Improvement of rat islet viability during transplantation: validation of pharmacological approach to induce VEGF overexpression:

Delayed and insufficient revascularization during islet transplantation deprives islets of oxygen and nutrients, resulting in graft failure. Vascular endothelial growth factor (VEGF) could play a critical role in islet revascularization. We aimed to develop pharmacological strategies for VEGF overexpression in pancreatic islets using the iron chelator deferoxamine (DFO), thus avoiding obstacles or safety risks associated with gene therapy. Rat pancreatic islets were infected in vivo using an adenovirus (ADE) encoding human VEGF gene (4.10(8) pfu/pancreas) or were incubated in the presence of DFO (10 mumol/L). In vitro viability, functionality, and the secretion of VEGF were evaluated in islets 1 and 3 days after treatment. Infected islets or islets incubated with DFO were transplanted into the liver of syngenic diabetic rats and the graft efficiency was estimated in vivo by measuring body weight, glycemia, C-peptide secretion, and animal survival over a period of 2 months. DFO induced transient VEGF overexpression over 3 days, whereas infection with ADE resulted in prolonged VEGF overexpression lasting 14 days; however, this was toxic and decreased islet viability and functionality. The in vivo study showed a decrease in rat deaths after the transplantation of islets treated with DFO or ADE compared with the sham and control group. ADE treatment improved body weight and C-peptide levels. Gene therapy and DFO improved metabolic control in diabetic rats after transplantation, but this effect was limited in the presence of DFO. The pharmacological approach is an interesting strategy for improving graft efficiency during transplantation, but this approach needs to be improved with drugs that are more specific

In vitro assessment of human islet vulnerability to instant blood mediated inflammatory reaction (IBMIR) and its use to demonstrate a beneficial effect of tissue culture

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or pre-cultured (24 h, 48 h) human islets were incubated in either ABO compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37 degrees C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess beta cell function, glucose stimulated insulin secretion (GSIS) assay was carried out. The extent of islet beta cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48 h pre-cultured islets. Freshly isolated islets had significantly higher TF content than 24 h and 48 h pre-cultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5 fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when pre-cultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extra-islet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24 h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation and proinsulin release

Wiskott-Aldrich syndrome protein deficiency in innate immune cells leads to mucosal immune dysregulation and colitis in mice

BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich Syndrome protein (WASP), a hematopoietic-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T-cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2 deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared to RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients, and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin (IL)-10. Administration of IL-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of IL-10, and homeostasis of regulatory T cells

Wiskott-Aldrich syndrome protein-mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pD

Genome-wide identification and phenotypic characterization of seizure-associated copy number variations in 741,075 individuals

Copy number variants (CNV) are established risk factors for neurodevelopmental disorders with seizures or epilepsy. With the hypothesis that seizure disorders share genetic risk factors, we pooled CNV data from 10,590 individuals with seizure disorders, 16,109 individuals with clinically validated epilepsy, and 492,324 population controls and identified 25 genome-wide significant loci, 22 of which are novel for seizure disorders, such as deletions at 1p36.33, 1q44, 2p21-p16.3, 3q29, 8p23.3-p23.2, 9p24.3, 10q26.3, 15q11.2, 15q12-q13.1, 16p12.2, 17q21.31, duplications at 2q13, 9q34.3, 16p13.3, 17q12, 19p13.3, 20q13.33, and reciprocal CNVs at 16p11.2, and 22q11.21. Using genetic data from additional 248,751 individuals with 23 neuropsychiatric phenotypes, we explored the pleiotropy of these 25 loci. Finally, in a subset of individuals with epilepsy and detailed clinical data available, we performed phenome-wide association analyses between individual CNVs and clinical annotations categorized through the Human Phenotype Ontology (HPO). For six CNVs, we identified 19 significant associations with specific HPO terms and generated, for all CNVs, phenotype signatures across 17 clinical categories relevant for epileptologists. This is the most comprehensive investigation of CNVs in epilepsy and related seizure disorders, with potential implications for clinical practice

Perfluorocarbons, new tool for pancreatic islets preservation in vitro

La transplantation d'îlots pancréatiques est une thérapie proposée aux diabétiques de type 1 afin de restaurer une sécrétion physiologique d'insuline. Cette thérapie se heurte à une perte importante d'îlots en culture due à l'hypoxie. Des émulsions de perfluorocarbures (PFCs) ont été testées dans cette étude afin d'apporter l'oxygène nécessaire aux îlots.Les émulsions de PFCs provoquent un décollement des lignées de cellules b à l'origine de la formation de pseudo-îlots et ainsi qu une absence d'adhésion des îlots pancréatiques de rat au support. Cet effet s'accompagne d'une diminution de l'hypoxie cellulaire, responsable, sur les îlots contrôles, d'une fibrose interstitielle irréversible. De plus, les émulsions de PFCs sont capables de préserver in vitro la fonctionnalité des îlots jusqu'à 5 jours après isolement. Ces données suggèrent que les émulsions de PFCs pourraient être un nouvel outil permettant de préserver les îlots pancréatiques in vitro mais offrant également de grandes potentialités quant à leur utilisation in vivo lors de la transplantation d'îlots libres ou d'îlots encapsulés.Islet transplantation proved a viable route to insulin independence for type 1 diabetic subjects. However, hypoxia enhances an important loss of pancreatic islets in culture. Perfluorocarbons (PFCs) serve also as oxygen "reservoirs" for harvested organs in pancreas organ transplantation. The aim of this work was to study effect of PFCs emulsions on b cells lines and on pancreatic islets in vitro.PFCs emulsion was not toxic but decreased cellular adhesion and enhanced cells detachment which triggered to pseudo-islets formation. Validation of PFCs emulsions on rat pancreatic islets also showed an anti-adhesive effect. Moreover, PFCs emulsions protected islets from an irreversible interstitial fibrosis in islets control linked to hypoxia. Consequently, PFCs emulsions preserved islets functionality for at least 5 days in vitro.Taken together, these data suggest that PFCs emulsions could be a new tool to preserve islets in vitro and would be very useful regarding islets transplantation

Perfluorocarbons, new tool for pancreatic islets preservation in vitro

La transplantation d'îlots pancréatiques est une thérapie proposée aux diabétiques de type 1 afin de restaurer une sécrétion physiologique d'insuline. Cette thérapie se heurte à une perte importante d'îlots en culture due à l'hypoxie. Des émulsions de perIslet transplantation proved a viable route to insulin independence for type 1 diabetic subjects. However, hypoxia enhances an important loss of pancreatic islets in culture. Perfluorocarbons (PFCs) serve also as oxygen "reservoirs" for harvested organs

Les perfluorocarbures un nouvel outil pour la conservation des îlots pancréatiques in vitro

[non disponible] But de l'étude : Toutes ces données montrent le rôle essentiel de l'hypoxie cellulaire dans l'échec à la transplantation d'îlots pancréatiques. Mon projet de thèse s'est développé dans le cadre d'un programme européen regroupant 18 équipes de 7 nationalités différentes intitulé BARP+ (BioARtificial Pancreas+ NMP3-CT-2003-505614) ayant pour objectif de développer et de valider un pancréas bioartificiel utilisant la technique de macroencapsulation. Mon rôle était de développer de nouveaux outils de lutte contre l'hypoxie cellulaire présente au sein de l’îlot au cours de son isolement et accentuée lors de son confinement dans les modules d'encapsulation. La collaboration avec le laboratoire CNRS UPR 22 à l'Institut Charles Sadron (ICS), dirigée par Marie-Pierre Krafft a permis d'utiliser de nouveaux outils, les émulsions de perfluorocarbures. Comme présenté ci-dessus, ces molécules permettent de lutter efficacement contre l'hypoxie cellulaire induite par le prélèvement d'organes et leur utilisation aujourd'hui en clinique est largement validée. Cependant, leur utilisation in vitro dans la préservation des îlots pancréatiques en luttant contre l'hypoxie n'a pas fait l'objet d'études concluantes. L'hypothèse que nous nous proposons d'explorer s'inscrit donc dans un cadre conceptuel largement validé dans le cas de la préservation des organes : l'utilisation des émulsions de perfluorocarbures comme transporteurs d'oxygène dans la lutte contre l'hypoxie des îlots pancréatiques