Ulrika Johansson

Inflammatory mediators expressed in human islets of Langerhans : implications for islet transplantation

Expression of immune modulating mediators in human Islets of Langerhans could have important implications for development of autoimmunity in type 1 diabetes and influence the outcome of clinical islet transplantation. Islets obtained from five donors were analyzed at various times after isolation using cDNA array technology. The Atlas Human Cytokine/Receptor and Hematology/Immunology nylon membranes representing 268 genes and 406, respectively, were used and the relative expression of each gene analyzed. Of the 51 gene products identified, high mRNA expression of MCP-1, MIF, VEGF, and thymosin beta-10 was detected in all islet samples. IL-8, IL-1-beta, IL-5R, and INF-gamma antagonist were expressed in islets cultured for 2 days. IL-2R was expressed in islets cultured for more than 6 days. In conclusion, several inflammatory mediators were expressed in isolated islets, particularly at an early stage after isolation, indicating that a few days of culture could be beneficial for the outcome of islet transplantation.

Formation of Composite Endothelial Cell-Mesenchymal Stem Cell-Islets; a novel approach to promote islet revascularization

OBJECTIVE—Mesenchymal stem cells (MSCs) contribute to endothelial cell (EC) migration by producing proteases, thereby paving the way into the tissues for ECs. MSCs were added to our previously described composite EC islets as a potential means to improve their capacity for islet angiogenesis. RESEARCH DESIGN AND METHODS—Human islets were coated with primary human bone marrow–derived MSCs and dermal microvascular ECs. The capacity of ECs, with or without MSCs, to adhere to and grow into human islets was analyzed. The survival and functionality of these composite islets were evaluated in a dynamic perifusion assay, and their capacity for angiogenesis in vitro was assessed in a three-dimensional fibrin gel assay. RESULTS—ECs proliferated after culture in MSC-conditioned medium, and MSCs improved the EC coverage threefold compared with EC islets alone. Islet survival in vitro and the functionality of the composite islets after culture were equal to those of control islets. The EC-MSC islets showed a twofold increase in total sprout formation compared with EC islets, and vascular sprouts emanating from the EC-MSC–islet surface showed migration of ECs into the islets and also into the surrounding matrix, either alone or in concert with MSCs. CONCLUSIONS—EC proliferation, sprout formation, and ingrowth of ECs into the islets were enhanced by MSCs. The use of composite EC-MSC islets may have beneficial effects on revascularization and immune regulation. The technique presented allows for pretreatment of donor islets with recipient-derived ECs and MSCs as a means of improving islet engraftment.

Composite islet-endothelial cell grafts : a novel approach to counteract innate immunity in islet transplantation.

An instant blood‐mediated inflammatory reaction (IBMIR) is elicited when islets come in contact with blood after intraportal transplantation. In contrast, endothelial cells (EC) readily tolerate contact with blood. A conceivable strategy to overcome IBMIR would be to create composite islet‐EC grafts. Human islets were co‐cultured with primary human aortic endothelial cells (HAEC) for 2–7 days to obtain 50–90% coverage. HAEC‐coated islets were exposed to ABO‐identical blood and analyzed with regard to clotting time, signs of inflammation and cell infiltration. Composite islet‐HAEC graft survival was assessed after transplantation to athymic (nu/nu) nude mice. Exposed to blood, HAEC‐coated islets induced less activation of coagulation and complement compared to control islets. Also, platelet and leukocyte consumption in blood was decreased. Clots with entrapped HAEC‐coated islets showed less infiltration of CD11b+ cells. The extent of protection correlated to the level of HAEC coverage. Transplanted composite grafts stained positive for insulin and PECAM‐1 demonstrating presence of both islets and HAEC within the islet graft 7 weeks after transplantation. Composite islet‐HAEC grafts reduce all components of IBMIR. Refinement of the technique will allow introduction of composite islet‐EC grafts in clinical islet transplantation, using autologous EC expanded in vitro and kept frozen until allogeneic islets become available for that specific recipient.

Anchoring of Vascular Endothelial Growth Factor to Surface-Immobilized Heparin on Pancreatic Islets: Implications for Stimulating Islet Angiogenesis

In pancreatic islet transplantation, early revascularization is necessary for long-term graft function. We have shown in in vitro and in vivo models that modification with surface-attached heparin protects the islets from acute attack by the innate immune system of the blood following intraportal islet transplantation. In this study, we have investigated the ability of an immobilized conjugate composed of heparin to bind the angiogenic growth factor vascular endothelial growth factor-A (VEGF-A) as a means of attracting endothelial cells (ECs) to induce angiogenesis and revascularization. We analyzed the capacity of VEGF-A to bind to immobilized heparin and how this affected the proliferation and adherence of ECs to both artificial glass surfaces and islets. Quartz crystal microbalance with dissipation monitoring and slot-blot demonstrated the binding of VEGF-A to heparin-coated surfaces upon which ECs showed protein-dependent proliferation. Also, ECs cultured on heparin-coated glass surfaces exhibited effects upon focal contacts. Heparinized islets combined with VEGF-A demonstrated unaffected insulin release. Further, covering islets with heparin also increased the adhesion of ECs to the islet surface. Immobilized heparin on the islet surface may be a useful anchor molecule for achieving complete coverage of islets with angiogenic growth factors, ultimately improving islet revascularization and engraftment in pancreatic islet transplantation.

Preparatory studies of composite mesenchymal stem cell islets for application in intraportal islet transplantation

Abstract Background. Low engraftment and adverse immune reactions hamper the success rate of clinical islet transplantation. In this study, we investigated the capacity of human mesenchymal stem cells (MSCs) to adhere to human islets of Langerhans and their effects in immune modulation and during blood interactions in vitro. Methods. Composite MSC–islets were formed by suspension co-culture, and the phenotype was evaluated by confocal microscopy. Islet function was assessed by dynamic insulin release in response to glucose in vitro. Mixed lymphocyte–islet reactions (MLIR) and the tubing blood loop model were utilized as in vitro tools to analyse the effect of MSCs on the innate and adaptive immune reactions triggered by the islets. Results. MSCs rapidly adhered to islets and spread out to cover the islet surface. Insulin expression and secretion were sustained with the MSC coating. MSC-coated islets showed unaffected reactions with blood in vitro in comparison to control islets. Furthermore, MSCs suppressed lymphocyte proliferation induced by islet cells in MLIR. Conclusion. We conclude that it is possible to create composite MSC–islets to enable delivery of the MSCs by utilizing the adhesive capacity of the MSCs. This could have beneficial immunosuppressive effects in optimizing pancreatic islet transplantation.

Bioallethrin causes permanent changes in behavioural and muscarinic acetylcholine receptor variables in adult mice exposed neonatally to DDT

We recently reported changes in the density of muscarinic acetylcholine receptors in cerebral cortex of mice treated neonatally with DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane) and receiving bioallethrin as adults. We also found behavioural aberrations in adult mice treated with bioallethrin, whether neonatally treated with DDT or the vehicle. To ascertain whether these changes were permanent, 10-day-old mice received an oral dose of DDT (0.5 mg/kg body weight) and at the age of 5 months they received bioallethrin orally (0.7 mg/kg body weight/day; 7 days). The animals were investigated at the age of 7 months. Here we report muscarinic acetylcholine receptor changes, additional behavioural disturbances and learning disabilities in mice receiving DDT as neonates and bioallethrin as adults, whereas the behavioural disturbances in mice receiving vehicle as neonates and bioallethrin as adults had diminished and changes in proportions of high- and low-affinity binding sites had developed. No changes in the density of nicotinic acetylcholine receptors were noted for any of the treated groups. In conclusion, exposure of neonates to DDT leads to increased susceptibility in adults to a short-acting pesticide with similar neurotoxic action. An adult exposure to this short-acting pesticide to mice neonatally exposed to DDT leads to irreversible muscarinic acetylcholine receptor changes and behavioural disturbances with additional changes 2 months after the exposure.

Neonatal exposure to DDT induces increased susceptibility to pyrethroid (bioallethrin) exposure at adult age. — Changes in cholinergic muscarinic receptor and behavioural variables

We have recently reported that DDT and the pyrethroid bioallethrin cause similar changes in the brain muscarinic cholinergic receptors (MAChR) and behavioural disturbances in the neonatal and adult mouse when given to neonatal mice during the peak of rapid brain growth. In the present study the interaction between neonatal and adult exposure to DDT and bioallethrin, respectively, is explored. Ten-day-old NMRI mice received a single low oral dose of DDT (0.5 mg/kg body wt). At adult age (5 months) the mice received bioallethrin 0.7 mg/kg body wt./day per os for 7 days. Mice used as controls received a 20% fat emulsion vehicle. The spontaneous behavioural tests revealed significant differences, both in mice treated neonatally with DDT and receiving bioallethrin as adults and in mice receiving the vehicle as neonates and bioallethrin as adults, compared with their corresponding controls. However, the behavioural changes developed in mutually opposite directions. Significant changes in MAChR, assayed in the P2 fraction of the cerebral cortex by using the muscarinic antagonist, quinuclidinyl benzilate ([3H]QNB) and agonist carbachol, was only observed in animals receiving DDT as neonates and bioallethrin as adults. The present study indicates an increased susceptibility in the cholinergic muscarinic receptors and a different behaviour reaction in animals already exposed to DDT (at a physiologically relevant dose), when again exposed to a similar neurotoxic agent as adults.

Low-dose effects of paraoxon in adult mice exposed neonatally to DDT: changes in behavioural and cholinergic receptor variables

This study revealed increased susceptibility in adult mice, exposed neonatally to a low dose of DDT (1,1,1-trichloro-2,2-bis( p-chlorofenyl)ethane), to develop changes in behaviour and cholinergic muscarinic receptors when exposed as adults to the organophosphorus insecticide paraoxon. 10-day-old NMRI male mice were given a single oral dose of DDT (0.5 mg/kg body weight). At the age of 5 months, paraoxon was administered by gavage as a single dose (0.7 or 1.4 mg/kg body weight) every 2nd day for 1 week. These doses caused approximately 15% and 45% inhibition of acetylcholinesterase respectively, 48 h after the last exposure. 24 h after the last paraoxon administration, a spontaneous motor activity test revealed no differences between any of the adult paraoxon-treated mice and their corresponding controls, though when the test was performed again 2 months later, mice exposed neonatally to DDT and given paraoxon as adults had developed changes in spontaneous behaviour. The density of muscarinic cholinergic receptors was significantly increased in this group. No significant changes were seen in either behaviour or muscarinic receptors in mice exposed neonatally to the vehicle and receiving paraoxon as adults and there were no significant differences in the muscarinic or nicotinic subpopulations investigated, between any of the treatment groups. These results show that a dose of paraoxon not having any effect in vehicle-treated animals can cause effects in animals neonatally exposed to DDT.