Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Successful ABO-incompatible liver transplantation using A2 donors.

INTRODUCTION: The longer waiting time for a liver graft among patients with blood group O makes it necessary to expand the donor pool for these patients. We herein have reported our experience with ABO-incompatible liver transplantation using A(2) donors to blood group O recipients. PATIENTS AND METHODS: Between 1996 to 2005, 10 adult blood group O recipients received 10 A(2) cadaveric grafts. Mean recipient age was 52 +/- 7.7 years (mean +/- SD). The initial immunosuppression was induction with antithymocyte globulin (n = 2), interleukin-2-receptor antagonists (n = 3), or anti-CD20 antibody (rituximab, n = 1), followed by a tacrolimus-based protocol. No preoperative plasmapheresis, immunoadsorption, or splenectomies were performed. RESULTS: Patient and graft survival was 10/10 and 8/10, respectively, at 8.5 months median follow-up (range 10 days to 109 months). Two patients were retransplanted because of bacterial arteritis (n = 1) and portal vein thrombosis (n = 1). The six acute rejections, which occurred in four patients, were all reversed by steroids or increased tacrolimus dosages. The pretransplant anti-A titers against A(1) red blood cells were 1:128 (NaCl technique) and 1:8 to 1024 (IAT technique). The maximum postoperative titers were 1:64 to 4000 (NaCl) and 1:256 to 32000 (IAT). CONCLUSION: The favorable outcome of A(2) to O grafting, with a patient survival of 10/10 and graft survival of 8/10, makes it possible to consider this blood group combination also in nonurgent situations. There was no hyperacute rejection or increased rate of rejections. Anti-A/B titer changes seem to not play a significant role in the monitoring of A(2) to O liver transplantation.

Characterization of the humoral immune response in two paediatric patients transplanted with split livers from ABO-incompatible living-related donors: appearance of cytomegalovirus-induced ABO antibodies.

Two blood group O paediatric patients, 12 and 6 months old, were transplanted with liver segments from their blood group A2Le (a(-)b+) Se and blood group A1Le (a(-)b+) Se fathers, respectively. Recipient anti-A antibody titres were reduced prior to transplantation by blood exchange. Both patients had rejection episodes in the post-transplant period that were reversed by anti-rejection therapy. No anti-A antibody titre rise occurred concomitant with these rejections. Postoperatively both patients had cytomegalovirus (CMV) infections, and simultaneous with these infections, a strong increase in anti-A antibody titres was seen, but no rejection occurred. The anti-A antibody titre increase seemed to be specific for A antigens, because the anti-B and anti-alphaGal (anti-pig) antibody titres did not show any changes. CMV infection is a serious cause of morbidity and mortality in immunosuppressed patients, and the virus can influence glycosylation of infected cells. Whether this can explain the importance of the infection in relation to the increase in titre remains to be elucidated.

Histo-blood group A antigen expression in pig kidneys--implication for ABO incompatible pig-to-human xenotransplantation.

OBJECTIVE: There is a relative shortage of donor organs for clinical transplantation, and the use of animal organs is being considered. A clinical trial was performed connecting pig kidneys to the circulation of a dialysis patient. MATERIAL AND METHODS: A pig kidney was, after plasmapheresis, extracorporeally connected to the circulation of a volunteer dialysis patient. The patient was of blood group B and the pig of blood group A. RESULTS: The experiment gave rise to a strong humoral immune response where the xenoantibodies were shown to be of immunoglobulin G (IgG), IgM and IgA immunoglobulin classes, recognizing mainly the Gal alpha1-3Gal epitope and the anti-A antibodies was exclusively of IgM type, recognizing the blood group A trisaccharide. Immunohistological examinations of blood group A pig kidneys revealed that blood group A antigens are located in the distal tubules, thin and thick tubules of Henle and the epithelium of the collecting ducts but absent in proximal tubules, glomeruli, large vessels and capillaries. In the perfused kidney, a patchy destruction of tubular cells was found and these segments stained positive for blood group A antigens and had a codeposition of human IgM antibodies and complement components. Tubular segments which were apparently normal were all negative for blood group A antigens but strongly expressed the Gal alpha1-xenoantigen. CONCLUSION: In this patient, challenged simultaneously with carbohydrate antigen epitopes representing both the ABO and the xenobarrier, the humoral immune response differed concerning the immunoglobulin classes induced. The low remaining anti-A titre after plasmapheresis was probably sufficient to cause destruction of A antigen-positive tubular cells, while the corresponding Gal alpha1-xenoantigen-positive cells were structurally intact. This case confirms that in future xenotransplantation, matching for the ABO system has to be undertaken in the same way as in human allotransplantation.

Lack of antibody production against Hanganutziu-Deicher (H-D) antigens with N-glycolylneuraminic acid in patients with porcine exposure history.

The significance of non-alphagalactosyl antigens remains unclear in pig-to-primate xenotransplantation. Hanganutziu-Deicher (H-D) antigens with terminal N-glycolylneuraminic acid (NeuGc) are widely expressed on endothelial cells of mammalian species, with the exception of humans. As baboons and monkeys also express H-D antigens, a pig-to-non-human primate experimental model cannot resolve the question of whether H-D antigens can elicit a potent humoral response in human recipients. The purpose of this study was to elucidate the clinical significance of H-D antigens by examining the sera from patients who have been previously exposed to porcine tissue. After the digestion of porcine aortic endothelial cells (PAEC) by neuraminidase, NeuGc and N-acetylneuraminic acid (NeuAc) were quantitated by HPLC. IgG and IgM antibody levels against H-D antigens were measured by NeuGc-GM3-coated ELISA plates in the sera of patients who had undergone ex vivo kidney perfusion 1 to 3 weeks and 2 years previously (n=2) or had been injected with fetal porcine islets 2 months previously (n= 10). HPLC determined that 9.7x 10(7) NeuAc and 6.3x 10(7) NeuGc residues per cell were released from PAEC by neuraminidase, while 25.7x 10(7) NeuAc and an undetectable level of NeuGc were released from human aortic endothelial cells (HAEC). No significant elevation of IgG or IgM antibody levels against NeuGc-GM3 was observed in sera from patients with a history of porcine exposure. Considering the active production of antibody against the foreign galactosyl antigens after pig-to-human xenotransplantation, some production of antibodies against the equally foreign H-D antigens would be expected, because large amounts of NeuGc terminated saccharides are present in the pig endothelial cell surface. However, no production of antibodies directed to H-D antigens could be found in patients exposed to porcine tissue. Further studies are warranted to explain why H-D antigens do not elicit a significant antibody production.

Distribution of the Galalpha1-3Gal antigen in cultured adult and fetal porcine pancreatic islet cells: an immunoelectron microscopic study.

The distribution of the Galalpha1-3Gal antigen (Galalpha) in cultured adult porcine islets (API) and fetal porcine pancreatic islet-like cell clusters (ICC) was studied using immunoelectron microscopy. API and ICC were cultured for 1 and 5 days, respectively, and immunogold labeled using human affinity isolated anti-Galalpha1-3Gal antibody, GS-IB4 lectin and antibodies against islet pancreatic hormones, vimentin, and von Willebrand factor. Differentiated endocrine cells were Gala-negative, but, in ICC, some immature endocrine cells were slightly Gala-positive. The Gala-expression in API was much weaker compared to ICC. In both API and ICC, the Gala antigen was expressed on duct epithelial cells, acinar cells, and endothelial cells. In ICC, strong Gala expression was observed on flattened cells covering their surfaces. These cells were identified as centroacinar cells originating from intra-islet ducts. In conclusion, although mature endocrine cells of cultured API and ICC lack the Gala-xenoantigen, several other cellular compounds are strongly Gala positive, which may contribute to xenorejection of these grafts.

Blocking of human anti-pig xenoantibodies by soluble GAL alpha 1-3Gal and Gal alpha 1-2Gal disaccharides; studies in a pig kidney in vitro perfusion model.

Depletion of anti-pig xenoantibodies reduces cell cytotoxicity of human serum to pig endothelial cells and lymphocytes. The aim of this study was to test, in a pig kidney xenoperfusion model, the ability of soluble alpha Gal terminated disaccharides to prevent the hyperacute rejection process in an organ. Porcine kidneys were perfused with whole human blood lacking saccharide and blood supplemented with Gal alpha 1-3GAL, Gal alpha 1-2Gal and lactose. Parameters evaluated were, urine production, renal blood flow, vascular resistance, renal clearance, blood cell counts, xenoantibody titers, complement activation and histopathology. The blood flow was higher in the Gal alpha 1-3Gal (155 +/- 31 ml/min x 100 g-1 kidney tissue) group compared to Gal alpha 1-2Gal (138 +/- 16), lactose (92 +/- 78) and controls (69 +/- 16), When calculated as percent of the blood flow value at 1 min, the blood flow at 30 min was 157% for the Gal alpha 1-3Gal and for 187% the Gal alpha 1-2Gal. The corresponding values for the lactose and control groups were 102% and 74%, respectively. Urine production in the lactose/control groups was lower (0.7 ml/min x 100 g-1 kidney tissue) compared to Gal alpha 1-3Gal (3.0) and Gal alpha 1-2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Gal alpha alpha 1-groups during the perfusions. An increase in urine potassium excretion was found in the Gal alpha alpha 1-groups while a reduction occurred in the lactose/control experiments. An initial 40-50% reduction in platelet count was observed in all groups while the leukocyte count showed a continuous decrease. Immunohistochemistry revealed less deposition of IgM, IgG, C3 and C1 q in the Gal alpha alpha 1-saccharide groups compared to the lactose/control groups. Soluble Gal alpha a1-disaccharides improved both functional and histological parameters. However, significant pathological changes were still present indicating that this approach to inhibit HAR must be used in combination with additional therapeutic approaches such as solid phase xenoantibody immunoadsorption and blocking of complement activation.

alpha-Gal epitopes in animal tissue glycoproteins and glycolipids.

alpha-Gal terminated saccharides are present on the cell surface both as glycolipids and glycoproteins in all mammals except Old World monkeys and humans. The structural diversity among identified saccharides terminated by this epitope in animal tissues is steadily increasing. The majority of these saccharides have the alpha-Gal linked to lactosamine but other core saccharides exist. The alpha-Gal terminated saccharides are recognized by the immune system as a specific antigen and antibodies directed to the alpha-Gal, which do not cross-react with the classic blood group B trisaccharide, are found in man and Old World monkeys. Similar to other complex carbohydrate cell surface antigens, the alpha-Gal epitope is heterogeneously distributed in different organs and in different cells within an organ. It is present on the vascular endothelium and it is the primary target for human naturally occurring antibodies following pig to primate/man xenotransplantation leading to hyperacute rejection of the graft. Important for the future will be to further structurally characterize this antigen system, its cellular/subcellular distribution, and to identify possible of additional glycosyltransferases, related to the already described alpha 1,3galactosyltransferase that may explain the structural diversity. Such information will be of importance in the studies of, for example, the pathogenesis of autoimmune diseases and for the production of genetically modified pigs to prevent xenograft rejection.

Physiologic incompatibilities in discordant xenotransplantation.

At present, several incompatibilities between pig and human, not only from the immunologic point of view but also regarding physiological and molecular systems, have been identified. It is anticipated that this is only the tip of the iceberg regarding this topic. However, there are also many systems that are compatible. Strategies have been outlined, and many tools, such as gene modification, are available to solve the problems. Therefore, the statement by Keith Reemtsma in the foreward to the latest issue of the monography Xenotransplantation "The important question is not whether xenotransplantation will succeed, but rather how and under what circumstances xenografts will provide predictable enough results to warrent clinical application" will hopefully be a reality in the future.

Extracorporeal ("ex vivo") connection of pig kidneys to humans. III. Studies of plasma complement activation and complement deposition in the kidney tissue.

The complement system is one of the important factors involved in the hyperacute rejection of xenografts. This report deals with the activation of the complement system in a clinical trial where pig kidneys were extracorporeally connected to two volunteer dialysis patients who were pretreated with plasmapheresis in order to substantially reduce anti-pig xenoantibodies. The clinical data of the perfusion experiments and the patients humoral immune response to pig xenoantigens have been reported in detail (Xenotransplantation 1996; 3:328-339, 340-353). Three consecutive daily plasmapheresis treatments of the patients reduced the plasma complement protein (C3, C4, and C5) concentrations to 8-27% of the baseline values. The perfusion of the pig kidney connected to patient 1 was terminated at 65 min due to graft rejection and this patient was not hemodynamically affected by the experiment. The second experiment was terminated at 15 min due to an anaphylactic like reaction of the patient. In patient 1 a slight reduction of plasma C3, C4, and C5 and an increase of C5a and SC5b-9 occurred, while C3a decreased during the perfusion. Patient 2 had an increase of all complement parameters, most prominent for C4d and SC5b-9, which occurred concomitant with the appearance of the anaphylactic like side effects. In general, plasma levels of PMN elastase, IL6 and IL8 increased in both patients during the perfusion. Immunohistochemical investigation of the kidney tissues revealed deposition of human complement factors C1q, C4c, and C3c in a congruent pattern with the vasculature of the kidney in patient 1. In kidney 2 only trace amounts of C1q and C3c were found. Both kidneys were negative for properdin. Therefore, in this experimental set up with extracorporeal connection of pig kidneys to the human circulation the human complement cascade is activated mainly through the classical pathway.

Heterogeneous expression of Gal alpha1-3Gal xenoantigen in pig kidney: a lectin and immunogold electron microscopic study.

BACKGROUND: The Gal alpha1-3Gal antigen (Gal alpha) is the primary target for human natural anti-pig xenoantibodies. The presence of Gal alpha has been shown in porcine endothelial cells (ECs) using light microscopy, whereas the expression of Gal alpha in other cell structures in the porcine kidney is only partially characterized. METHODS: Immunogold electron microscopy of pig kidney cryosections was performed using Griffonia simplicifolia isolectin B4 and affinity isolated human anti-Gal alpha1-3Gal antibodies. RESULTS: The most intense expression of Gal alpha was found on the apical and basolateral portions of the plasma membrane of the proximal convoluted tubule segments 1 and 2 cells, whereas segment 3 and 4 cells were negative. A strong staining was found in peritubular capillary ECs and in the inner medullary and papillary collecting duct cells. Moderate labeling of ECs and subendothelium was observed in large blood vessels, whereas glomerular ECs reacted weakly. Additionally, glomerular parietal epithelial cells, connecting tubule cells, and some cortical collecting duct cells were labeled. Among interstitial cells, a part of type-1 cells and all type-2 cells were labeled, whereas others were negative. CONCLUSIONS: By immune electron microscopy, a detailed information of the Gal alpha antigen distribution in porcine nephrons and blood vessels has been revealed, which clarifies conflicting data obtained by light microscopy. In addition, expression of the Gal alpha antigen in the renal interstitial cells was documented for the first time. These data are of importance for the understanding of xenoantibody-mediated hyperacute rejection, for interpretation of pig kidney xenograft biopsies, and for generating transgenic pigs lacking the Gal alpha epitope.