Helen Barlow

Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons.

BACKGROUND The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. METHODS Transgenic pigs were produced by microinjection of gene constructs for human complement regulatory proteins CD55 and CD59 and the enzyme alpha1,2-fucosyltransferase (H-transferase, HT), which reduces expression of the major xenoepitope galactose-alpha1,3-galactose (alphaGal). Kidneys from CD55/HT and CD55/CD59/HT transgenic pigs were transplanted into nephrectomised, nonimmunosuppressed adult baboons. RESULTS In several lines of transgenic pigs, CD55 and CD59 were expressed strongly in all tissues examined, whereas HT expression was relatively weak and did not significantly reduce alphaGal. Control nontransgenic kidneys (n=4) grafted into baboons were hyperacutely rejected within 1 hr. In contrast, kidneys from CD55/HT pigs (n=2) were rejected after 30 hr, although kidneys from CD55/CD59/HT pigs (n=6) maintained function for up to 5 days. In the latter grafts, infiltration by macrophages, T cells, and B cells was observed at days 3 and 5 posttransplantation. The recipients developed thrombocytopenia and abnormalities in coagulation, manifested in increased clotting times and an elevation in the plasma level of the fibrin degradation product D-dimer, within 2 days of transplantation. Treatment with low molecular weight heparin prevented profound thrombocytopenia but not the other aspects of coagulopathy. CONCLUSIONS Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Changes In Cell Surface Glycosylation In α1,3-galactosyltransferase Knockout And α1,2-fucosyltransferase Transgenic Mice

BACKGROUND Inactivation of the alpha1,3-galactosyltransferase (GalT) gene by homologous recombination (knockout [KO] mice) and competition for the enzymes N-acetyllactosamine substrate by transgenically expressed alpha1,2-fucosyltransferase (H-transferase) are two genetic approaches to elimination of the Gal alpha1,3Gal (alphaGal) epitope, which is the major xenoantigen in pigs against which humans have preformed antibodies. Such genetic manipulations often have unpredictable results. METHODS A panel of 19 selected lectins was used to characterize the changes in cell surface glycosylation in GalT KO and H-transferase transgenic mice, compared with nontransgenic littermate controls. RESULTS GalT KO mice showed complete elimination of the alphaGal epitope, as reported previously. Surprisingly, however, this was associated with only a modest increase in N-acetyllactosamine residues and had little other effect on the pattern of lectin binding. In contrast, the pattern of lectin binding to H-transferase transgenic mouse cells was more profoundly disturbed and indicated, in addition to the expected expression of H substance and suppression of the alphaGal epitope, that there was a marked reduction in alpha2,3-sialylation and exposure of the normally cryptic antigens, sialylated Tn and Forssman antigens. Similar changes in lectin reactivity with porcine aortic endothelial cells were induced by neuraminidase treatment. CONCLUSIONS Lectins were able to bind underlying carbohydrate structures (sialylated Tn and Forssman antigens) that are normally cryptic antigens on H-transferase transgenic mouse spleen and cardiac endothelial cells, probably as a consequence of the reduction in the electronegativity of the cell surface due to reduced sialylation. As humans have preformed anti-Tn and anti-Forssman antibodies, it is possible that these structures may become targets of the xenograft rejection process, including hyperacute rejection.

Protective Effects of Recombinant Human Antithrombin III in Pig-to-Primate Renal Xenotransplantation

Delayed rejection of pig kidney xenografts by primates is associated with vascular injury that may be accompanied by a form of consumptive coagulopathy in recipients. Using a life‐supporting pig‐to‐baboon renal xenotransplantation model, we have tested the hypothesis that treatment with recombinant human antithrombin III would prevent or at least delay the onset of rejection and coagulopathy. Non‐immunosuppressed baboons were transplanted with transgenic pig kidneys expressing the human complement regulators CD55 and CD59. Recipients were treated with an intravenous infusion of antithrombin III eight hourly (250 units per kg body weight), with or without low molecular weight heparin. Antithrombin‐treated recipients had preservation of normal renal function for 4–5 days, which was twice as long as untreated animals, and developed neither thrombocytopenia nor significant coagulopathy during this period. Thus, recombinant antithrombin III may be a useful therapeutic agent to ameliorate both early graft damage and the development of systemic coagulation disorders in pig‐to‐human xenotransplantation.

Control of IBMIR in neonatal porcine islet xenotransplantation in baboons.

The instant blood‐mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose‐α1,3‐galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti‐thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal‐deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet‐poor plasma. Recipients of GM NICC developed a variable anti‐pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell‐mediated rejection, although scattered insulin‐positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long‐term graft survival may require more effective immunosuppression or further donor genetic modification.

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/μl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/μl and then decreased at 10 ng/μl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/μl to maximise integration and efficiency rates in pigs.

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice.

BACKGROUND Complement activation plays a pivotal role in hyperacute xenograft rejection. In humans, activation of complement is regulated by a number of cell surface regulatory proteins. Membrane cofactor protein (CD46) is one such regulator that protects cells by acting as a cofactor for the factor I-mediated cleavage of C3b and C4b. Transgenic animals expressing human CD46 may provide organs that are resistant to complement attack. However, attempts to generate mice expressing human CD46 using cDNA-based constructs have been largely unsuccessful. METHODS Transgenic mice expressing a glycosylphosphatidyl inositol (GPI)-linked form of CD46 were generated by microinjection of a hybrid CD46/CD55 cDNA under the control of the human intercellular adhesion molecule-2 promoter. Expression of CD46-GPI on the vascular endothelium was determined by immunohistochemistry. The ability of CD46-GPI to protect mouse tissues from human complement attack was determined using an ex vivo isolated perfused heart model. RESULTS Three founder animals expressing CD46-GPI were identified. Histological analysis showed strong and uniform expression of CD46-GPI on the vascular endothelium of all organs examined. Ex vivo perfusion of transgenic mouse hearts with human plasma showed a reduction in C3c deposition and a slightly prolonged function compared with controls. CONCLUSIONS High-level expression of CD46-GPI was achieved in transgenic mice by using a modified cDNA-based construct. The CD46-GPI was functional, providing some protection from complement-mediated damage in the ex vivo model, and may be useful in xenotransplantation if expressed in combination with CD55 and CD59.

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.

Long-Term Function of Genetically Modified Porcine Neonatal Islet Xenografts in Baboons

Introduction In xenotransplantation the stringent pig-to-baboon islet xenograft preclinical model is a difficult one with the maximum period of normoglycemia reported to only 1 day. Previously we have shown that neonatal islet cell clusters (NICC) from GTKO/CD55-CD59-HT pigs prevent both hyper acute rejection (HAR) and the instant blood mediated inflammatory response (IBMIR) in immunosuppressed baboons. However, the maturation and function of the NICC were not examined in detail in the initial study, because (i) the recipients were not diabetic, and (ii) the standard clinical-based immunosuppression used was insufficient to prevent biopsy-proven rejection within 1 month. In the current study, therefore, we used streptozotocin-induced diabetic recipients and changed to a costimulation blockade-based immunosuppressive regimen. Aims To achieve long-term normoglycemia in diabetic baboons transplanted with neonatal pig islets, and to investigate the effect of ceasing immunosuppression. Materials and Methods: Five diabetic baboons received intraportal infusion of NICC (10,000-50,000 IEQ/kg) from 1-5 day old GTKO/CD55-CD59-HT piglets. From day -3 recipients were treated with anti-CD2 induction and subsequently maintenance with oral tacrolimus, anti-CD154 and belatacept, which were progressively ceased. Graft survival and function was followed by daily blood sugar levels (BSL), IVGTT, OGTT and immunohistochemical analysis of liver biopsies taken at various time points over the study period. Results and Discussion None of the baboons exhibited signs of thrombosis associated with IBMIR, with no change to platelet counts, vWF, fibrinogen or D-dimer levels from baseline. Recipients developed normal fasting BSL and had normal IVGTT and OGTT, with porcine insulin and C-peptide secreted in response to glucose bolus stimulus. All animals have become normoglycaemic off all exogenous insulin. Liver biopsies revealed strong positive staining for insulin, glucagon and somatostatin in xenografts. One recipient receiving 50,000 IEQ/kg was insulin-independent for >7 months, including 7 weeks after the last drug (belatacept) was ceased. A second recipient receiving 10,000 IEQ/kg took longer to achieve insulin independence, but remained insulin independent >18 months, including 6-months off all immunosuppression. The fourth and fifth animals are being followed out past 6-months and 2-months post transplant. Conclusion We have confirmed that GTKO/CD55-CD59-HT neonatal islets are protected from IBMIR in the baboon model. More importantly, we have demonstrated for the first time long-term survival and function of porcine islets in baboons. The costimulation blockade-based immunosuppression permitted maturation of the islets such that the dose required to achieve normoglycemia (10,000 IEQ/kg) was lower than that reported in any NHP model. Remarkably, this recipient remained normoglycemic for 6 months after immunosuppression was ceased. Bristol-Myers Squibb Company for donation of Belatacept,. Keith Reimann at NIH Nonhuman Primate Reagent Resource for supply of Anti CD154 antibody.