Yannick D. Muller

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Unique arrangement of alpha- and beta-cells in human islets of Langerhans

OBJECTIVE It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells. RESEARCH DESIGN AND METHODS Human pancreas specimens and isolated islets were processed for histology. Sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells. RESULTS In small human islets (40–60 μm in diameter), β-cells had a core position, α-cells had a mantle position, and vessels laid at their periphery. In bigger islets, α-cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets. As a consequence of this organization, the ratio of β-cells to α-cells was constantly higher in the core than in the mantle part of the islets, and decreased with increasing islet diameter. This core-mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets. Three-dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates, folded with different degrees of complexity and bordered by vessels on both sides. In epithelial plates, most β-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non–β-cells. CONCLUSIONS Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between β- and α-cells, while permitting homologous contacts between β-cells.

Transplantation of mesenchymal stem cells for the treatment of liver diseases, is there enough evidence?☆

Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been extensively investigated in small animal models to treat both acute and chronic liver injuries. Mechanisms of action are not clearly elucidated but may include their ability to differentiate into hepatocyte-like cells, to reduce inflammation, and to enhance tissue repair at the site of injury. This approach is controversial and evidence in large animals is missing. Side effects of MSC infusion such as the contribution to a fibrotic process have been reported in experimental settings. Nevertheless, MSCs moved quickly from bench to bedside and over 280 clinical trials are registered, of which 28 focus on the treatment of liver diseases. If no severe side-effects were observed so far, long-term benefits remain uncertain. More preclinical data regarding mechanisms of action, long term safety and efficacy are warranted before initiating large scale clinical application. The proposal of this review is to visit the current state of knowledge regarding mechanisms behind the therapeutic effects of MSCs in the treatment of experimental liver diseases, to address questions about efficacy and risk, and to discuss recent clinical advances involving MSC-based therapies.

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice

BACKGROUND & AIMS Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. METHODS Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. RESULTS MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL- and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. CONCLUSIONS MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis.

Influence of donor age on islet isolation and transplantation outcome

BACKGROUND It has been suggested that the age of human organ donors might influence islet isolation and transplantation outcome in a negative way due to a decrease of in vivo function in islets isolated from older donors. METHODS We retrospectively analyzed 332 islet isolations according to donor age. We determined isolation outcome by islet yields, transplantation rates, and [beta]-cell function in vitro. Transplanted patients were divided into two groups depending on donor age (n=25 and n=31 patients for <=45- and >45-year-old donors, respectively). We assessed islet graft function by C-peptide/glucose ratio, [beta] score, secretory units of islets in transplantation index, and insulin independence rate at 1, 6, and 12 months after transplantation. RESULTS There was no difference in islet yields between the two groups (251,900+/-14,100 and 244,600+/-8400 islet equivalent for <=45- and >45-year-old donors, respectively). Transplantation rates and stimulation indices were similar in both groups as well. All islet graft function parameters were significantly higher at 1-month follow-up in patients who had received islets from younger donors. At 6-month follow-up after second or third injection and at 12-month follow-up, secretory units of islets in transplantation indices and C-peptide/glucose ratios were significantly higher in patients with donors aged 45 years or younger. CONCLUSIONS These data suggest that, despite similar outcomes of the isolation procedure, islet graft function is significantly influenced by donor age. These results may have important consequences in the definition of pancreas allocation criteria.

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8+ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Anti-CD154 mAb and Rapamycin Induce T Regulatory Cell Mediated Tolerance in Rat-to-Mouse Islet Transplantation

Background Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4+CD25+Foxp3+ T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. Methodology/Principal Findings C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0–28 d) or late (100–128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. Conclusions/Significances These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

Islet Autotransplantation After Extended Pancreatectomy for Focal Benign Disease of the Pancreas

Background. Extended pancreatectomy is associated with the risk of surgical diabetes. Islet autotransplantation is successful in the prevention of diabetes after pancreas resection for chronic pancreatitis (CP), with insulin independence rates of 50% at 1 year. The aim of the present study is to demonstrate the safety and efficiency of islet autotransplantation after extended left pancreatectomy for benign disease. Methods. Between 1992 and 2009, 25 patients underwent extended pancreatectomy and islet autotransplantation for benign disease. Of these, 15 patients were operated for focal lesions located at the neck of the pancreas (14 benign tumors and 1 traumatic pancreatic section), the remainder being CP cases. After unequivocal diagnosis of benignity, the rest of the pancreas was processed and infused into the portal vein. Metabolic results were analyzed and isolation results were compared with those obtained from patients with CP or donors with brain death (DBD). Results. There was no mortality and a low morbidity (Streptococcus mitis bacteremia in 1 patient), no portal thrombosis or pancreatic fistula occurred. Median follow-up was 90 months. Actuarial patient survival was 100% at 10 years. Actuarial insulin independence was 94% at 10 years. All patients had positive basal and stimulated C-peptide levels and normal HbA1c. Mean islet yields were 5455 IEQ/gram vs. 1457 in CP (P=0.001) and 3738 in DBD (P=0.003). Conclusions. Islet autotransplantation after extensive pancreatic resection for benign disease is a safe and successful procedure. Islet yields after isolation, which are equivalent to the live donor situation, are significantly better than those from DBD donors.

Computer-Assisted Digital Image Analysis to Quantify the Mass and Purity of Isolated Human Islets Before Transplantation

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 (r2≥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<0.0001) when calculated using individual sphere volumes compared with the Ricordi algorithm. Measurement of red glass microspheres showed identical values comparing standard manual and computerized analyses. Conclusions. Computer-assisted digital image analysis is an objective and a reliable method for analyzing pancreatic islets before transplantation.

T regulatory cells in xenotransplantation

Abstract:  The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long‐term graft survival. In this regard, the induction of donor‐specific tolerance through isolation and expansion of xenoantigen‐specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4+CD25+Foxp3+ T cells, CD8+CD28− Treg, double negative CD4−CD8− T cells, and natural killer Treg. Although only studied in vitro so far, human CD4+CD25+Foxp3+ Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8+CD28− Treg and double negative CD4−CD8− Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4+Vα14+ Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4+CD25+Foxp3+ Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.