Atousa Aminian

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.

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.

Knock out of alpha1,3-galactosyltransferase or expression of alpha1,2-fucosyltransferase further protects CD55- and CD59-expressing mouse hearts in an ex vivo model of xenograft rejection.

BACKGROUND Organs from transgenic animals with high-level endothelial expression of the human complement regulatory factors CD55 and CD59 are significantly protected from human complement-mediated injury. Elimination or reduction of the major xenoepitope alphaGal, achieved by knocking out the alpha1,3-galactosyltransferase gene (Gal KO) or expressing human alpha1,2-fucosyltransferase (H transferase or HTF), also affords protection, although to a lesser degree. In this study, we examined whether the protection provided by strong CD55 and CD59 expression can be augmented by the Gal KO or HTF modifications. METHODS Hearts from four groups of mice (wild type, CD55/CD59, CD55/CD59/HTF, and CD55/CD59/Gal KO) were perfused ex vivo with 40% human plasma. Mean heart work for each group was compared over a 60-min period. RESULTS Wild-type hearts ceased to function effectively within 15 min of plasma addition. CD55/CD59 hearts displayed prolonged survival and maintained approximately 10% maximum work at the end of perfusion. Introduction of Gal KO or HTF onto the CD55/CD59 background resulted in a further prolongation, with work maintained at 20-30% of the maximum level. CONCLUSIONS We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

Transgenic expression of human α1,2-fucosyltransferase (H-transferase) prolongs mouse heart survival in an ex vivo model of xenograft rejection

BACKGROUND The expression of human alpha1,2-fucosyltransferase (H-transferase, HT) has been proposed as an alternative strategy to alpha1,3-galactosyltransferase (GT) gene knockout, which is not currently feasible in pigs, to reduce the galactose-alpha1,3-galactose (Gal) epitope expression. HT expression has recently been shown in transgenic mice and pigs to significantly reduce Gal expression on a variety of cells; however, its ability to do so on endothelial cells and its effectiveness at prolonging xenograft survival are yet to be determined. METHODS HT-transgenic, Gal knockout (Gal KO) mice, and mice containing both genetic modifications (HT-transgenic/Gal KO) were tested for H-substance and Gal expression on splenocytes and endothelial cells by flow cytometric analysis. In addition, the hearts of these mice were perfused ex vivo with 6% human plasma, and the effect on cardiac function was determined. RESULTS AND CONCLUSION H-substance expression was detected on both splenocytes and endothelial cells of HT-transgenic mice. The level of H-substance expression was not affected by the presence or absence of GT in the cells, consistent with HT being dominant over GT. The ability of HT expression to reduce Gal expression was highly variable depending on the cell type. Gal expression on splenocytes was almost completely eliminated, whereas on endothelial cells, substantial Gal remained despite a 70% reduction. When perfused ex vivo with human plasma, hearts from HT-transgenic, Gal KO, and HT-transgenic/Gal KO mice demonstrated a similar prolongation in survival, compared with wild-type controls. Therefore, as far as hyperacute rejection is concerned, HT expression may be as effective as Gal KO in protecting against xenoantibody and complement mediated injury. However, the effect of residual Gal on non-hyperacute rejection responses remains to be determined.

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.

High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone

Abstract: High‐level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement‐mediated injury in an ex vivo perfusion model. In this study we examine whether co‐expression of CD55 provides additional protection. CD55/CD59 double‐transgenic mice were generated by co‐injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM‐2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild‐type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high‐level endothelial co‐expression of CD55 and CD59 provides greater protection from human complement‐mediated injury in this model than expression of CD59 alone.

High-level Endothelial Expression Of Human Cd59 Prolongs Heart Function In An Ex Vivo Model Of Xenograft Rejection

BACKGROUND Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

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.

Involvement Of Both The Classical And Alternate Pathways Of Complement In An Ex Vivo Model Of Xenograft Rejection

BACKGROUND It is now generally accepted that complement activation is critical for the hyperacute rejection of xenografts. Activation of the classical pathway as the result of the interaction of xenoreactive IgM xenoantibodies with the vascular endothelium has been observed in all species combinations examined to date. A number of studies using a variety of species combinations have also implicated alternate pathway involvement; however, these studies do not enable a conclusion to be drawn as to whether the alternate pathway can be activated in the complete absence of classical pathway activation. METHODS In this study, human plasma was depleted of both Clq and factor D and then reconstituted with purified Clq or factor D to restore the classical and alternate complement pathways, respectively. The ability of these modified plasmas to prosecute hyperacute rejection was then examined using an ex vivo isolated mouse heart perfusion model based on the Langendorff system. RESULTS AND CONCLUSIONS In the mouse to human species combination, both the classical and alternate pathways of complement are independently capable of initiating complement activation and mediating xenograft rejection.

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.