Publications by authors named "Mathieu Armanet"

24 Publications

  • Page 1 of 1

Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells.

Nat Commun 2020 11 25;11(1):5982. Epub 2020 Nov 25.

August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Rosselló 149-153, 08036, Barcelona, Spain.

Expanding the mass of pancreatic insulin-producing beta cells through re-activation of beta cell replication has been proposed as a therapy to prevent or delay the appearance of diabetes. Pancreatic beta cells exhibit an age-dependent decrease in their proliferative activity, partly related to changes in the systemic environment. Here we report the identification of CCN4/Wisp1 as a circulating factor more abundant in pre-weaning than in adult mice. We show that Wisp1 promotes endogenous and transplanted adult beta cell proliferation in vivo. We validate these findings using isolated mouse and human islets and find that the beta cell trophic effect of Wisp1 is dependent on Akt signaling. In summary, our study reveals the role of Wisp1 as an inducer of beta cell replication, supporting the idea that the use of young blood factors may be a useful strategy to expand adult beta cell mass.
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http://dx.doi.org/10.1038/s41467-020-19657-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689468PMC
November 2020

Regulated expression and function of the GABA receptor in human pancreatic beta cell line and islets.

Sci Rep 2020 08 10;10(1):13469. Epub 2020 Aug 10.

Institut Cochin, INSERM U1016, CNRS UMR 8104, Université de Paris, 123 bd du Port-Royal, 75014, Paris, France.

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABA receptor compared to human islet. In ECN90 cells, baclofen, a specific GABA receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABA receptor in human islets modulates insulin secretion.
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http://dx.doi.org/10.1038/s41598-020-69758-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417582PMC
August 2020

Correction to: Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features.

Stem Cell Res Ther 2020 05 21;11(1):190. Epub 2020 May 21.

Laboratory of Cell Therapy for Diabetes, Institute of Regenerative Medicine and Biotherapy, Univ. Montpellier, CHU Montpellier, Montpellier, France.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s13287-020-01717-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240903PMC
May 2020

Characterization of immortalized human islet stromal cells reveals a MSC-like profile with pancreatic features.

Stem Cell Res Ther 2020 04 17;11(1):158. Epub 2020 Apr 17.

Laboratory of Cell Therapy for Diabetes, Institute of Regenerative Medicine and Biotherapy, Univ. Montpellier, CHU Montpellier, Montpellier, France.

Background: Mesenchymal stromal cells (MSCs) represent an interesting tool to improve pancreatic islet transplantation. They have immunomodulatory properties and secrete supportive proteins. However, the functional properties of MSCs vary according to many factors such as donor characteristics, tissue origin, or isolation methods. To counteract this heterogeneity, we aimed to immortalize and characterize adherent cells derived from human pancreatic islets (hISCs), using phenotypic, transcriptomic, and functional analysis.

Methods: Adherent cells derived from human islets in culture were infected with a hTERT retrovirus vector and then characterized by microarray hybridization, flow cytometry analysis, and immunofluorescence assays. Osteogenic, adipogenic, and chondrogenic differentiation as well as PBMC proliferation suppression assays were used to compare the functional abilities of hISCs and MSCs. Extracellular matrix (ECM) gene expression profile analysis was performed using the SAM (Significance Analysis of Microarrays) software, and protein expression was confirmed by western blotting.

Results: hISCs kept an unlimited proliferative potential. They exhibited several properties of MSCs such as CD73, CD90, and CD105 expression and differentiation capacity. From a functional point of view, hISCs inhibited the proliferation of activated peripheral blood mononuclear cells. The transcriptomic profile of hISCs highly clusterized with bone marrow (BM)-MSCs and revealed a differential enrichment of genes involved in the organization of the ECM. Indeed, the expression and secretion profiles of ECM proteins including collagens I, IV, and VI, fibronectin, and laminins, known to be expressed in abundance around and within the islets, were different between hISCs and BM-MSCs.

Conclusion: We generated a new human cell line from pancreatic islets, with MSCs properties and retaining some pancreatic specificities related to the production of ECM proteins. hISCs appear as a very promising tool in islet transplantation by their availability (as a source of inexhaustible source of cells) and ability to secrete a supportive "pancreatic" microenvironment.
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http://dx.doi.org/10.1186/s13287-020-01649-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165390PMC
April 2020

The flanking peptides issue from the maturation of the human islet amyloid polypeptide (hIAPP) slightly modulate hIAPP-fibril formation but not hIAPP-induced cell death.

Biochimie 2020 Mar 12;170:26-35. Epub 2019 Dec 12.

Sorbonne Université, Ecole Normale Supérieure, PSL University, CNRS, Laboratoire des Biomolécules (LBM), 4 Place Jussieu, F-75005, Paris, France. Electronic address:

Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic β cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic β-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.
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http://dx.doi.org/10.1016/j.biochi.2019.12.005DOI Listing
March 2020

Implication of glycogen synthase kinase 3 in diabetes-associated islet inflammation.

J Endocrinol 2020 01;244(1):133-148

Université Paris Diderot, BFA, UMR 8251, CNRS, Team 'Biologie et Pathologie du Pancréas Endocrine', Paris, France.

Islet inflammation is associated with defective β cell function and mass in type 2 diabetes (T2D). Glycogen synthase kinase 3 (GSK3) has been identified as an important regulator of inflammation in different diseased conditions. However, the role of GSK3 in islet inflammation in the context of diabetes remains unexplored. In this study, we investigated the direct implication of GSK3 in islet inflammation in vitro and tested the impact of GSK3 inhibition in vivo, on the reduction of islet inflammation, and the improvement of glucose metabolism in the Goto-Kakizaki (GK) rat, a spontaneous model of T2D. GK rats were chronically treated with infra-therapeutic doses of lithium, a widely used inhibitor of GSK3. We analyzed parameters of glucose homeostasis as well as islet inflammation and fibrosis in the endocrine pancreas. Ex vivo, we tested the impact of GSK3 inhibition on the autonomous inflammatory response of non-diabetic rat and human islets, exposed to a mix of pro-inflammatory cytokines to mimic an inflammatory environment. Treatment of young GK rats with lithium prevented the development of overt diabetes. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines in the islets. It decreased islet fibrosis and partially restored the glucose-induced insulin secretion in GK rats. Studies in non-diabetic human and rat islets exposed to inflammatory environment revealed the direct implication of GSK3 in the islet autonomous inflammatory response. We show for the first time, the implication of GSK3 in islet inflammation and suggest this enzyme as a viable target to treat diabetes-associated inflammation.
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http://dx.doi.org/10.1530/JOE-19-0239DOI Listing
January 2020

Conventional and Neo-antigenic Peptides Presented by β Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.

Cell Metab 2018 12 2;28(6):946-960.e6. Epub 2018 Aug 2.

INSERM, U1016, Cochin Institute, 75014 Paris, France; CNRS, UMR8104, Cochin Institute, 75014 Paris, France; Paris Descartes University, Sorbonne Paris Cité, 75014 Paris, France; Assistance Publique Hôpitaux de Paris, Service de Diabétologie, Cochin Hospital, 75014 Paris, France. Electronic address:

Although CD8 T-cell-mediated autoimmune β cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by β cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known β cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8 T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by β cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
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http://dx.doi.org/10.1016/j.cmet.2018.07.007DOI Listing
December 2018

Modeling human pancreatic beta cell dedifferentiation.

Mol Metab 2018 04 8;10:74-86. Epub 2018 Feb 8.

INSERM U1016, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014 Paris, France. Electronic address:

Objective: Dedifferentiation could explain reduced functional pancreatic β-cell mass in type 2 diabetes (T2D).

Methods: Here we model human β-cell dedifferentiation using growth factor stimulation in the human β-cell line, EndoC-βH1, and human pancreatic islets.

Results: Fibroblast growth factor 2 (FGF2) treatment reduced expression of β-cell markers, (INS, MAFB, SLC2A2, SLC30A8, and GCK) and activated ectopic expression of MYC, HES1, SOX9, and NEUROG3. FGF2-induced dedifferentiation was time- and dose-dependent and reversible upon wash-out. Furthermore, FGF2 treatment induced expression of TNFRSF11B, a decoy receptor for RANKL and protected β-cells against RANKL signaling. Finally, analyses of transcriptomic data revealed increased FGF2 expression in ductal, endothelial, and stellate cells in pancreas from T2D patients, whereas FGFR1, SOX,9 and HES1 expression increased in islets from T2D patients.

Conclusions: We thus developed an FGF2-induced model of human β-cell dedifferentiation, identified new markers of dedifferentiation, and found evidence for increased pancreatic FGF2, FGFR1, and β-cell dedifferentiation in T2D.
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http://dx.doi.org/10.1016/j.molmet.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985229PMC
April 2018

MondoA Is an Essential Glucose-Responsive Transcription Factor in Human Pancreatic β-Cells.

Diabetes 2018 03 27;67(3):461-472. Epub 2017 Dec 27.

INSERM U1016, Cochin Institute, Paris, France

Although the mechanisms by which glucose regulates insulin secretion from pancreatic β-cells are now well described, the way glucose modulates gene expression in such cells needs more understanding. Here, we demonstrate that MondoA, but not its paralog carbohydrate-responsive element-binding protein, is the predominant glucose-responsive transcription factor in human pancreatic β-EndoC-βH1 cells and in human islets. In high-glucose conditions, MondoA shuttles to the nucleus where it is required for the induction of the glucose-responsive genes arrestin domain-containing protein 4 (ARRDC4) and thioredoxin interacting protein (TXNIP), the latter being a protein strongly linked to β-cell dysfunction and diabetes. Importantly, increasing cAMP signaling in human β-cells, using forskolin or the glucagon-like peptide 1 mimetic Exendin-4, inhibits the shuttling of MondoA and potently inhibits TXNIP and ARRDC4 expression. Furthermore, we demonstrate that silencing MondoA expression improves glucose uptake in EndoC-βH1 cells. These results highlight MondoA as a novel target in β-cells that coordinates transcriptional response to elevated glucose levels.
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http://dx.doi.org/10.2337/db17-0595DOI Listing
March 2018

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

Sci Rep 2016 05 11;6:25765. Epub 2016 May 11.

INSERM, U1016, Institut Cochin, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, 75014, France.

Acute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation.
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http://dx.doi.org/10.1038/srep25765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863151PMC
May 2016

Cell rearrangement in transplanted human islets.

FASEB J 2016 Feb 3;30(2):748-60. Epub 2015 Nov 3.

*Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland; Cell Therapy Unit, Hôpital Saint Louis, Assistance Publique-Hôpitaux de Paris, and University Paris 7, Paris, France; and Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche Scientifique 1138, Centre de Recherches des Cordeliers, Paris, France.

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central β cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and β cells into islet-like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti-VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.
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http://dx.doi.org/10.1096/fj.15-273805DOI Listing
February 2016

Proteasome dysfunction mediates high glucose-induced apoptosis in rodent beta cells and human islets.

PLoS One 2014 18;9(3):e92066. Epub 2014 Mar 18.

CNRS UMR 5203, INSERM U661, and Montpellier 1 & 2 University, Institute of Functional Genomics, Montpellier, France; Laboratory for Diabetes Cell Therapy, Institute for Research in Biotherapy, University Hospital St-Eloi, Montpellier, France; Department of Endocrinology-Diabetes-Nutrition, University Hospital Lapeyronie, Montpellier, France.

The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0092066PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958412PMC
December 2014

Fetal PGC-1α overexpression programs adult pancreatic β-cell dysfunction.

Diabetes 2013 Apr 28;62(4):1206-16. Epub 2012 Dec 28.

INSERM, UMRS 872, Cordeliers Research Center, Paris, France.

Adult β-cell dysfunction, a hallmark of type 2 diabetes, can be programmed by adverse fetal environment. We have shown that fetal glucocorticoids (GCs) participate in this programming through inhibition of β-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1α represses genes important for β-cell development and function. More precisely, PGC-1α inhibited the expression of the key β-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1α complex to the Pdx1 promoter. To explore PGC-1α function, we generated mice with inducible β-cell PGC-1α overexpression. Mice overexpressing PGC-1α exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased β-cell mass, and β-cell hypotrophy. Interestingly, PGC-1α expression in fetal life only was sufficient to impair adult β-cell function whereas β-cell PGC-1α overexpression from adult age had no consequence on β-cell function. Altogether, our results demonstrate that the GR and PGC-1α participate in the fetal programming of adult β-cell function through inhibition of Pdx1 expression.
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http://dx.doi.org/10.2337/db12-0314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609553PMC
April 2013

A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion.

J Clin Invest 2011 Sep 25;121(9):3589-97. Epub 2011 Aug 25.

Université Pierre et Marie Curie-Paris 6, Biotechnology and Biotherapy Team, Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière (CRICM), UMRS 975, Paris, France.

Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.
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http://dx.doi.org/10.1172/JCI58447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163974PMC
September 2011

Assessment of human islet labeling with clinical grade iron nanoparticles prior to transplantation for graft monitoring by MRI.

Cell Transplant 2010 17;19(12):1573-85. Epub 2010 Aug 17.

Cell Isolation and Transplantation Center, Geneva University Hospitals and University of Geneva, School of Medicine, 4 rue Gabrielle-Perret-Gentil, Geneva 14, Switzerland.

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80-90% purity) were incubated for 24 h with various concentrations of SPIOs (14-280 μg/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perl's (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 ± 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.
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http://dx.doi.org/10.3727/096368910X515863DOI Listing
June 2011

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

Diabetes 2010 May 25;59(5):1202-10. Epub 2010 Feb 25.

Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland.

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 mum in diameter), beta-cells had a core position, alpha-cells had a mantle position, and vessels laid at their periphery. In bigger islets, alpha-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 beta-cells to alpha-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 beta-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non-beta-cells.

Conclusions: Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between beta- and alpha-cells, while permitting homologous contacts between beta-cells.
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http://dx.doi.org/10.2337/db09-1177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857900PMC
May 2010

Regulated laminin-332 expression in human islets of Langerhans.

FASEB J 2009 Dec 10;23(12):4046-55. Epub 2009 Aug 10.

Department of Surgery, Cell Isolation and Transplantation Center, Geneva University Hospitals and University of Geneva, 1211 Geneva-4, Switzerland.

Laminin-332 (LN-332) is a basement membrane component known to exert a beneficial effect on rat pancreatic beta cells in vitro. In this work, we analyzed the expression of LN-332 in human islets, its expression after inflammatory insults by cytokines, and the molecular mechanisms responsible for this effect. By Western blotting and RT-PCR, we showed that LN-332 was expressed in isolated human islets. By immunofluorescence on pancreas sections, we observed that labeling was confined to endocrine cells in islets. Confocal microscopy analysis on isolated islet cells revealed that labeling was granular but did not colocalize with hormone secretory granules. LN-332 was most abundant in cultured islets compared to freshly isolated islets and was found in culture medium, which suggests that it was secreted by islets. When islets were exposed to interleukin (IL)-1beta, expression and secretion of LN-332 increased as compared to control. No effect was observed with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited culture- and IL-1beta-induced LN-332 expression in islets. These results show that LN-332, known to have some beneficial effect on beta cells in vitro, is produced and secreted by endocrine islet cells and is up-regulated by stressing conditions such as culture and IL-1beta-exposure.
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http://dx.doi.org/10.1096/fj.08-127142DOI Listing
December 2009

Immunomodulation by blockade of the TRANCE co-stimulatory pathway in murine allogeneic islet transplantation.

Transpl Int 2009 Sep 15;22(9):931-9. Epub 2009 May 15.

Cell Isolation and Transplantation Center, University of Geneva School of Medicine, Geneva, Switzerland.

We explore herein the effect of TNF-related activation-induced cytokine (TRANCE) co-stimulatory pathway blockade on islet survival after allograft transplantation. Expression of TRANCE on murine C57Bl/6 (B6) CD4+ T cells after allogeneic activation was analyzed by fluorescence-activated cell sorter (FACS). The effect of a TRANCE receptor fusion protein (TR-Fc) and anti-CD154 antibody (MR1) on B6 spleen cell proliferation after allogeneic activation was assessed by mixed lymphocyte reaction (MLR). Three groups of B6 mice were transplanted with allogeneic islets (DBA2): Control; short-term TR-Fc-treatment (days 0-4); and prolonged TR-Fc-treatment (days -1 to 13). Donor-specific transfusion (DST) was performed at the time of islet transplantation in one independent experiment. Transplantectomy samples were analyzed by immunohistochemistry. TRANCE expression was upregulated in stimulated CD4+ T cells in vitro. In MLR experiments, TR-Fc and MR1 both reduced spleen cell proliferation, but less than the combination of both molecules. Short-course TR-Fc treatment did not prolong islet graft survival when compared with controls (10.6 +/- 1.9 vs. 10.7 +/- 1.5 days) in contrast to prolonged treatment (20.7 +/- 3.2 days; P < 0.05). After DST, primary non function (PNF) was observed in half of control mice, but never in TR-Fc-treated mice. Immunofluorescence staining for Mac-1 showed a clear decrease in macrophage recruitment in the treated groups. TRANCE-targeting may be an effective strategy for the prolongation of allogeneic islet graft survival, thanks to its inhibitory effects on co-stimulatory signals and macrophage recruitment.
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http://dx.doi.org/10.1111/j.1432-2277.2009.00892.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858391PMC
September 2009

Computer-assisted digital image analysis to quantify the mass and purity of isolated human islets before transplantation.

Transplantation 2008 Dec;86(11):1603-9

Department of Surgery, Cell Isolation and Transplantation Center, Geneva University Hospitals and University of Geneva, Geneva, Switzerland.

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<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.
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http://dx.doi.org/10.1097/TP.0b013e31818f671aDOI Listing
December 2008

The role of macrophage migration inhibitory factor in mouse islet transplantation.

Transplantation 2008 Nov;86(10):1361-9

Islet Transplant Program, Alberta Diabetes Institute, University of Alberta, Edmonton AB, Canada.

Background: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells.

Methods: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice.

Results: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups.

Conclusion: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.
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http://dx.doi.org/10.1097/TP.0b013e31818bdbefDOI Listing
November 2008

Blockade of beta1 integrin-laminin-5 interaction affects spreading and insulin secretion of rat beta-cells attached on extracellular matrix.

Diabetes 2006 May;55(5):1413-20

Department of Genetic Medicine and Development, University Medical Center, 1 rue Michel-Servet, 1211 Geneva-4, Switzerland.

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic beta-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor beta1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on beta-cell function and spreading. When expression of two well-known laminin-5 ligands, beta1 and beta4 integrin, was assessed by Western blot and RT-PCR, only the beta1 integrin was detected in beta-cells. Anti-beta1 integrin antibody reduced the spreading of beta-cells on 804G matrix. Blockade of the interaction between beta1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti-beta1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti-beta1 integrin and -laminin-5 antibodies interfere with spreading of beta-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of beta1 integrins by laminin-5 is an important component of normal beta-cell function.
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http://dx.doi.org/10.2337/db05-1388DOI Listing
May 2006

Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion.

J Biol Chem 2005 Nov 29;280(44):36824-32. Epub 2005 Aug 29.

Department of Cell Physiology and Metabolism, University Medical Center, Geneva University Hospitals, CH-1211 Geneva 4, Switzerland.

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.
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http://dx.doi.org/10.1074/jbc.M505560200DOI Listing
November 2005

Positron-emission tomography imaging of early events after transplantation of islets of Langerhans.

Transplantation 2005 Feb;79(3):353-5

Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals, Geneva, Switzerland.

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.
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http://dx.doi.org/10.1097/01.tp.0000149501.50870.9dDOI Listing
February 2005

Comparative analysis and validation of different assays for glycopeptide susceptibility among methicillin-resistant Staphylococcus aureus strains.

J Microbiol Methods 2004 May;57(2):231-9

Division of Infectious Diseases, University Hospitals of Geneva, CH-1211, 24 rue Micheli-du-Crest, 1211 Geneva 14, Switzerland.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibiting intermediate susceptibility to glycopeptides (GISA) is challenging for clinical microbiology laboratories. We compared three different screening assays for evaluating trends in decreased glycopeptide susceptibility during two periods. Ninety four and ninety five consecutive MRSA blood isolates from 189 bacteremic patients collected during periods A (1989-1994) and B (1999-2001), respectively, were screened in parallel for vancomycin or teicoplanin susceptibility by glycopeptide-containing brain-heart infusion agar (BHIA) tests, Etest MICs performed at a standard (0.5 McFarland) or high (2.0 McFarland) inoculum on Mueller-Hinton (MHA) or BHIA, respectively. Any MRSA isolate yielding <50 CFU (representing <10(-6) of the plated inoculum) on either BHIA containing 2 mg/l of vancomycin (V2-BHIA) or 5 mg/l of teicoplanin (T5-BHIA) was considered as fully susceptible to vancomycin or teicoplanin, respectively. The proportion of MRSA isolates yielding>50 CFU on either V2-BHIA or T5-BHIA significantly (P<0.01) increased from 7/94 (7.4 %) or 8/94 (8.5%) in period A to 16/95 (16.8 %) or 14/95 (14.7%) in period B, respectively. Vancomycin Etests MICs on MHA were of lower sensitivity (<30% and<65%), but high specificity (100% and 99%) on periods A and B isolates, respectively, compared to those on V2-BHIA. Vancomycin Etests MICs on BHIA were of a higher sensitivity (57% and 81%) but lower specificity (91% and 65%) compared to those on V2-BHIA, on periods A and B isolates, respectively, reflecting an unexpectedly high number of false positive isolates on period B isolates (28/95). Screening of decreased susceptibility to vancomycin or teicoplanin on V2-BHIA or T5-BHIA, respectively, may represent simple low-cost alternatives to Etest MICs, minimising the risk of missing potential GISA isolates.
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http://dx.doi.org/10.1016/j.mimet.2004.01.012DOI Listing
May 2004