through addition of FET. When the implants were pre-incubated with human anti-Gal antibodies, Gal-positive tissue had strongly enhanced calcification compared with Gal-negative tissue and any tissue not pre-incubated with such antibodies. These interesting results need to be discussed in more detail. What puzzled us is the methodologic concept chosen in this work: 1. The tissue samples were implanted sub-cutaneously. The au- thors explain that this method is adequate because it is tradi- tionally used and empirically well validated. This argument is not convincing, because the environment is simply physiolog- ically different. Manji et al 4 delivered distinct data for xeno- graft calcification by valve implantation in rats into the infra- renal aortic position compared with former sub-cutaneous models. 2. As correctly reviewed by Lila et al, humans and Old World monkeys are the only mammals who lack the -Gal epitope but display high anti-Gal titers. Thus, rats are the wrong model to use if the immunologic role of -Gal is to be addressed. Rats express the Gal epitope themselves and do not have anti-Gal antibodies. Therefore, a specific immune response to -Gal is hardly possible. 3. The preincubation with human anti-Gal antibodies is an ap- proach toward an immunologic linkage, but the implicated assumption of the authors—that antibodies can accomplish effector functions in a xenogenic system such as human anti- bodies in a rat organism—is unsustainable. This may be pos- sible, but the actual scenario that is taking place can never be defined clearly, and was not even tried in the present study. So far, the presented results lack an explanation. Never- theless, they are at hand; it would be restrictive not to consider them. We know that Gal epitopes and the respec- tive antibodies can differ slightly amongst each other, 5 and this, perhaps, could be a possible explanation favoring an immunologic background. From our point of view, how- ever, this is unlikely and would have to be proven. Because immunologic reasons for the significant difference in calci- fication of -Gal–negative tissue must be questioned, other possible explanations should be evaluated. We are content that our results are encouraging researchers to seek for deeper insights concerning -Gal and its role in biopros- thetic heart valve destruction, but emerging obstacles should be overcome rather than evaded. Disclosure statement None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to dis- close. Andreas Mangold, MD Hendrik Jan Ankersmit, MD Department of Thoracic Surgery Medical University of Vienna Wien, Austria References 1. Lila N, McGregor C, Carpentier S, Rancic J, Byrne G, Carpentier A. Gal knockout pig pericardium: new source of material for heart valve bioprostheses. J Heart Lung Transplant 2010;29:538-43. 2. Konakci KZ, Bohle B, Blumer R, et al. Alpha-gal on bioprostheses: xenograft immune response in cardiac surgery. Eur J Clin Invest 2005;35:17-23. 3. Mangold A, Szerafin T, Hoetzenecker K, et al. Alpha-gal specific IgG immune response after implantation of bioprostheses. Thorac Cardiovasc Surg 2009; 57:191-5. 4. Manji R, Zhu L, Nijjar N, et al. Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and fail from xenograft rejection. Circulation 2006;114:318-27. 5. Galili U. Xenotransplantation and ABO incompatible transplantation: the sim- ilarities they share. Transfus Apher Sci 2006;35:45-58. Rat Model in the Study of the Role of -Gal in Heart Valve Bioprostheses Calcification In the comments of Mangold and Ankersmit concerning our study of the use of -1,3-galactosyltransferase (Gal) knockout pig pericardium as a new source of material for heart valve bioprostheses (HVB), they underline their own contribution, 1 published in 2005, as forming the basis of our work. This publication made an important contribution to the field, because it showed there was an induced antibody re- sponse to -Gal after implantation of a BHV, but we think it is an over-statement to suggest that it is the basis of our work. We began engineering Gal-deficient pigs in 2001 and pre- sented the modified cell line that gave rise to our Gal-deficient pigs in 2003. 2 At that time, there was evidence of antibody- enhanced tissue calcification from several groups 3 and a grow- ing awareness of the potential for an immunologic mechanism to contribute to valve calcification despite tissue fixation. Mangold and Ankersmit also claim that they have “de- scribed . . . for the first time and definitively disproved this often-raised assertion that the fixation process of biopros- thetic valves is abolishing anti-genicity of the tissue.” We completely agree, as indicated in the very first sentence of our article, which states, “despite glutaraldehyde fixation some immunogenicity persists.” Our report builds on the previous results of Human and Zilla, 3 who showed that a pre-formed human antibody can initiate calcification of fixed tissues. They showed that sensitized rabbit sera to ho- mogenates of glutaraldehyde-fixed porcine aorta enhanced cal- cification in a rabbit sub-cutaneous implant model compared with pre-immune sera. Our report confirms that graft-specific antibody may stim- ulate calcification but, importantly, extends this finding to show that a pre-formed physiologically relevant human anti- body, namely anti-Gal, is equally capable of accelerating cal- cification. This implies that in humans, there is no need for HVB sensitization to initiate calcification. This is a critical observation, because the affinity and toxicity of pre-formed and induced antibody may differ significantly. Regarding the methodology, we would like to make the following remarks: 1. We are aware that sub-cutaneous implantation in rats for few months differs from implantation of HVB in aortic position; however, our 40-year experience dem- onstrates that the sub-cutaneous model is convenient, 912 The Journal of Heart and Lung Transplantation, Vol 29, No 8, August 2010