Anthony J. F. d’Apice

The impact of purinergic signaling on renal ischemia-reperfusion injury.

Background. Adenosine provides renovascular protection in mouse models of ischemia-reperfusion injury (I/RI) through purinergic members of the G protein-coupled receptor family, such as the adenosine 2A receptor (A2AR). Ectonucleotidases CD39 and CD73 are integral vascular and immune nucleotidases that regulate extracellular adenosine signaling. Current investigation of CD39 and CD73 in renal I/RI has primarily focused on their respective roles in ischemic preconditioning. Methods. In this study, we established a unilateral renal I/RI model and investigated the role of adenosine generation versus nucleotide removal in mediating protection in renal I/RI using mice deficient in CD39, CD73 or A2AR, thereby sequentially disrupting ectonucleotidase cascade and adenosinergic signaling. Results. Compared with wild-type mice, Cd73 null mice showed reduced levels of serum creatinine and urea, apoptosis of renal cells, and histologic damage after I/RI. Deletion of CD39 was associated with severe renal injury. Administration of apyrase, a soluble form of CD39, decreased global apoptosis and I/RI induced renal injury in wild-type mice. Apyrase treatment also improved renal histology to some extent in A2AR null mice. Conclusion. The relative protective effect of CD73 deletion in renal I/RI may reflect an effect of AMP accumulation. Deletion of CD39 showed deleterious effects and administration of soluble CD39 exerted renal protection, which is partially mediated by A2AR. The protective effect conferred by apyrase suggests that supplementing CD39 NTPDase activity may be a useful therapeutic strategy in renal transplantation.

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement

BACKGROUND It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

Gene-modified pigs

These days no one would consider doing a trial of pig-to-primate heart or kidney xenotransplantation without genetic modification (GM) of the donor. However, quite the opposite approach has become the norm in pancreatic islet cell xenotransplantation. Hering and Larsen and their associates have clearly shown that GM is not essential, having achieved >6 month reversal of diabetes in cynomolgus monkeys using genetically unmodified (GUM) pig islets [1,2]. This iconoclastic approach is probably due to them not being born and bred in the school of disappointment which has been xenotransplantation, with its focus hitherto on vascularized organs, hyperacute rejection (HAR) and acute vascular rejection. Consequently, they have not endured the last two decades’ struggle to overcome these barriers by a variety of genetic modifications aimed to de-pig and/or humanize the donor. In other words they are not burdened by the traditional baggage of the field. The islet graft equivalent of HAR is the instant blood-mediated inflammatory reaction (IBMIR) [3,4], and they were quite familiar with that from human islet allotransplantation. While a nuisance, it is not completely fatal; it and other peri-graft events just kill 70 to 80% of the islets. But, more importantly, 20 to 30% survive and function. With the unlimited supply of islets available from pigs, the absolute number of islets surviving can be brought up to a curative level simply by increasing the dose from about 10 000 IEQ/kg to about 50 000 IEQ/kg. The question then is: is there anything in the traditional baggage which might add to this rudely rampant pre-clinical success? The answer is undoubtedly a resounding: Yes! This confidence is based on three factors which are not addressed by the success of GUM islets. First, transplantation of islets, particularly in very large number into the liver via the portal circulation, risks portal vein thrombosis and portal hypertension [5,6]. GM has the potential to minimize both the number of islets required and the risk of thrombosis. Second, future recipients will include young juvenile-onset diabetics, often children who will face up to 70 to 80 yr of treatment if they are to be given a normal life expectancy. This will not be achieved by a transplant which requires any immunosuppressive regimen in use today, let alone those used in these successful pre-clinical studies and which are not yet clinic-ready, particularly as they employ an anti-CD154mAb. Wide clinical applicability of this therapy will necessitate an approach which ultimately has little or no systemic impact on the recipients, particularly as diabetes and immunosuppression are an unhappy blend. GM donors offer this possibility. Third, and as an extension of the second point, GM has far greater capacity than has been employed to date, and has the potential to make xenotransplantation not only successful but also superior to allotransplantation. This will flow from an array of new GM technologies and the fact that a pig can be GM while a human donor is GUM. In addition, pig islets are likely to be resistant to ongoing autoimmune disease, unlike alloislets.

Anti-Xenograft Immune Responses in α1,3-Galactosyltransferase Knock-Out Mice

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

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.

On the need for porcine embryonic stem cells to produce Gal KO pigs expressing multiple transgenes to advance xenotransplantation research

In reviewing the transgenesis literature in 2001, we concluded that while gene targeting and nuclear transfer of somatic cells bypassed the need for porcine embryonic stem cells (pESCs) to generate a1,3 galactosyltransferase knockout (Gal KO) pigs, the limited lifespan of these cells in culture prevented us from performing multiple rounds of homologous recombination to produce Gal KO pigs expressing multiple transgenes inserted as knockins in the space of one generation [1]. In particular, we concluded that a cell type ideally pESCs would still be required if such animals were to be produced without having to resort to generations of breeding and that this was a major technical hurdle because it had been suggested that as many as 10 genes may need to be examined before clinical trials of xenotransplantation could be contemplated [reviewed in 2]. Almost 10 years on from our initial review, the question needs to be asked: just how far are we from achieving this goal?

Overcoming Hyperacute Xenograft Rejection With Transgenic Animals

SummaryXenotransplantation offers an alternative source of organs to solve the current critical shortage of donor organs required for patients with end-stage kidney, heart and liver disease. For social, ethical and logistical purposes, pigs appear to be the most appropriate donor animal.The immunological barriers to xenotransplantation are greater than in allotransplantation because of the presence of preformed natural antibodies in the serum of the recipient. The rapid binding of antibody to donor endothelial cells is followed by complement activation, cell damage and vascular thrombosis. Antirejection therapies aimed at reducing the level of antibody, complement activity and cell-mediated immunity in the recipient may result in a significant increase in complications such as infections and malignancies compared with allotransplantation. Transgenic technology may permit modification of the donor organ, enabling it to evade the rapid antibody- and complement-mediated destruction.The main strategies to prevent xenotransplant rejection have been to reduce expression of ‘Gal’, the major target epitope for natural antibody, and to inhibit complement activation. Transgenic animals expressing membrane-bound inhibitors of the complement pathway and enzymes that compete for Gal synthesis have been generated. Both approaches provide limited protection, and preliminary experiments in vitro suggest that a combination approach may reduce antibody- and complement-mediated cellular damage.