Reto M. Baertschiger

Low Concentration of Interleukin-1β Induces FLICE-Inhibitory Protein–Mediated β-Cell Proliferation in Human Pancreatic Islets

High glucose concentrations have a dual effect on β-cell turnover, inducing proliferation in the short-term and apoptosis in the long-term. Hyperglycemia leads to β-cell production of interleuking (IL)-1β in human pancreatic islets. Fas, a death receptor regulated by IL-1β, is involved in glucose-induced β-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-1β at low concentrations may participate in the mitogenic actions of glucose through the Fas-FLIP pathway. Thus, exposure of human islets to low IL-1β concentrations (0.01–0.02 ng/ml) stimulated proliferation and decreased apoptosis, whereas increasing amounts of IL-1β (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-1β was monophasic. Low IL-1β also induced the IL-1 receptor antagonist (IL-1Ra), suppression of which by RNA interference abrogated the beneficial effects of low IL-1β. The Fas antagonistic antibody ZB4 and small interfering RNA to FLIP prevented low IL-1β–stimulated β-cell proliferation. Consistent with our in vitro results, IL-1β knockout mice displayed glucose intolerance along with a decrease in islet Fas, FLIP, Pax4, and PDX-1 transcripts. These findings indicate that low IL-1β levels positively influence β-cell function and turnover through the Fas-FLIP pathway and that IL-1Ra production prevents harmful effects of high IL-1β concentrations.

Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver.

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.

Improved survival of fulminant liver failure by transplantation of microencapsulated cryopreserved porcine hepatocytes in mice.

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.

Expectations and strategies regarding islet transplantation: metabolic data from the GRAGIL 2 trial

Background. 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. Methods. 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%. Results. 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. Conclusion. 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.

Sequential Kidney/Islet Transplantation: Efficacy and Safety Assessment of a Steroid-Free Immunosuppression Protocol

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.

Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes

Abstract:  Background:  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).

Mesenchymal stem cells derived from human exocrine pancreas express transcription factors implicated in beta-cell development.

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.Abbreviations: Ab - antibodies, API - alpha1-proteinase inhibitor, bmMSC - bone marrow-derived mesenchymal stem cells, BSA - bovine serum albumin, CK19 - cytokeratin 19, DMEM - Dulbeccos Modified Eagle Medium, EMT - epithelial to mesenchymal transition, FCS - fetal calf serum, FITC - fluorescein isothiocyanate, HGF - hepatocyte growth factor, hMSC - human mesenchymal stem cells, Ig - immunoglobulin, ITS - insulin-transferrin-selenium, MSC - mesenchymal stem cells, PBS - phosphate-buffered saline, PE - phycoerythrin, pMSC - pancreas-derived mesenchymal stem cells, RT - room temperature, RT-PCR - reverse transcription polymerase chain reaction

Quality of life after islet transplantation: data from the GRAGIL 1 and 2 trials.

Background  Rigorous assessment of health‐related quality of life (HRQL) is mandatory to establish the benefits of islet transplantation.

Transplantation of Encapsulated Hepatocytes During Acute Liver Failure Improves Survival Without Stimulating Native Liver Regeneration

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.

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice.

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.