Dual Islet Transplantation Modeling of the Instant Blood-Mediated Inflammatory Reaction

Abstract

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig-to-primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof-of-concept that this model can serve to properly control experiments comparing modified xenoislet preparations. Wild-type (WT) and α1,3-galactosyltransferase knockout (GTKO) neonatal porcine islets (NPIs) were studied in non-immunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at one and 24 hours after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages, and platelets. Insulin, complement, antibody, neutrophils, macrophages, and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 hours in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction

“…One of the major characteristics of IBMIR after intraportal transplantation is the recruitment of neutrophils and macrophages to sites of islet engraftment (30). To test our hypothesis that AAT protects islet grafts from IBMIR, livers of recipients were collected 6 h PT for histological analysis.…”

“…Cytokines produced by infiltrated leukocytes and endocrine islet cells in IBMIR play a crucial role in the destruction of islet grafts (17,21,30,40). Cytokine-induced β-cell failure and death are key events that lead to diabetes (41).…”

Early barriers to neonatal porcine islet engraftment in a dual transplant model

“…Hemilivers were divided into 5 sections each and preserved in 10% neutral buffered formalin for histological evaluation, as described. (27) Tissue samples were embedded in paraffin and underwent subsequent sectioning and hematoxylin and eosin (H&E) staining for morphologic observation. Tissue slides also underwent independent staining for standard hematoxylin and eosin, as well as galactose-α1,3-galactose (Gal, Enzo Life Sciences, Farmingdale, NY, USA) for one and 24 hour assessments, porcine specific CD44 (PORC24A IgG2a, Washington State College of Veterinary Medicine, Pullman, WA, USA) for 7 day post infusion assessments, insulin (Sigma-Aldrich, St. Louis, MO, USA), C3d (Abcam, Cambridge, MA, USA), C4d (American Research Products, Waltham, MA, USA), IgG (Sigma-Aldrich, St. Louis, MO, USA), IgM (KPL, Gaithersburg, MD, USA), neutrophil (neutrophil elastase, Dako, Carpinteria, CA, USA), IFN-gamma (Abcam, Cambridge, MA, USA), macrophage (CD68, Dako, Carpinteria, CA, USA), natural killer (NK) cell (CD3-CD8+, CD3, Abcam, Cambridge, MA, USA; CD8, Abcam, Cambridge, MA, USA), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, Roche Diagnostics GmbH, Mannheim, Germany), cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA) and platelets (CD61, Dako, Carpinteria, CA, USA).…”

“…Quantitative immunohistochemical analysis was performed using Aperio Imagescope (Leica Biosystems, Vista, CA, USA) digital pathology software. (27) Individual slides were scanned and islets were manually selected and analyzed using an optimized positive pixel algorithm to obtain a percent pixel positivity of the individual stain per islet. The sum of individual islets within a hemiliver was compiled to compute a percent positive staining representative of each hemiliver, termed the positivity index.…”

“…(27) Xenotransplantation is a species-specific endeavor, making preclinical (e.g. pig to primate) graft survival models laborious and resource-intensive.…”