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Baertschiger RM, Buhler LH. Xenotransplantation Literature Update January–February, 2006. Xenotransplantation 2006; 13: 272–276. © Blackwell Munksgaard, 2006

The immunosuppressive (IS) regimen based on sirolimus/low-dose tacrolimus is considered a major determinant of success of the Edmonton protocol. This regimen is generally considered safe or even protective for the kidney. Herein, we analyzed the impact of the sirolimus/low-dose tacrolimus combination on kidney function. The medical charts of islet transplant recipients with at least 6 months follow up were reviewed. There were five islet-after-kidney and five islet transplantation alone patients. Serum creatinin, albuminuria, metabolic control markers and graft function were analyzed. Impairment of kidney function was observed in six of 10 patients. Neither metabolic markers nor IS drugs levels were significantly associated with the decrease of kidney function. Although a specific etiology was not identified, some subsets of patients presented a higher risk for decline of kidney function. Low creatinin clearance, albuminuria and long-established kidney graft were associated with poorer outcome.

AIM: to study whether sensitization to pig antigens results in humoral and/or cellular sensitization to alloantigens in baboons, and thus increases the risks of organ allotransplantation after xenotransplantation. Serum from baboons that were naive (n = 4), sensitized to Gal alpha 1,3Gal (Gal) antigens (n = 2), or sensitized to Gal + non-Gal pig antigens (n = 2) were tested by flow cytometry for the presence of immunoglobulin G (IgG) and IgM antibodies that bind to pig or baboon peripheral blood mononuclear cells (PBMC). Two allosensitized baboons were used as positive controls. The same 10 sera were tested in a complement-mediated cytotoxicity assay to detect cytotoxic antibodies against pig, allo and self-PBMC. The T-cell responses of the same baboons to allogeneic and pig PBMC stimulators in mixed lymphocyte reaction (MLR) were studied. All baboon sera contained cytotoxic antibodies that bound to pig PBMC. Binding and cytotoxicity were higher in xenosensitized baboons, particularly in those sensitized to Gal + non-Gal antigens (P < 0.001). None of the naive or xenosensitized baboon sera bound to baboon PBMC. Serum from allosensitized baboons showed anti-baboon IgG and IgM binding, but there was no increase in binding to pig PBMC or in cytotoxicity to pig cells. The MLR response to pig stimulators in baboons sensitized to non-Gal pig antigens was greater than that of naive or Gal-sensitized baboons (P < 0.001), but there was no increase in the response to baboon cells. In baboons, no in vitro evidence that a previous pig xenograft might endanger the outcome of a subsequent allograft was documented.

The aim of our study was to assess cell trafficking and early events after intraportal islet transplantation. Sprague-Dawley rat islets were incubated for various times, with various concentrations of 2-[F]fluoro-2deoxy-D-glucose (FDG), and in presence of various glucose concentrations. FDG-labeled syngeneic islets or FDG alone were injected in rats. Radioactivity was measured in the liver and in various organs by positron-emission tomography for 6 hours. FDG uptake increased with incubation time or FDG concentration and decreased in presence of glucose. In vivo, all islets implanted in the liver, with an uptake 4.4 times higher than controls (44.2% vs. 10.1%, P=0.02). Radioactivity in the liver decreased at the same rate after injection of labeled-islets and FDG alone. Ex vivo labeling of islets and imaging of posttransplant early events were feasible. Islets engrafted exclusively in the liver. No islet loss could be demonstrated 6 hours after transplantation.

On the basis of the recommendation of the American Diabetes Association’s Panel on Ethical Scientific Programs (ESP), the American Diabetes Association, the publisher of Diabetes, is issuing this expression of concern to alert readers to questions about the reliability of the data in the above-cited article. After readers contacted the journal about the potential republication of images from this article, the ESP reviewed the following issues

The aim of this study was to evaluate the effects of intraperitoneal transplantation of encapsulated human hepatocytes on liver metabolism and regeneration of mice with acute liver failure. Primary human hepatocytes were immortalized using lentiviral vectors coding for antiapoptotic genes and microencapsulated using alginate-polylysine polymers. In vitro, immortalized human hepatocytes showed low, but stable, synthetic and catabolitic functions over time, when compared to primary hepatocytes. In vivo, mice with acute liver failure and transplanted with encapsulated immortalized human hepatocytes had a significantly improved survival and biochemical profile, compared to mice transplanted with empty capsules. Serum levels of cytokines implicated in liver regeneration were lower in mice transplanted with hepatocytes compared to mice receiving empty capsules. This decrease was significant for IL-6 and HGF at 3 h. Measurement of liver regeneration showed no significant difference between mice transplanted with hepatocytes compared to control groups. Intraperitoneal transplantation of encapsulated immortalized hepatocytes significantly improved survival of mice with acute liver failure by providing metabolic support and without modifying liver regeneration. The lower levels of cytokines implicated in liver regeneration suggest that the metabolic support provided by the encapsulated hepatocytes reduced the inflammatory stress on the liver and herein decreased the regenerative trigger on residual hepatocytes. These data emphasize that metabolic function and regeneration of hepatocytes are two distinct aspects that need to be studied and approached separately during acute liver failure.

Background Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration. Methods We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes. Results At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment. Conclusion IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.

Purpose Multifocal panhepatic hepatoblastoma (HB) without extrahepatic disease is generally considered as an indication for total hepatectomy and liver transplantation. However, after initial chemotherapy, downstaging of the tumor sometimes allows complete macroscopic resection by partial hepatectomy. This procedure is no longer recommended because of the risk of persistent viable tumor cells in the hepatic remnant. We report our experience with conservative surgery in such cases. Method Between 2000 and 2005, 4 children were consecutively referred to our unit with multinodular pan-hepatic HBs (classification PRETEXT IV of the International Society of Pediatric Oncology Liver Tumor Study Group SIOPEL). Three of them had extrahepatic disease at diagnosis. All patients were treated according to SIOPEL 3 and 4 protocols. Results Extrahepatic metastases were still viable in 2 of 3 patients after initial chemotherapy. These patients eventually died of tumor recurrence. In the 2 patients without residual extrahepatic disease, liver tumors had regressed, and complete macroscopic excision of hepatic tumor remnants could be achieved by conservative surgery. These 2 children are alive and well and free of tumor 7 years after diagnosis. Conclusions Conservative surgery may be curative in some multinodular PRETEXT IV HB patients, with a good response to preoperative chemotherapy and complete excision of all macroscopic tumor remnants. However, because of the lack of reliable predictors of sterilization of the microscopic disease in the residual liver, with subsequent poor prognosis, total hepatectomy and liver transplantation remain currently recommended in patients with multinodular PRETEXT IV HB without extrahepatic disease, even though some of these children are probably overtreated.

There was an error in affiliation 3 for author Sophie Clement. Affiliation 3 should be: Division of Clinical Pathology, Medical School of Geneva, Geneva, Switzerland.

The aim of this study was to establish hepatocyte isolation in pigs, and to evaluate function of isolated hepatocytes after encapsulation, cryopreservation, and transplantation (Tx) in a mouse model of fulminant liver failure (FLF). After isolation, porcine hepatocytes were microencapsulated with alginate-poly-L-Lysine-alginate membranes and cryopreserved. In vitro, albumin production of free and encapsulated hepatocytes were measured by enzyme linked-immunoadsorbent assay. In vivo, encapsulated hepatocytes were transplanted into different groups of mice with FLF and the following experimental groups were performed: group 1, Tx of empty capsules; group 2, Tx of free primary porcine hepatocytes; group 3, Tx of fresh encapsulated porcine hepatocytes; group 4, Tx of cryopreserved encapsulated porcine hepatocytes. In vitro, fresh or cryopreserved encapsulated porcine hepatocytes showed a continuous decreasing metabolic function over 1 week (albumin and urea synthesis, drug catabolism). In vivo, groups 1 and 2 showed similar survival (18% and 25%, respectively, p > 0.05). In groups 3 and 4, Tx of fresh or cryopreserved encapsulated porcine hepatocytes significantly increased survival rate to 75% and 68%, respectively ( p < 0.05). Primary porcine hepatocytes maintained metabolic functions after encapsulation and cryopreservation. In mice with FLF, Tx of encapsulated xenogeneic hepatocytes significantly improved survival. These results indicate that porcine hepatocytes can successfully be isolated, encapsulated, stored using cryopreservation, and transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.

OBJECTIVES: Transplantation of in vitro generated islets or insulin-producing cells represents an attractive option to overcome organ shortage. The aim of this study was to isolate, expand, and characterize cells from human exocrine pancreas and analyze their potential to differentiate into beta cells. METHODS: Fibroblast-like cells growing out of human exocrine tissue were characterized by flow cytometry and by their capacity to differentiate into mesenchymal cell lineages. During cell expansion and after differentiation toward beta cells, expression of transcription factors of endocrine pancreatic progenitors was analyzed by reverse transcription polymerase chain reaction. RESULTS: Cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to mesenchymal stem cells from bone marrow. A culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, nestin, Ngn3, Pdx1, and NeuroD. Activin A and hepatocyte growth factor induced an expression of insulin, glucagon, and glucokinase. CONCLUSIONS: Proliferating cells with characteristics of mesenchymal stem cells and endocrine progenitors were isolated from exocrine tissue. Under specific conditions, these cells expressed little insulin. Human pancreatic exocrine tissue might thus be a source of endocrine cell progenitors.

Organ transplantation has encountered great development during the 80's. However, the number of organ donations and transplantations performed stabilized during the 90ies, with a concomitant increase of patients on the waiting list. Xenotransplantation, i.e. the use of animal organs for transplantation to humans, is one among various alternatives to human organ donation. Xenotransplantation offers several advantages, e.g. it would be possible to transplant all patients at an early stage of their disease. The main barriers to xenotransplantation are the strong immunological responses that human develop against animal antigens and zoonoses. To overcome these hurdles, genetically modified pigs have been engineered by cloning and could allow the initiation of new clinical trials in a near future.

Whether islet transplantation should be aimed at restoring insulin independence or providing adequate metabolic control is still debated. The GRAGIL2 trial was designed as a phase 1-2 study where primary outcome was the rate of insulin independence, and secondary outcome was the success rate defined by a composite score based upon basal C-peptide, HbA1c, hypoglycemic events, and exogenous insulin needs.C-peptide negative type 1 brittle diabetic patients experiencing severe hypoglycemia were eligible to receive a maximum of two islet preparations totalizing 10,000 IE/kg or more, with a threshold of 5,000 IE/kg for the first infusion, according to the Edmonton protocol, within the Swiss-French GRAGIL multicentric network. A sequential analysis with a triangular test was performed in every five patients after 6- and 12-month follow-up. Maximal inefficiency was set at 40% and minimal efficiency at 66%.From September 2003 to October 2005, 10 patients were included. Median waiting time was 6.7 months (first injection) and 9 weeks (second injection). All but one patient received 11,089+/-505 IE/kg: one received a single graft of 5398 IE/kg. At 6 months, insulin independence and composite success rates were 6 of 10 and 6 of 10, respectively. At 12 months, insulin independence was observed in 3 of 10 patients and success in 5 of 10 patients.Based upon our sequential analysis settings, islet transplantation failed to achieve the primary goal, insulin independence, but tended to succeed in reaching the secondary goal, successful metabolic control. Currently it appears to be a successful biological closed-loop glucose control method for brittle diabetes.

High glucose concentrations have a dual effect on beta-cell turnover, inducing proliferation in the short-term and apoptosis in the long-term. Hyperglycemia leads to beta-cell production of interleuking (IL)-1beta in human pancreatic islets. Fas, a death receptor regulated by IL-1beta, is involved in glucose-induced beta-cell apoptosis. Fas engagement can be switched from death signal to induction of proliferation when the caspase 8 inhibitor, FLICE-inhibitory protein (FLIP), is active. Here, we show that IL-1beta at low concentrations may participate in the mitogenic actions of glucose through the Fas-FLIP pathway. Thus, exposure of human islets to low IL-1beta concentrations (0.01-0.02 ng/ml) stimulated proliferation and decreased apoptosis, whereas increasing amounts of IL-1beta (2-5 ng/ml) had the reverse effects. A similarly bimodal induction of FLIP, pancreatic duodenal homeobox (PDX)-1, and Pax4 mRNA expression, as well as glucose-stimulated insulin secretion, was observed. In contrast, Fas induction by IL-1beta was monophasic. Low IL-1beta also induced the IL-1 receptor antagonist (IL-1Ra), suppression of which by RNA interference abrogated the beneficial effects of low IL-1beta. The Fas antagonistic antibody ZB4 and small interfering RNA to FLIP prevented low IL-1beta-stimulated beta-cell proliferation. Consistent with our in vitro results, IL-1beta knockout mice displayed glucose intolerance along with a decrease in islet Fas, FLIP, Pax4, and PDX-1 transcripts. These findings indicate that low IL-1beta levels positively influence beta-cell function and turnover through the Fas-FLIP pathway and that IL-1Ra production prevents harmful effects of high IL-1beta concentrations.

International audience; Recent updates of the Edmonton trial have shown that insulin independence is progressively lost in approximately 90% of islet transplant recipients over the first 5 years. Early prediction of islet graft injury could prompt the implementation of strategies attempting to salvage the transplanted islets. We hypothesize that islet damage is associated with the release and detection of insulin mRNA in the circulating blood. Whole blood samples were prospectively taken from 19 patients with type 1 diabetes receiving 31 islet transplants, immediately prior to transplantation and at regular time-points thereafter. After RNA extraction, levels of insulin mRNA were determined by quantitative reverse tran-scriptase-polymerase chain reaction. All patients exhibited a primary peak of insulin mRNA immediately after transplantation, without correlation of duration and amplitude with graft size or outcome. Twenty-five subsequent peaks were observed during the follow-up of 17 transplantations. Fourteen secondary peaks (56%) were closely followed by events related to islet graft function. Duration and amplitude of peaks were higher when they heralded occurrence of an adverse event. Peaks of insulin mRNA can be detected and are often associated with alterations of islet graft function. These data suggest that insulin mRNA detection in the peripheral blood is a promising method for the prediction of islet graft damage.

Aim Islet transplantation is gaining recognition as a therapeutic option for selected diabetic patients. The immunosuppressive regimen based on sirolimus/low-dose tacrolimus is considered a major breakthrough that allowed considerable improvement in graft survival. A high incidence of side effects associated with such a regimen has been reported in the literature, but this immunosuppressive protocol is generally considered safe or even protective to the kidney. Herein, we analyze the impact of the sirolimus/low-dose tacrolimus–based protocol on kidney function. Patients and Methods Five islet-after-kidney and 5 islet-transplant-alone patients were enrolled and followed up. Renal function was assessed by the periodic measurement of serum creatinine and by the presence of albuminuria. Metabolic control markers and graft function were followed, as well as immunosuppressive whole blood trough levels. Results Kidney function significantly decreased in 6 of 10 patients. Neither metabolic markers nor immunosuppressive drugs levels were significantly associated with the decreased kidney function. Conclusion Although a specific etiology was not identified, subsets of patients presented a higher risk for decrease of kidney function. The presence of low creatinine clearance, albuminuria, and long-established kidney graft were associated with poorer outcomes.

The aim of this study was to evaluate in vitro and in vivo functions of isolated hepatocytes after immortalization, cryopreservation, encapsulation and xenotransplantation into mice with fulminant liver failure (FLF).Rat and human hepatocytes were isolated from normal liver tissue by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors coding for SV 40 large T antigen, Bmi-1 and telomerase. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 micron alginate-PLL-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or freshly isolated) were cultured in albumin-free medium and albumin production was measured by enzyme-linked immunosorbent assay (ELISA). In vivo, a model of FLF was established in C57/BL6 mice by acetaminophen administration (700 mg/kg i.p.) followed 15 h later by a 30% hepatectomy. Microencapsulated (cryopreserved or freshly isolated) hepatocytes were transplanted intraperitoneally to mice with FLF and the following experimental groups were performed: group 1 (n = 10) Tx of empty capsules; group 2 (n = 12) Tx of free primary rat hepatocytes; group 3 (n = 12) Tx of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) Tx of fresh encapsulated rat hepatocytes; group 5 (n = 9) Tx of cryopreserved encapsulated IHH; group 6 (n = 10) Tx of fresh encapsulated IHH. Animals were killed at regular intervals and histopathology of microcapsules and liver tissue was obtained.In vitro, cryopreserved or fresh encapsulated rodent hepatocytes showed a progressively decreasing albumin secretion over 1 week in culture. In contrast, cryopreserved or fresh encapsulated IHH showed minimal, but stable albumin secretion. In vivo, FLF was achieved by combination of acetaminophen with 30% hepatectomy, resulting in a reproducible survival of 23% +/- 5%. In groups 1 and 2, survival rates were not improved significantly compared with untreated mice. In groups 3 and 4, Tx of cryopreserved or fresh encapsulated rat hepatocytes significantly increased survival rate to 66% and 80%, respectively (P0.01). In groups 5 and 6, Tx of cryopreserved or fresh encapsulated IHH improved survival to 50% and 55%, respectively (P0.05). Histopathology revealed that encapsulated hepatocytes were viable up to 2 weeks post-Tx.Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation short-term. IHH showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes loose specific functions after immortalization. After induction of FLF in mice, intraperitoneal Tx of encapsulated (primary or immortalized, fresh or cryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naïve and genetically modified hepatocytes can successfully be encapsulated, stored using cryopreservation, and be transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.

International audience; AIM: We sought to investigate the feasibility of 18F-FDG-leukocyte imaging to detect islet rejection. METHODS: Two thousand Sprague-Dawley (SD, syngeneic group) or Lewis (allogeneic group) islet equivalents were intraportally injected into SD rat recipients. Four and 7 days after transplantation, 10(8) 18F-FDG-labeled splenocytes were injected into the jugular vein. Splenocytes were harvested from naïve or sensitized (12 days after intraportal transplantation of 2000 Lewis IEQ) SD rats. Positron emission tomography (PET) imaging was started 5 minutes after splenocyte infusion and performed hourly for 4 hours. RESULTS: One hour after splenocyte injection, FDG was mainly detected in the heart and lungs. It was then further distributed to other organs, and from the second hour, the highest tracer concentration was located in the abdomen. Liver FDG uptake was similar between syngeneic, allogeneic, and sensitized allogeneic groups at 4 and 7 days after islet transplantation. DISCUSSION: No islet rejection was detected by 18F-FDG-leukocyte imaging. The amount of transplanted tissue was only few millilitres and the additional related inflammation in case of rejection is small and difficult to detect. The liver showed a relatively high spontaneous tracer uptake; the related background prevented detection of a potential increase in tracer uptake in cases of islet rejection.

BACKGROUND: Accurate determination of islet purity and mass before transplantation is an essential part of quality control. The standard method is based on manual evaluation of these parameters and thus subjective and prone to errors. Therefore, we developed a computerized approach aimed at evaluating more objectively the number and purity of isolated human islets. METHODS: Islets were isolated and purified from human pancreata according to a standard method. For each preparation, two samples were dithizone stained. One sample was analyzed manually by microscopy, following the standard procedure, and the other was digitally photographed for both digital manual and computerized analyses. Computerized analysis was performed using the MetaMorph and ImageJ softwares to automatically quantify purity and size of islets. Islet equivalent (IEQ) number was calculated using the Ricordi algorithm or considering the individual volume of each islet. Computerized analysis was validated using calibrated red glass microspheres. RESULTS: When digital manual and computerized analyses were compared, mean values of total islet number, IEQ number calculated using the Ricordi algorithm, and purity were similar. Comparisons of individual values showed good correlations (r>or=0.89). By standard manual analysis, total islet number and purity were higher and IEQ number similar compared with digital manual and computerized analyses. IEQ number was 10% lower (P

Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.

International audience; The aim of this study was to assess the efficiency and safety of the Edmonton immunosuppression protocol in recipients of islet-after-kidney (IAK) grafts. Fifteen islet infusions were administered to 8 patients with type 1 diabetes and a functioning kidney graft. Immunosuppression was switched on the day of transplantation to a regimen associating sirolimus-tacrolimus-daclizumab. Insulin-independence was achieved in all patients for at least 3 months, with an actual rate of 71% at 1 year after transplantation (5 of 7 patients). After 24-month mean follow-up, five have ongoing insulin independence, 11-34 months after transplantation, with normal HbA1c, fructosamine and mean amplitude of glycemic excursions (MAGE) values. Results of arginine-stimulation tests improved over time, mostly after the second islet infusion. Severe adverse events included bleeding after percutaneous portal access (n=2), severe pneumonia attributed to sirolimus toxicity (n=1), kidney graft loss after immunosuppression discontinuation (n=1), reversible humoral kidney rejection (n=1) and fever of unknown origin (n=1). These data indicate that the Edmonton approach can be successfully applied to the IAK setting. This procedure is associated with significant side effects and only patients with stable function of the kidney graft should be considered. The net harm versus benefit has not yet been established and will require further studies with larger numbers of enrolled subjects.