Alain Belcourt

Prevention of adhesion and promotion of pseudoislets formation from a β-cell line by fluorocarbon emulsions

Transplantation of pancreatic islets from cadaveric human donors is a promising therapy for type 1 diabetes. By reducing hyper/hypoglycaemic events, this therapy can strongly improve the quality of life of patients. There have been substantial advances in the islet-transplantation protocol (the Edmonton Protocol) through an improved islet-isolation method and the use of steroid-free immunosuppressants. Nevertheless, several difficulties continue to limit this approach. These include the gradual loss of islet graft functionality, side effects related to the use of immunosuppressants and, last but not least, the small number of potential donors. An alternate approach to solving the problem of the shortage of human islets consists of the transplantation of b-cell lines or b-cell surrogates. For this purpose, sources of b cells, such as pig and mouse pancreas and human and mouse embryonic stem cells are being investigated. Established bcell lines, such as the mouse insulinoma-6 (MIN6) cells offer several advantages over isolated islet material. The native pancreatic islet tissue is composed of various cell types (a, b and d cells). Furthermore, the sensitivity to glucose of b cells from freshly isolated islets exhibits considerable variability. In contrast, the MIN6 cell line provides a homogenous b-cell population whose insulin secretion responds physiologically to glucose stimulation. Under classical culture conditions, MIN6 cells adhere to uncoated, negatively charged tissue culture plastic to form mono ACHTUNGTRENNUNGlayers, but when grown on collagen-derived substrates, they form islet-like structures (pseudoislets) after 6–8 days of culture. Due to a lack of cell-to-cell contact, b cells from mono ACHTUNGTRENNUNGlayers are considerably less responsive to glucose and other nutrients than b cells arranged as pseudoislets. Hence, pseudoislets offer a better experimental model for studies of b-cell functionality than monolayer cells. In this study, we report that a perfluorocarbon (PFC) emulsion is able to prevent the adhesion of MIN6 cells onto negatively charged tissue culture plastic. Furthermore, the cells were seen to rapidly aggregate into well-formed pseudoislets. Owing to the specific properties of PFCs mentioned below, this approach to pseudoislet formation has the advantage of being nonimmunogenic, in contrast to the collagen-derived substrate approach. Owing to exceptional biological inertness, excellent spreading properties, high oxygen solubility and extremely low solubility in water, PFCs are being investigated for various biomedical applications. These include, in particular, intravascular oxygen transport, diagnosis, lung-surfactant replacement and drug delivery. Within the framework of the present investigation, the oxygen-carrying capacity of PFCs is also expected to prevent the hypoxia that contributes to the failure of post-transplant survival. This study represents an initial step whose aim is to establish the effects of PFCs on the viability and morphology of the MIN6 cell line. A PFC emulsion stabilized with Pluronic F-68 has been shown to reduce the endothelial adherence of sickled red cells. In that study, however, the reduction of adherence was also obtained with a PFC-free aqueous solution of Pluronic F68. The result was, therefore, attributed to a lubricating effect of the surfactant. In the present study, two hydrocarbon (HC) oil emulsions (Intralipid@ and a soybean oil (SBO) emulsion) that use the same surfactant as the PFC emulsion, provided the appropriate controls for assessing the effect of the PFC. Other studies have shown a modification of cell adhesion by using either pure liquid PFCs (cell culture at a liquid/liquid interface) or PFC-functionalized solid substrates. In both studies, a tendency to reduction of cell adhesion and promotion of spherical cell shape (reduced cell spreading) was observed. In the liquid/liquid system, cell growth was in the form of island monolayers, rather than in a continuous sheet monolayer of cells. In the present study, a 70% w/v-concentrated perfluorooctyl bromide (C8F17Br) emulsion, emulsified with egg yolk phospholipids (EYP, 4.67% w/v) and stabilized by a semifluorinated alkane, C6F13C10H21 (F6H10, 3.27% w/v), was used. [27] This PFC emulsion has a small average droplet size (~80 nm average diameter after terminal heat sterilization) and a narrow particlesize distribution. The mean diameter of the PFC droplets was only 100 nm after five months of storage at 4 8C. This PFC emulsion as well as the HC emulsions (Intralipid and a 45% w/v SBO emulsion) used as controls were diluted with an equal volume of twice-concentrated culture medium (DMEM) and added to the culture wells (see Experimental Section). The MIN6 cells were then incubated in DMEM containing various percentages (v/v) of these 1:1 diluted emulsions. For clarity, the results are expressed in terms of % v/v of the 1:1 diluted emulsions in the culture medium as well as, in the case of the PFC emulsion, % w/v of PFC in the total DMEM+emulsion volume. The effect of the PFC and HC emulsions on MIN6 cell cultures was investigated by counting the number of cells that remained adhered to the substrate. This number was compared with that obtained for control experiments in which the cells [a] Dr. M. Sanchez Dominguez, Dr. M. P. Krafft Syst mes Organis s Fluor s Finalit s Th rapeutiques (SOFFT). Institut Charles Sadron (CNRS) 6 rue Boussingault. 67083 Strasbourg, Cedex (France) Fax: (+33)388-414-099 E-mail : krafft@ics.u-strasbg.fr [b] E. Maillard, Dr. S. Sigrist, Dr. A. Belcourt Centre Europ en d’Etude du Diab te (CeeD) 1 boulevard Ren Leriche 67200 Strasbourg (France) Fax: (+33)390-201-219 E-mail : severine.sigrist@wanadoo.fr

Influence of acinar tissue contamination on encapsulated pancreatic islets: morphological and functional studies.

BACKGROUND The present study concerns the influence of acinar contamination on pancreatic islets encapsulated in an artificial membrane (AN 69). METHODS Pure, handpicked pancreatic islets were contaminated by the addition of acinar tissue (ratio, 1:1). The morphological aspect and insulin release of both pure (n=12) and contaminated (n=12) encapsulated islets were assessed after 10 days of culture or implantation in the peritoneal cavity of rats. RESULTS After implantation, the encapsulated islets, irrespective of their purity, were totally altered, whereas the morphological aspect of the cultivated islets remained intact only in the absence of acinar tissue contamination. This contamination induced a significant decrease in the stimulation index of insulin release. The stimulation index decreased by 42% for fresh islets and by 52% and 34% for cultivated and implanted islets, respectively, without modification in their basal release of insulin. CONCLUSIONS The acinar tissue proved detrimental to the encapsulated implanted and cultivated islets.

Surface treatment of polycarbonate films aimed at biomedical application

Aiming to encapsulate pancreatic islets, a biocompatible polycarbonate membrane (What-man) was treated with plasma argon in order to improve its surface properties. The argon plasma treatment decreased the hydrophobicity of the membrane by fixing polyvinylpyrrolidone (PVP) at the surface. The water angle contact decreased from 47° to 20° after this treatment, while the structure and pore diameter were preserved. The treatment also increased significantly the water permeability from 62 ± 8 ml/min to 200 ± 29 ml/min (P < 0.001). ToF-SIMS analyses revealed that the argon plasma treatment of the membrane allowed the installation of an uniform PVP layer at the surface. The concentration equilibrum in glucose was reached after 8 h diffusion for the treated membrane, while it was only 32.4 ± 8.6% (P < 0.01) for the untreated membrane. The biocompatibility of the polycarbonate membrane was assessed after one month of implantation in rats and proved to be unaffected by the surface treatment. In conclusion, the present study provided sufficient information to establish a relationship between the physicochemical modifications of the PVP-plasma-treated polycarbonate membrane and the improvement in its permeability.

Physicochemical and biological studies of corona-treated artificial membranes used for pancreatic islets encapsulation: Mechanism of diffusion and interface modification

The artificial AN69 membrane (Hospal), a synthetic copolymer composed of acrylonitrile and sodium methallyl sulphonate suitable for pancreatic islet encapsulation, was submitted to physicochemical treatment (Corona discharge) to improve its insulin permeability. X-ray photoelectron spectroscopy (XPS) analysis of the AN69 membrane indicated the presence of up to two molecular layers of glycerol at its surface while the surface energies revealed the presence of hydrophilic sites (-SO3Na/glycerol) located at the membrane surface and acrylonitrile hydrophobic groups inside the material. The Corona discharges decreased the number of glycerol molecules at the membrane surface and from a biological point of view, produced a threefold increase in insulin diffusion. Furthermore, the biocompatibility of the treated membrane was preserved after 1 year of intraperitoneal implantation. The increase in insulin permeability should result from a decrease of the membrane polarity and of a steric hindrance in pores. Thus, Corona discharge treatment may serve to optimize the properties of artificial membranes used for pancreatic islets encapsulation.

Markers for risk of type 1 diabetes in relatives of Alsacian patients with type 1 diabetes.

Background: The cytotoxic T lymphocyteassociated antigen 4 gene (CTLA-4) encode the T cell receptor involved in the control of T cell proliferation and mediates T cell apoptosis. The receptor protein is a specific T lymphocyte surface antigen that is detected on cells only after antigen presentation. Thus, CTLA-4 is directly involved in both immune and autoimmune responses and may be involved in the pathogenesis of multiple T cell-mediated autoimmune disorders. There is polymorphism at position 49 in exon 1 of the CTLA-4 gene, providing an A-G exchange. Moreover, we assessed the CTLA-4 49 (Thr/Ala) polymorphism in diabetic patients and first-degree relatives as compared to control subjects. Research design and methods: Three loci (HLA-DQB1, DQA1 and CTLA-4) were analysed in 62 type 1 diabetic patients, 72 firstdegree relatives and 84 nondiabetic control subjects by means of PCR-RFLP. Results: A significant enrichment in DQB1 alleles encoding for an amino acid different from Asp in position 57 (NA) and DQA1 alleles encoding for Arg in position 52 was observed in diabetic subjects and first-degree relatives as compared to controls. The genotype and allele frequencies of these polymorphisms in type 1 diabetic patients and firstdegree relatives differed significantly from those of controls (p< 0.001 and 0.05 respectively). CTLA-49 Ala alleles frequencies were 75.8% in type 1 diabetic patients and 68.1% in first-degree relatives in comparison to 35.7% in control subjects. The Ala/Ala genotype conferred a relative risk of 18.8 (p < 0.001). Conclusion: The CTLA-4 49 Ala allele confers an increased risk of type 1 diabetes, independent of age and HLA-DQ genetic markers.

Reduction of Macrophage Activation after Antioxidant Enzymes Gene Transfer to Rat Insulinoma INS-1 Cells

BACKGROUND After transplantation, islet damage occurs through oxidative stress and host immune rejection mediated in part by macrophage activation. We investigated the influence of the overexpression of catalase (CAT) and Cu/Zn superoxide dismutase (Cu/Zn SOD) by rat insulinoma INS-1 beta cells exposed to oxidative stress on their viability and murine macrophage activation. METHODS INS-1 cells were infected with adenoviral vectors containing CAT (AdCAT) or Cu/Zn SOD (AdSOD) genes. After 72 hours, noninfected and infected INS-1 cells were exposed to oxidative stress and their viability was assessed using a colorimetric assay. Murine peritoneal exudate macrophages (mPEM) incubated with the supernatant of infected and stressed INS-1 cells were tested for chemotaxis and cytokine release (TNF-alpha, IL-alpha and IFN-gamma). RESULTS After infection, AdCAT and AdSOD gene transfer protected INS-1 cells from the toxicity of different oxidative reagents. The exposure of non-infected INS-1 cells to oxidative stress stimulated mPEM chemotaxis. INS-1 cells infection with AdCAT or AdSOD reduced significantly mPEM chemotaxis from 2.41 +/- 0.31 to 1.61 +/- 0.17 and from 2.53 +/- 0.24 to 1.27 +/- 0.14 respectively (n = 5; p < 0.05). Cytokine release by mPEM was stimulated after exposure to stressed noninfected INS-1 cell supernatant. CAT and Cu/Zn SOD overexpression by infected INS-1 cells decreased significantly the release of TNF-alpha from 268.18 +/- 30.18 to 81.40 +/- 23.58 pg/ml and from 446.96 +/- 75.47 to 20.37 +/- 2.38 pg/ml respectively (n = 6; p < 0.001). The overexpression of these enzymes also reduced significantly the release of IL-1beta and IFN-gamma. CONCLUSIONS CAT or Cu/Zn SOD gene transfer to INS-1 cells preserved them from oxidative damage and reduced the macrophage activation induced by these pancreatic cells. Therefore, protection of pancreatic beta cells against oxidative injury by antioxidant enzymes gene transfer is an effective approach to overcome the deleterious actions of macrophages in pancreatic islet transplantation.

Surface analysis of an encapsulation membrane after its implantation in mini-pigs.

The biocompatibility of membranes aiming at being a part of a bioartificial pancreas has been tested. For that purpose, we have studied a polycarbonate membrane surface after its implantation in mini-pigs. The membranes were made hydrophilic by an argon plasma surface treatment followed by a dipping in a hydrophilic polymer solution. Two polymers were tested: polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC). To test their biocompatibility, an encapsulation device for pig Langerhans islets, with external membranes treated as described above, was implanted in different mini-pigs. The pigs received no further treatment. The devices were explanted after in vivo exposure and the membranes were analysed by XPS (x-ray photoelectron spectroscopy) and ToF-SIMS (time-of-flight secondary ion mass spectrometry). After this time, the substrate with the PVP or HPMC treatment was still detected on the different samples. The surface treatment signal, however, was attenuated. This is explained by the detection of other components partly covering the surface. XPS and ToF-SIMS analyses revealed the presence of biological molecules on the two faces of the membrane: the outside face in contact with the biological environment and the inside face in contact with the device. ToF-SIMS images show the inhomogeneity of the biological molecules on the membrane surface. In conclusion, biological molecules adhered to the encapsulation membrane surface after implantation but the surface treatments remained unaltered.