Reply to Mangold and Ankersmit

Abstract

We appreciate the comments [1] of Drs Mangold and Ankersmit on our article [2]. Our group proved that genipin fixation is a novel alternative to conventional glutaraldehyde fixation, and the addition of decellularization, organic solvent treatment and detoxification prevent calcification of glutaraldehyde/genipinfixed bovine pericardium. We also found that the titer of anti-α-Gal(Galα1-3Galβ1-4GlcNAc-R) antibodies (immunoglobulin G) increases after the implantation of bovine pericardium to rabbit, and in vivo calcification results are consistent with the increase in anti-α-Gal antibodies titer [2]. Although the cause and significance of the xenoreactive antibody response between the different species in α-Gal concordant animal model have not been exactly known, some possible suggestions may exist: (i) by a species-specific expression of these conjugated compounds including α-Gal epitopes; (ii) a tissue-specific expression of these conjugated compounds including α-Gal epitopes and (iii) the differences in the fine specificity of natural anti-Gal antibodies in various species which recognize various ‘facets’ of the α-Gal epitope in its three-dimensional form. A species-specific expression of these conjugated compounds including α-Gal epitopes has been well demonstrated for so long. Thyroglobulin, fibrinogen and immunoglobulins from various species express varying amounts of Galα1-3Galβ14GlcNAc residues between 0.01 and 11 residues per molecule [3]. Rabbit red blood cells contain a range of glycolipids of varying lengths that terminate with the Galα1-3Galβ1-4GlcNAc-R structure. These glycolipids vary in size by increments of five monosaccharides, and each increment forms an additional branch. Although rat did not contain Galα1-3Galβ1-4GlcNAc neutral glycosphingolipids in the kidney, pig and sheep contained sialic acid-containing glycolipids (gangliosides) with the Galα1-3Galβ1-4GlcNAc-R structure, most likely the same branched, ganglioside identified in bovine red blood cells [4]. Thymus tissue from sheep, pig and rabbit also contained a range (compounds with 5–11 sugars) of neutral glycolipids terminating with the Galα1-3Galβ1-4GlcNAc-R structure demonstrating a species-specific expression of these compounds. Although the kidney and thymus from sheep, pig and rabbit express Galα1-3Galβ1-4GlcNAc-R glycolipids, these glycolipids were not detected in brain tissue demonstrating a tissue-specific expression of these compounds [5]. The detailed analysis of N-linked glycans from porcine kidney demonstrated that Galα1-3Galβ14GlcNAc-R termini were found on a variety of complex bi-, triand tetra-antennary N-glycans [6]. As we evaluated recently the immune response in the implantation of bovine pericardium to mice in which the α1,3-galactosyltransferase gene was knocked out (Gal/ mice), the titer of anti-α-Gal antibodies much increased. But interestingly, we also observed the increase in anti-α-Gal antibodies in wild-type mouse and rat as well as rabbit, although much less than Gal/ mice. We think that further transplantation or implantation experiments are needed to validate more exact xenoreactive immunologic responses between the different species.