apoptosis detection indicated that islet cell apoptosis was correlated with the time of leucocyte infiltration, but not with the time of antibody binding

apoptosis detection indicated that islet cell apoptosis was correlated with the time of leucocyte infiltration, but not with the time of antibody binding. results indicate that there is a cell-mediated, species-specific accelerated rejection after re-transplantation of xenogeneic islets. Introduction Transplantation of pancreatic islets is a potential treatment for patients with insulin-dependent (type 1) diabetes. The preliminary results of islet transplantation are encouraging and the number of insulin-independent patients is increasing with improvements in the technique of islet isolation and with the use of new immune-suppressive drugs.1C3 As islet transplantation becomes more successful, the scarcity of human tissue appears AC710 Mesylate as a potential limit to the procedure, and other animal species are being considered as sources of tissue.4C6 However, two major questions have to be addressed in xenogeneic islet transplantation. First, xeno-antigens trigger immune rejection, destroying the grafted islets. Second, animal viruses may be transmitted to humans. It has been reported that immune tolerance can be induced by injection of donor islet antigens into recipient’s thymus,7,8 and long-term survival of encapsulated xenogeneic islets has been achieved in a large animal model.9 Also, prolonged survival of genetically engineered islets has been described.10 This progress in immune-tolerance and immune-isolation as well as in genetic engineering make it likely that xenogeneic islets will be transplanted to humans in the future. In addition, the fear that animal viruses can be transmitted to humans has been eased recently.11 Therefore, it is reasonable to believe that the immune-rejection of xenografts will be overcome and animal virus infection may be avoided with genetically engineered animals in the future. In theory, because of the unrestricted supply of tissues, xenogeneic islet transplantation could be performed repeatedly even if the xenograft has been rejected. However, little is known on the functional consequence of islet re-transplantation in sensitized recipients, although accelerated rejection may be expected. In this study, we investigated the survival of re-grafted rat islets in mice that had been sensitized with rat, pig or human islet grafts, with attention to the rejection mechanisms. Materials and methods Islet isolation and purification Rat islets Islets were isolated from SpragueCDawley (SD) and Lewis male rats (350 g; BRL, Basel, Switzerland) using the intraductal collagenase digestion technique described previously.12 Briefly, 10 ml of Hank’s balanced salt solution (HBSS) with 2 mg/ml collagenase type XI (Sigma, St Louis, MO) were injected into the pancreatic duct. After pancreatectomy, the pancreata were digested AC710 Mesylate in a 37 water-bath for 19 min. The isolated islets were further purified by Euro-Ficoll (Sigma) gradient centrifugation. The purified islets were washed three times and then resuspended in HBSS for transplantation. Pig islets Pancreata were obtained from female large white pigs (more than 200 kg) in a local slaughterhouse (Orbe, Switzerland). Islet isolation was performed within 4 hr after pancreas procurement using a modified automated method described previously.13 Briefly, after preparation of the pancreas and distension with Liberase PI (Roche, Basel, Switzerland), digestion was performed in a modified digestion chamber AC710 Mesylate at 37 until the appearance of free islets. Islet purification was performed by Euro-Ficoll AC710 Mesylate gradient centrifugation on a cell separator (Cobe 2991, Cobe, Lakewood, CO). The purified islets were washed three times and resuspended in HBSS for transplantation. Human islets Human pancreata were obtained from multiorgan heart-beating donors and kept in University of Wisconsin solution at 4 for transport (cold ischaemia time <8 hr). Islet digestion and isolation were performed using a semiautomated method adapted from Ricordi apoptosis detection in grafted isletsTo detect apoptotic AC710 Mesylate cells, commercial kits were used according to the manufacturer's protocol (TACS terminal deoxynucleotide (TdT) apoptosis detection kit, R & D, Minneapolis, MN). Briefly, serial frozen sections were cut at 4 m intervals, fixed with 37% formaldehyde and permeabilized with proteinase K solution. Samples were then incubated in the quenching solution and then in the TdT labelling buffer. Samples were incubated with 50 l of labelling reaction mix at 37 for 1 hr. The slides were incubated in the TdT stop buffer and then were incubated with 50 l of Mouse monoclonal to CD16.COC16 reacts with human CD16, a 50-65 kDa Fcg receptor IIIa (FcgRIII), expressed on NK cells, monocytes/macrophages and granulocytes. It is a human NK cell associated antigen. CD16 is a low affinity receptor for IgG which functions in phagocytosis and ADCC, as well as in signal transduction and NK cell activation. The CD16 blocks the binding of soluble immune complexes to granulocytes.This clone is cross reactive with non-human primate streptavidinChorseradish peroxidase (HRP) solution for 10 min and counterstained in methyl green solution. The slides were then sequentially.