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Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

ABSTRACT: The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an ?1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n?=?5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.

SUBMITTER: Bottino R

PROVIDER: S-EPMC4169326 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

Bottino R R Wijkstrom M M van der Windt D J DJ Hara H H Ezzelarab M M Murase N N Bertera S S He J J Phelps C C Ayares D D Cooper D K C DK Trucco M M

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 20140912 10

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-dia ...[more]

PMID: 25220221

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Project description:BACKGROUND:Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti-CD154 monoclonal antibody (mAb) in pig-to-non-human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti-CD40 (2C10R4) mAb-based studies have not been reported. METHODS:Nine streptozotocin (STZ)-induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen-free (DPF) miniature pigs. They were treated with anti-CD40 mAb-based immunosuppressive regimen and were divided into 3 groups: anti-CD40 only group (n = 2), belatacept group (anti-CD40 mAb+belatacept, n = 2), and tacrolimus group (anti-CD40 mAb+tacrolimus, n = 5). All monkeys received anti-thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C-peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted. RESULTS:All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow-up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C-peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti-CD40 only, belatacept, and tacrolimus groups, respectively. Anti-?Gal IgG levels in serum and the number of interferon-? secreting T cells in peripheral blood mononuclear cells did not increase in most animals. CONCLUSION:These results showed that anti-CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti-CD40 mAb was not as effective as anti-CD154 mAb in terms of preventing early islet loss.

Project description:Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL-6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL-6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non-human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined. Pig islets were isolated from designated pathogen-free (DPF) SNU miniature pigs and transplanted via portal vein into five streptozotocin-induced diabetic monkeys. One group (n = 2, basal group) was treated with anti-thymoglobulin (ATG), anti-CD40 antibody (2C10R4), sirolimus, and tacrolimus, and the other group was additionally given tocilizumab on top of basal immunosuppression (n = 3, Tocilizumab group). To confirm IL-6 blocking effect, C-reactive protein (CRP) levels and serum IL-6 concentration were measured. Scheduled biopsy of the margin of the posterior segment right lobe inferior of the liver was performed at 3 weeks after transplantation to assess the degree of revascularization of the transplanted islets. Immunohistochemical staining using anti-insulin, anti-CD31 antibodies, and lectin IB4 was conducted to find the origin of endothelial cells in the islet graft. CRP significantly increased at 1~2 days after transplantation in Basal group, but not in Tocilizumab group, and higher serum IL-6 concentration was measured in latter group, showing the biological potency of tocilizumab. In Basal group, well-developed endothelial cells were observed on the peri- and intraislet area, whereas the number of CD31+ cells in the intraislet space was significantly reduced in Tocilizumab group. Finally, new endothelial cells in the pig islet graft were positive for CD31, but not for lectin IB4, suggesting that they are originated from the recipient monkey. Our results demonstrated that tocilizumab can delay revascularization of the transplanted islet, although this effect had no significant correlation to the overall islet graft survival. In the pig to NHP islet xenotransplantation model, the endothelial cells from recipient monkey form new blood vessels in and around pig islets.

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Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

Publications

Pig-to-monkey islet xenotransplantation using multi-transgenic pigs.

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