Warfarin or Low-Molecular-Weight Heparin Therapy does not Prolong Pig-To-Primate Cardiac Xenograft Function

Microvascular thrombosis is a prominent feature in cardiac delayed xenograft rejection (DXR). We investigated the impact of warfarin or low-molecular-weight heparin (LMWH) anti-coagulation on xenograft function using a heterotopic pig-to-primate model. Donor hearts were from CD46 transgenic pigs and baboon immunosuppression included tacrolimus, sirolimus, anti-CD20 and TPC, an alpha-galactosyl-polyethylene glycol conjugate. Three groups of animals were studied. Group 1 (n = 9) was treated with warfarin, Group 2 (n = 13) with LMWH and Group 3, received no anti-coagulant drugs. The median duration of xenograft function was 20 days (range 3-62 days), 18 days (range 5-109 days) and 15 days (range 4-53 days) in Groups 1 to 3 respectively. Anti-coagulation achieved the targeted international normalized prothrombin ratio (INR) and anti-factor Xa levels consistent with effective in vivo therapy yet, no significant impact on median xenograft function was observed. At rejection, a similar histology of thrombosis and ischemia was apparent in each group and the levels of fibrin deposition and platelet thrombi in rejected tissue was the same. Anti-coagulation with warfarin or LMWH did not have a significant impact on the onset of DXR and microvascular thrombosis. However, a role for specific anti-coagulant strategies to achieve long-term xenograft function cannot be excluded.     

Survey of glycoantigens in cells from α1-3galactosyltransferase knockout pig using a lectin microarray

Miyagawa S, Takeishi S, Yamamoto A, Ikeda K, Matsunari H, Yamada M, Okabe M, Miyoshi E, Fukuzawa M, Nagashima H. Survey of glycoantigens in cells from α1‐3galactosyltransferase knockout pig using a lectin microarray.
Xenotransplantation 2010; 17: 61–70. © 2010 John Wiley & Sons A/S. Abstract: Background: Glycoantigens represent major obstacles to successful xenotransplantation. Even after the α1‐3galactosyltransferase (GalT) gene knockout (GalT‐KO) pigs were produced, non‐Gal antigens continue to be present. This study reports on lectin blot analyses for endothelial cells (EC) and fibroblasts from GalT‐KO pigs. Methods: Differences in glycoantigens that are produced on cell surfaces in humans and pigs were surveyed. Differences between ECs and fibroblasts from wild‐type and GalT‐KO pigs were also examined. EC and fibroblasts from GalT‐KO pigs (heterozygous and homozygous) with N‐acetylglucosaminyltransferase‐III (GnT‐III), a wild‐type EC from the sibling, human EC lines, HUVEC (human EC from umbilical veins), & HAOEC (human EC from aortas), and human fibroblast line were used. EC and fibroblasts were cultured in gelatin‐coated dishes for several days. After sonication and centrifugation, the supernatant protein from each cell was labeled with Cy3, applied to a lectin array and scanned with an SC Profiler, and analyzed using an Array Pro Analyzer. Results: The pig EC showed higher signals in Euonymus Europaeus (EEL) & Griffonia simplicifolia I‐B₄ (GSI‐B4), binds α‐Gal, and in Wisteria Floribunda (WFA), Helix pomatia (HPA), Glycine max (SBA), & Griffonia simplicifolia I‐A₄ (GSI‐A4), binds GalNAc including the Thomsen‐Friedenreich precursor (Tn)‐antigen, while the human EC showed strong signals in Ulex europaeus I (UEA‐I), Maackia amurensis (MAL), Erythrina cristagalli (ECA), & Trichosanthes japonica I (TJA‐I) instead. The EC from the GalT‐KO pig signals for EEL & GSI‐B4 disappeared and those for Bauhinia purpurea alba (BPL), HPA, SBA, & GSI‐A4 were greatly diminished as well, while it up‐regulated signals for Sambucus Nigra (SNA), Sambucus sieboldiana (SSA), & TJA‐I, bind α2‐6 sialic acid, compared to the wild‐type pig EC. Concerning fibroblasts, the signals for HPA, SBA, & GSI‐A4 were the most intense in the wild‐type, and the intensities for homozygous‐KO were less, approaching those of humans. In addition, the order of the intensities, as detected by Arachis hypogaea (PNA) & Maclura pomifera (MPA), binding Galβ1‐2GalNAc, indicates that the Thomsen‐Friedenreich (T)‐antigen is likely present on pig fibroblasts. Conclusion: It is possible that the T‐antigen and Tn‐antigen related to GalNAc are non‐Gal antigens, but, fortunately, not only α‐Gal but also GalNAc were found to be decreased in the KO‐pig.     

Complement regulation in the GalT KO era

Miyagawa S, Yamamoto A, Matsunami K, Wang D, Takama Y, Ueno T, Okabe M, Nagashima H, Fukuzawa M. Complement regulation in the GalT KO era.
Xenotransplantation 2010; 17: 11–25. © 2010 John Wiley & Sons A/S. Abstract: A number of institutes have reported on the successful production of α‐galactosyltransferase knockout (GalT‐KO) pigs. After producing such pigs, hyperacute rejection appeared to no longer be a problem. However, acute vascular rejection (AVR)/acute humoral xenograft rejection (AHXR) is defined as a rejection that begins within 24 h after transplantation and gradually destroys the graft. The origin of AVR/AHXR continues to be a controversial topic, but is generally thought to be initiated by xeno‐reactive antibodies, including non‐Gal antibodies and subsequent activation of the graft endothelium, the complement and the coagulation systems. The complement is activated via the classical pathway by non‐Gal antigens and ischemia‐reperfusion injury, via the alternative pathway, especially on islets, and via the lectin pathway. Therefore the complement system is still an important recognition and effector mechanism of AVR/AHXR. In addition, quite recently, based on the relationship between complement and coagulation systems, a new pathway has been proposed. All complement regulatory proteins (CRPs) have the ability to regulate complement activation in different ways. Therefore, to effectively protect xenografts against AVR/AHXR, it appears reasonable to employ not only one but several CRPs including anti‐complement drugs. Non‐Gal antigens, such as the Hanganutziu‐Deicher antigen, is still present on GalT‐KO grafts. The further assessment of antigens continues to be an important issue in the area of clinical xenotransplantation. The above conclusions suggest that the expression of human CRPs on GalT‐KO grafts is necessary. Moreover, multilateral inhibition of complement activation is required in conjunction with the regulation of the coagulation system.