Hilton Gock

Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation

Extracellular nucleotides play an important role in thrombosis and inflammation, triggering a range of effects such as platelet activation and recruitment, endothelial cell activation, and vasoconstriction. CD39, the major vascular nucleoside triphosphate diphosphohydrolase (NTPDase), converts ATP and ADP to AMP, which is further degraded to the antithrombotic and anti-inflammatory mediator adenosine. Deletion of CD39 renders mice exquisitely sensitive to vascular injury, and CD39-null cardiac xenografts show reduced survival. Conversely, upregulation of CD39 by somatic gene transfer or administration of soluble NTPDases has major benefits in models of transplantation and inflammation. In this study we examined the consequences of transgenic expression of human CD39 (hCD39) in mice. Importantly, these mice displayed no overt spontaneous bleeding tendency under normal circumstances. The hCD39 transgenic mice did, however, exhibit impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism. Donor hearts transgenic for hCD39 were substantially protected from thrombosis and survived longer in a mouse cardiac transplant model of vascular rejection. These thromboregulatory manifestations in hCD39 transgenic mice suggest important therapeutic potential in clinical vascular disease and in the control of serious thrombotic events that compromise the survival of porcine xenografts in primates.

Genetic modification of pigs for solid organ xenotransplantation

Xenotransplantation of solid organs will only ever become a clinical reality with genetic modification of the pig, which is now widely accepted as the most likely donor species for humans. The understanding of the barriers to xenotransplantation has required advances in genetic technologies to resolve these problems. Hyperacute rejection has been overcome by overexpression of complement regulatory proteins or targeted disruption of the enzyme associated with the major carbohydrate xenoantigen. The subsequent barriers of disordered coagulation, induced antibody, and cell-mediated rejection remain challenging. The mechanisms for these incompatibilities are being deciphered, and multiple genetic manipulations to resolve these issues are currently in progress. Moreover, new technologies offer help to producing sizeable numbers of modified pigs in a timely manner. This article retraces the basis and foreshadows progress of the genetically modified pig for xenotransplantation as it advances toward the clinic.

Darbepoetin alfa administered monthly maintains haemoglobin concentrations in patients with chronic kidney disease not receiving dialysis: a multicentre, open-label, Australian study.

Aim:  Darbepoetin alfa, an erythropoiesis‐stimulating protein, has a longer serum half‐life than recombinant human erythropoietin, allowing less‐frequent administration. This study aimed to demonstrate that once‐monthly (QM) darbepoetin alfa administration would maintain haemoglobin (Hb) concentrations in subjects with chronic kidney disease (CKD) not receiving dialysis who had previously been administered darbepoetin alfa every 2 weeks (Q2W).

Hyperacute rejection of vascularized heart transplants in BALB/c Gal knockout mice

Abstract: Pig‐to‐primate vascularized xenografts undergo hyperacute rejection (HAR). This results from pre‐formed xenoreactive antibodies directed against galactose‐α1,3‐galactose (αGal) in the donor organ and activation of the complement cascade. We describe an in vivo murine model of HAR using a BALB/c mice system devoid of histocompatibility or complement differences between donor and recipient to investigate in isolation, the effects of αGal epitope and anti‐αGal antibody interactions in causing rejection of vascularized heart transplants. Gal KO mice were immunized with rabbit red blood cell membranes to induce high anti‐αGal antibody titers that were predominantly IgM by ELISA (enzyme‐linked immunosorbent assay). When αGal‐expressing mice hearts were transplanted heterotopically into these recipients (n = 12), 67% of grafts rejected within 24 h, the majority within 16 h with histological features of HAR. In contrast, none of the grafts in the non‐immunized Gal KO recipient control group (n = 11) underwent HAR. Interestingly, approximately 50% of the remaining grafts in both the immunized and non‐immunized Gal KO recipient group were rejected between 7 and 27 days by a rejection process characterized by a dense infiltrate of macrophage/monocytes, perivascular cuffing and tissue destruction similar to recent descriptions of delayed xenograft rejection (DXR). In addition, some grafts (21.5%) continued to survive in the immunized Gal KO recipients despite the presence of anti‐αGal antibody and normal complement activity and these showed well‐preserved myocardium when harvested whilst still functioning well at days 30 or 90. No rejection was seen when Gal KO donors were used in this system (n = 4), nor when αGal‐expressing BALB/c hearts were transplanted into αGal‐expressing BALB/c recipients (n = 5). This in vivo small animal model offers the opportunity to test a variety of strategies to overcome HAR prior to more resource intensive pig‐to‐primate studies, and may provide insights into the processes similar to DXR and accommodation.

Naturally acquired anti-αGal antibodies in a murine allograft model similar to delayed xenograft rejection

Abstract: Antibodies directed against galactose‐α1,3‐galactose (αGal) are believed to play an important a role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether α1,3‐galactosyltransferase‐deficient (Gal KO) mice can naturally acquire a sufficient anti‐αGal titre to cause the delayed type rejection of αGal‐expressing hearts. Gal KO mice of various ages were assessed for anti‐αGal antibody levels. αGal‐expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. Results: in Gal KO mice greater than 6‐month‐old, 64% had an anti‐αGal antibody titre above the background level. When wild‐type αGal‐expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti‐αGal antibodies with age. The titre in these mice was sufficient to cause a “delayed‐type” rejection of a significant proportion of αGal‐expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti‐αGal antibodies in the pathogenesis of DXR.

Fate of alphaGal +/+ pancreatic islet grafts after transplantation into alphaGal knockout mice.

Abstract:  Background:  Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose‐α1,3‐galactose (αGal) epitope on endothelial cell surfaces and therefore have preformed anti‐αGal antibodies. The role of these antibodies in rejection of non‐vascular xenografts remains controversial. This study investigared the role of anti‐αGal antibodies in rejection of non‐vascularized αGal+/+ grafts in αGal −/− mice.

Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation.

Thrombosis and inflammation are major obstacles to successful pig‐to‐human solid organ xenotransplantation. A potential solution is genetic modification of the donor pig to overexpress molecules such as the endothelial protein C receptor (EPCR), which has anticoagulant, anti‐inflammatory and cytoprotective signaling properties. Transgenic mice expressing human EPCR (hEPCR) were generated and characterized to test this approach. hEPCR was expressed widely and its compatibility with the mouse protein C pathway was evident from the anticoagulant phenotype of the transgenic mice, which exhibited a prolonged tail bleeding time and resistance to collagen‐induced thrombosis. hEPCR mice were protected in a model of warm renal ischemia reperfusion injury compared to wild type (WT) littermates (mean serum creatinine 39.0 ± 2.3 μmol/L vs. 78.5 ± 10.0 μmol/L, p < 0.05; mean injury score 31 ± 7% vs. 56 ± 5%, p < 0.05). Heterotopic cardiac xenografts from hEPCR mice showed a small but significant prolongation of survival in C6‐deficient PVG rat recipients compared to WT grafts (median graft survival 6 vs. 5 days, p < 0.05), with less hemorrhage and edema in rejected transgenic grafts. These data indicate that it is possible to overexpress EPCR at a sufficient level to provide protection against transplant‐related thrombotic and inflammatory injury, without detrimental effects in the donor animal.

Anti‐Gal antibody‐mediated skin graft rejection requires a threshold level of Gal expression

Abstract:  Background:  Despite overcoming xenograft hyperacute rejection (HAR), Gal (galactose‐α1,3‐galactose) expression may not be completely eliminated from the α1,3‐galactosyltransferase gene knockout (Gal KO) pig because of alternative galactosyltransferases. Whether low levels of “residual” Gal are still susceptible to either complement fixing or non‐complement fixing antibody beyond the HAR barrier remains unknown. Furthermore, it would be impossible to analyze the immune response specific to low‐level Gal in a xenograft setting given the multitude of xenoantigens that could induce a recipient response. To investigate this question, we therefore used a skin graft model in BALB/c mice where the sole difference between donor and recipient was the expression of Gal, where rejection is caused by passively administered anti‐Gal monoclonal antibody and where HAR does not occur.

Altered Glycosylation in Donor Mice Causes Rejection of Strain‐Matched Skin and Heart Grafts

Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO‐incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2‐fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell–mediated innate rejection crisis that affected 50–95% of the graft at 10–20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long‐term (>100 days). Experiments using “parked” grafts or MHC class II‐deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.

Efficacy of a non-vancomycin-based peritoneal dialysis peritonitis protocol

Background:  Peritonitis has a significant impact upon morbidity and mortality of peritoneal dialysis (PD) patients. Gram‐positive organisms account for the majority of infections and vancomycin is a cost effective broad‐spectrum antimicrobial treatment for PD peritonitis, but this may lead to the emergence of multiple antibiotic‐resistant organisms. The purpose of the present paper was to evaluate the efficacy of a non‐vancomycin‐based protocol comprising cephazolin and gentamicin, which was introduced in the present PD population as empirical treatment for peritonitis.