The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914.

BACKGROUND: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. METHODS: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. RESULTS: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. CONCLUSIONS: HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.

Anti-non-Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF-transgenic porcine hearts in cynomolgus monkeys.

BACKGROUND: Anti-Gal alpha 1-3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine-to-nonhuman primate transplantation. The role of anti-non-Gal antibodies in AHXR is less well defined. METHODS: Eleven cynomolgus monkeys received a heterotopic heart transplant from a human decay-accelerating factor transgenic pig, and maintenance immunosuppression with cyclosporin A or tacrolimus, steroids, mycophenolate sodium or mycophenolate mofetil, and in 10 animals the Gal-containing soluble glycoconjugate GAS914. Six ended with AHXR (6 to 78 day survival) and five did not show AHXR (9 to 36 day survival). Anti-Gal antibodies were depleted in vivo with GAS914, or in vitro with Gal-coated Sepharose beads. IgM- and IgG-class anti-non-Gal antibodies in serum depleted of anti-Gal antibodies were measured by flow cytometry using porcine endothelial target cells. RESULTS: Compared with pre-transplant values, all six recipients with AHXR showed a substantially higher level of anti-non-Gal IgM antibodies at rejection; in five animals there was also an increase in IgG-class antibodies. There was no relevant change in recipients without AHXR. AHXR at time of cessation of heart contraction could be preceeded by a steady increase in antibody level starting 2 to 3 weeks earlier. CONCLUSIONS: AHXR is invariably associated with increased circulating anti-non-Gal antibodies. These antibodies are not observed in recipients without AHXR, and five of six recipients with AHXR were adequately depleted of anti-Gal antibodies by maintenance GAS914. This indicates that anti-non-Gal antibodies play a significant role in the pathogenesis of AHXR. Also, the assessment of these antibodies could be used as an early monitor of AHXR.

Hemodynamic and hormonal responses to the sudden interruption of caval flow: insights from a prospective study of hepatic vascular exclusion during major liver resections.

UNLABELLED: Hepatic vascular exclusion (HVE) combines portal triad clamping and occlusion of the inferior vena cava. Although HVE has been performed for major liver resections during the last 2 decades, little is known about the mechanisms that explain its satisfactory hemodynamic tolerance. Consequently, we performed a comprehensive study of both hemodynamic and hormone responses to HVE. Twenty-two patients who underwent liver resection for secondary tumors developed in noncirrhotic livers were prospectively studied. Heart rate, arterial blood pressure, pulmonary artery pressure, mixed venous saturation, cardiac output, and left ventricular dimensions determined by transesophageal echocardiography were monitored in HVE patients. Blood concentrations of arginine vasopressin (AVP), epinephrine, norepinephrine, dopamine, and atrial natriuretic peptide and plasma renin activity (PRA) were measured before clamping; 5, 15, and 30 min after clamping; and 15 min after unclamping. Hemodynamic response to HVE was characterized by a significant (P < 0.05) decrease in left ventricular dimensions, fractional area change, and pulmonary artery pressure. We also observed a marked decrease in cardiac output (50%) and an increase in heart rate and systemic vascular resistance. After unclamping, there was peripheral vasodilation, assessed by a significant decrease in systemic vascular resistance from the preclamping value to unclamping. An acute and sustained increase in AVP and norepinephrine that returned to baseline after unclamping and the absence of modification in PRA concentrations were noted. The marked decrease in venous return that characterizes HVE is compensated for by an increase in vascular resistance secondary to an important activation of the AVP and sympathetic systems. The PRA system does not play an important role in maintaining arterial blood pressure during HVE. IMPLICATIONS: Hemodynamic and hormonal responses to the acute interruption of caval venous return to the heart were investigated in patients undergoing liver resection with hepatic vascular exclusion. A compensatory role for arginine vasopressin and sympathetic systems that provoked increased vascular resistance was demonstrated.