin in both the intact human pancreas and clinical human pancreatic islet grafts. Nestin was found to be expressed at different levels in the acinar component of human pancreatic biopsies depending on donor, as well as in ductal structures and islets to some degree. In islets, insulin-producing beta-cells rarely co-expressed the protein, and in the ducts a small percentage (1-2%) of cells co-expressed nestin and cytokeratin 19 (CK19) while most expressed only CK19 (90%) or nestin (5-10%) alone. Assessment of nestin expression in neonatal pancreatic sections revealed an increased number of islet-associated positive cells as compared with adult islets. Nestin immunoreactivity was also found in cells of the pancreatic vasculature and mesenchyme as evidenced by co-localization with smooth muscle actin and vimentin. Samples from post-islet isolation clinical islet grafts revealed a pronounced heterogeneity in the proportion of nestin-positive cells (<1-72%). Co-localization studies in these grafts showed that nestin is not co-expressed in endocrine cells and rarely (<5%) with cytokeratin-positive ductal cells. However, relatively high levels of co-expression were found with acinar cells and cells expressing the mesenchymal marker vimentin. In conclusion we have shown a diffuse and variable expression of nestin in human pancreas that may be due to a number of different processes, including post-mortem tissue remodeling and cellular differentiation. For this reason nestin may not be a suitable marker solely for the identification of endocrine precursor cells in the pancreas.

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AIM/HYPOTHESIS: Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes. METHODS: ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks. RESULTS: Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state. CONCLUSIONS/INTERPRETATION: Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.

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The immunoprotective nature of the testis has led to numerous investigations for its ability to protect cellular grafts. Sertoli cells (SCs) are at least partially responsible for this immunoprotective environment and survive allogeneic and xenogeneic transplantation. The ability of SCs to survive transplantation leads to the possibility that they could be engineered to deliver therapeutic proteins. As a model to test this hypothesis, we examined the ability of SCs that produce green fluorescent protein (GFP) to survive transplantation and continue expressing GFP. SCs were isolated from transgenic mice engineered to express GFP and transplanted as aggregates under the kidney capsule of severe combined immunodeficient (SCID) and Balb/c mice. Using this paradigm, it was possible to compare the survival of transgenic SCs directly in both immunodeficient and immunocompetent recipients. Fluorescence microscopy of the kidney capsule and immunohistochemistry of the grafts for GFP and GATA-4 revealed the presence of GFP-expressing SCs under the kidney capsule of SCID and Balb/c mice at both 30 and 60 days post-transplantation. In contrast, islets transplanted to Balb/c mice were rejected. Thus, SCs survive transplantation and continue to express GFP raising the possibility that SCs can be engineered using transgenic technology to produce proteins, such as insulin, factor VIII, or dopamine for the treatment of diabetes, hemophilia or Parkinson's disease, respectively.

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An immunoisolated collection of cells, which communicate and exchange essential factors, co-stimulatory hormones, as well as providing immunoprotection and immunomodulation, can be prepared, given existing scientific and medical know-how, within two decades. These "Bioartificial Organ Grafts" have advantages relative to isolated cell therapies, including beta-cell encapsulation for diabetes treatment, and xenotransplantation, which has a de facto moratorium. This paper documents that the majority of the research for the bioartificial organ grafts has been concluded, with the remaining hurdles minimum in comparison. The use of co-encapsulation and the induction of local immune-privilege will provide a more sensitive humoral hormonal response and graft survival, without systemic immunosuppression. A call for the staged implementation of bioartificial organ grafts, based on the best available medical practice, materials, tissue and technology available, is advocated. The implementation of bioartificial organ grafts can begin within the next two years, based on allografts succeeded by genetically modified human tissue, without the need to pass through a xenograft stage.

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The expression of Galalpha-(1,3)Gal (alphaGal) on porcine islet cells remains controversial. Several groups have reported that porcine islet endocrine cells do not express alphaGal while we have shown in neonatal porcine islets (NPI) that beta cells do express this antigen. We hypothesize that endocrine cells expressing alphaGal on NPI are less mature cells that may have originated from ductal cells and that expression of this antigen disappears as they develop into fully mature beta cells. Thus, we further examined alphaGal expression on various porcine islet cell preparations and correlated this with the proportion of cytokeratin 7 (CK7)-positive ductal cells. In vitro and in vivo expression of alphaGal and CK7 was significantly (P<0.05) higher in less mature NPI cells compared with matured NPI and adult porcine islet cells while the reverse was observed in the proportion of beta cells. Moreover, a significantly higher proportion of CK7-positive cells was detected in the Gal-expressing population compared with non-expressing cells. In contrast, a higher proportion of beta cells was observed in the Gal-negative population compared with the Gal-positive population. These data showed a reduced expression of alphaGal and CK7 as porcine islet cells mature into beta cells suggesting a possible role for alphaGal in the maturation of pancreatic endocrine beta cells.

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The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-N(G)-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

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The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (Lp) in the absence of cryoprotectant was calculated as 0.050 +/- 0.005, 0.071 +/- 0.006, and 0.300 +/- 0.016 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (mean +/- SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (Ps) values for 1 M DMSO were 0.89 +/- 0.12, 1.86 +/- 0.28, and 5.33 +/- 0.26 microm/min at 4 degrees C, 10 degrees C, and 22 degrees C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The Lp values for cells exposed to 1 M DMSO were 0.071 +/- 0.006, 0.084 +/- 0.008, and 0.185 +/- 0.014 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively. The activation energy for these values was 8.95 kcal/mol. The Ps values for 2 M DMSO were 1.11 +/- 0.13, 1.74 +/- 0.19, and 7.68 +/- 0.12 microm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The Lp values in the presence of 2 M DMSO were 0.070 +/- 0.006, 0.085 +/- 0.008, and 0.192 +/- 0.009 microm/min/atm at 4 degrees C, 10 degrees C, and 22 degrees C, respectively, with an activation energy of 9.40 kcal/mol. Solutions of 1 M EG gave Ps values of 1.01 +/- 0.13, 1.45 +/- 0.25, and 4.90 +/- 0.48 microm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding Lp values were 0.071 +/- 0.007, 0.068 +/- 0.006, and 0.219 +/- 0.012 microm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 +/- 0.15, 1.42 +/- 0.23, and 5.56 +/- 0.22 microm/min; the activation energy was 15.70. The Lp values for cells in the presence of 2 M EG were 0.068 +/- 0.008, 0.071 +/- 0.006, and 0.225 +/- 0.010 microm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements in the low temperature storage of islets from newborn pigs.

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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.

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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.

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