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Background: Pre-clinical demonstration of porcine islet graft function is necessary to support the clinical transplantation of pig islets. C-peptide concentration is an especially useful marker of insulin secretion, because its measurement is not confounded by the presence of exogenous insulin. To measure porcine C-peptide (PCP), researchers in the field exclusively used the Millipore (previously Linco Research) radioimmunoassay (RIA) until 2011, when Mercodia released an alternative enzyme-linked immunosorbent assay (ELISA). (At the end of 2013, the Millipore RIA was withdrawn from the market for commercial reasons.) In our current study, to directly compare these two assays, we performed validation studies on each. We also performed interlaboratory comparisons. Then, to determine the level of agreement between the assays, we analyzed the porcine serum C-peptide concentration measurement results obtained from each assay., Methods: Using pre-established method validation acceptance criteria, we determined and evaluated the detection limit, sensitivity, precision, linearity, and recovery of the two commercially available PCP assays described above (ELISA and RIA). After validation requirements were met, we performed a method comparison by determining C-peptide concentration in 60 serum samples collected from 31 normal, healthy adult Landrace pigs in the fasting state; a subset underwent an intravenous glucose challenge test, to stimulate the typical physiologic range of C-peptide. All analyses were performed according to manufacturer instructions. To compare the assays, we used Deming regression analysis., Results: Both assays met acceptance criteria. The RIA had a sensitivity of 0.1 ng/ml; it was linear to 2.9 ng/ml. The ELISA had a detection limit of 0.03 ng/ml; it was linear to 1.2 ng/ml. Recovery ranged from 89 to 113% with both assays. The coefficient of variability was 8% in interlaboratory comparisons. Deming regression analysis directly comparing both assays revealed significant correlation between them (before log-transformation: R2=0.9803, P<0.0001; after log-transformation: R2=0.9727, P<0.0001). Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. To transform ELISA data, we used the standard regression equation y=2.191x+0.1119 and the log-transformed regression equation y=0.8101x+0.7502. Both the transformed and the log-transformed (exponential) values approximated the measured RIA levels with a high degree of accuracy in the concentration range of 0 to 1.0 ng/ml., Conclusions: Porcine C-peptide concentration can be reliably measured in porcine serum samples with either assay (ELISA or RIA). However, the C-peptide results generated by these two assays are not equivalent. Therefore, assay bias must be considered before directly comparing pre-clinical studies that used either of these assays. We determined that harmonization between the assays is appropriate in a specific concentration range. Outside of that range, we do not know whether a linear correction function can be more broadly applied. The variation between the two assays may be related to calibration or reagent factors. To determine which assay is truly more accurate and to effectively compare interlaboratory results, we will need a traceable reference standard., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis., Methods: Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry., Results: The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM., Conclusions: Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: Endothelial damage is a critical step in the development of (xeno) transplantation-related and cardiovascular pathology. In humans, the amount of circulating endothelial cells (CEC) correlates to disease intensity and functions as a valuable damage marker. While (xeno) transplantation and cardiovascular research is regularly performed in porcine models, the paucity of antibodies against porcine endothelium epitopes hinders the use of CEC as damage marker., Objective: This study aimed to develop a method for porcine CEC detection using anti-human antibodies against porcine endothelium epitopes., Methods: Human umbilical vein endothelial cells (HUVEC, control) and their swine equivalent (SUVEC) were used to assess the cross-species immunoreactivity of fluorescently labeled anti-human CD31/CD51/CD54/CD62E/CD105/CD106/CD144/CD146/PAL-E/lectin-1/vWF antibodies by isotype-controlled fluorescence-activated cell sorting (FACS) and confocal microscopy. Next, reactivity was ascertained with mature porcine kidney-derived endothelial cells (PKEC), and a FACS-based whole blood CEC quantification method was employed using osmotic erythrolysis and CD105 and CD146 double staining after CD45 exclusion., Results: Of the 21 assayed antibodies, the MEM-229 clone of CD105 and P1H12 clone of CD146 showed immunoreactivity with SUVEC and PKEC. Double staining showed baseline porcine CEC count of 673.1 ± 551.4 CEC/ml, while the first 7.5 ml of drawn blood (representative of vascular damage) contained 1118 ± 661.4 CEC/ml (n = 14, P = 0.04). A second experiment (n = 5) including CD45 exclusion identified only 14.5 ± 10.8% double-positive CD105-146 events per ml blood., Conclusion: Porcine endothelium can be specifically labeled using anti-human CD146 and CD105 antibodies. These antibodies can therefore be used for the identification and quantification of CEC in porcine whole blood by FACS after osmotic erythrolysis., (© 2013 John Wiley & Sons A/S.)

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired., (© 2012 John Wiley & Sons A/S.)

The first blood transfusions in humans were xenotransfusions, carried out by Jean-Baptiste Denis beginning in 1667. Richard Lower, Matthäus Purmann and Georges Mercklin also experimented with the use of animal blood for transfusion until this practice was forbidden in 1670, after the death of one of Denis's patients. In the middle of the 19th century, xenotransfusion was rescued from oblivion by the work of Pierre Cyprien Oré. Franz Gesellius and Oscar Hasse fervently defended xenotransfusion, but Emil Ponfick and Leonard Landois stressed the potentially harmful effects of inter-species transfusion from 1874 onward. Xenotransfusion was abandoned completely following the discovery of blood groups by Karl Landsteiner in 1900. From 2000, because of progress in xenotransplantation and the need of blood supply, xenotransfusion is again being considered. Pigs are the best potential donors. The development of alpha-1,3-galactosyltransferase gene-knockout pigs has overcome the first hurdle to xenotransfusion. The main obstacle to porcine red blood cell transfusion is now the cellular response involving macrophages or natural killer cells.

Background: The use of porcine red blood cells has recently been proposed as a possible solution to the shortage of blood for human transfusion., Objectives: The purpose of this paper is to compare some ethical issues regarding xenotransfusion (XTF) with those relating to xenotransplantation (XT) of organs, tissues and cells., Materials and Methods: Various ethical concerns and viewpoints relating to XTF are discussed., Results: The main ethical obstacles to XT do not apply to XTF. It is much more ethically acceptable to raise pigs for regular blood collection as it doesn't damage the health of the animal. Porcine endogenous retrovirus infection, the major concern associated with XT, does not apply to XTF, since red blood cells have no DNA and have a very short lifespan. Clinical trials will be possible in humans once XTF has been demonstrated to be effective and harmless in non-human primates. Transgenesis is acceptable for pig blood donors because only a limited number of genes are involved, and these animals will never enter into the livestock gene pool or the food chain., Conclusion: Because the need for blood is less pressing than that for organs, tissues or cells, the use of animal blood for human transfusion is not an absolute necessity. However, it represents a real opportunity. The ability to gain access to an unlimited quantity of blood is a reasonable justification for XTF. Because its technical and ethical hurdles are less stringent, XTF could be the first large-scale clinical application of XT.