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Distribution of Non-Gal Antigens in Pig Cornea: Relevance to Corneal Xenotransplantation

Cohen, David; Miyagawa, Yuko MS; Mehra, Ruhina; Lee, Whayoung MD; Isse, Kumiko MD, PhD; Long, Cassandra BS; Ayares, David L. PhD; Cooper, David K. C. MD, PhD, FRCS; Hara, Hidetaka MD, PhD

doi: 10.1097/ICO.0000000000000069
Basic Investigation

Purpose: The aim of this study was to investigate the distribution of antigens other than galactose-α-1,3-galactose (Gal) (non-Gal) recognized by human and rhesus monkey serum antibodies in the α-1,3-galactosyltransferase gene-knockout (GTKO) pig cornea.

Methods: The distribution of non-Gal, specifically N-glycolylneuraminic acid (NeuGc), in the corneas from wild-type (WT) and GTKO pigs was identified. Corneal sections from WT and GTKO pigs were incubated with human or rhesus monkey serum to determine immunoglobulin (Ig)M and IgG binding to corneal tissue by means of fluorescent microscopy.

Results: Strong expression of NeuGc was found in all layers of both WT and GTKO pig corneas. In both humans and monkeys, antibody binding (IgG > IgM) to GTKO was found to be weaker than that to entire WT pig corneas, but in both, most antibody binding, especially IgG, was to the epithelium. There was weak diffuse antibody binding, especially of IgG, to the corneal stroma, suggesting binding to antigens expressed on collagen. There was no or minimal binding of IgM/IgG to the corneal endothelium.

Conclusions: Although the cornea is avascular, antibodies in primate serum can bind to pig antigens, especially on epithelial cells and stromal collagen. Although the binding to entire GTKO corneas was weaker than that to WT corneas, deletion of the expression of NeuGc and expression of human complement–regulatory proteins in the pig cornea will be important if prolonged clinical corneal xenograft survival is to be achieved.

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*Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA;

Department of Pathology, University of Pittsburgh, Pittsburgh, PA; and

Revivicor, Inc, Blacksburg, VA.

Reprints: Hidetaka Hara, Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Thomas E. Starzl Biomedical Sciences Tower, Room W1546, 200 Lothrop St, Pittsburgh, PA 15261 (e-mail:

David Cohen and Yuko Miyagawa contributed equally to this work.

Research on xenotransplantation at the University of Pittsburgh is funded in part by NIH Grants #1RO3A1096296-01 (H.H.), #IU19A1090959-01 (D.K.C.C.), #U01A1066331(D.K.C.C.), and #5P01 HL107152-02 (D.K.C.C.), by an Ocular Tissue Engineering and Regenerative Ophthalmology (OTERO) Postdoctoral Fellowship (W.L.), and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor Inc, Blacksburg, VA.

D. L. Ayares is an employee of Revivicor Inc. The other authors have no funding or conflicts of interest to disclose.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (

Received August 02, 2013

Accepted December 12, 2013

Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.