Eugen Koren

Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation.

Pig tissues were screened by immunofluorescence with lectins, mAb, and human natural antibodies for the presence of carbohydrate antigens, which may be potential targets for hyperacute vascular rejection in pig to man xenotransplantation. The unfucosylated monomorph linear B-antigen was found at the surface of all porcine vascular endothelial cells. This pig linear-B antigen reacts strongly with the anti-aGal isolectin B4 from Griffonia simplicifolia 1 and with human natural anti-αGal antibodies specifically purified by affinity chromatography on synthetic oligosaccharides containing the terminal nonreducing αGall±3βGal-R disaccharide. This antigenic activity is destroyed by treatment of pig tissues with α-galactosidase. The localization of this linear-B epitope on vascular endothelium and its reactivity with natural human anti-aGal antibodies suggest that it may play a major role in the hyperacute vascular rejection of pig to man organ xenografts. The lectin from Maackia amurensis reacting with αNeuAc2±3βGall±4GlcNAc/Glc was also positive on pig vascular endothelium, but we do not know yet whether there are human natural antibodies reacting with the carbohydrate recognized by this lectin. Epithelial cells of pig renal proximal convoluted tubules, respiratory epithelium, pancreatic ducts, and epidermis express the linear-B antigen, but they are less likely to trigger a hyperacute vascular rejection because they are not directly exposed to the blood. The genetically defined pig A+/A− system controls the expression of A and H antigens in pig epithelial cells from renal distal and collecting tubules, biliary ducts, pancreatic ducts, large bronchi, and digestive mucosa. The pig A antigen may trigger an immune response in human O or B recipients if they are transplanted with organs from A+ pigs, but the pig A antigen is probably not involved in the hyperacute vascular rejection of a xenograft because it is not expressed on vascular endothelium.

Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells.

Autoantibodies to ribosomal P-proteins are present in 12-16% of patients with systemic lupus erythematosus and are associated with neuropsychiatric disease. As the ribosomal P proteins are located in the cytoplasm, the pathogenic effects of their cognate autoantibodies are unclear. In this study affinity-purified anti-P autoantibodies were used to explore the cell surface of several types of human and animal cells. Immunofluorescence as well as EM immunogold analysis demonstrated, on the surface of human hepatoma cells, the presence of an epitope that is antigenically related to the immunodominant carboxy terminus of P-proteins. The presence of this epitope was also demonstrated on the surface of human neuroblastoma cells and, to a lesser extent, on human fibroblasts. Furthermore, the Western blot technique revealed in purified human and animal plasma membranes a 38-kD protein that is closely related or identical with ribosomal P0 protein. The availability of reactive P peptide on the surface of cells makes possible the direct effect of autoantibodies on the function and viability of cells that express this antigenic target. This delineates one of the possible impacts of anti-P antibodies in disease expression.

EVIDENCE THAT INTRAVENOUSLY ADMINISTERED α-GALACTOSYL CARBOHYDRATES REDUCE BABOON SERUM CYTOTOXICITY TO PIG KIDNEY CELLS (PK15) AND TRANSPLANTED PIG HEARTS

Methods of inhibiting the hyperacute antibody-mediated rejection that occurs when pig organs are transplanted into primates have been investigated using the baboon as a potential recipient. Baboons were treated with different regimens that included combinations of (1) splenectomy, (2) pharmacologic immunosuppression (CsA, cyclophosphamide, corticosteroids +/- methotrexate), and (3) intravenous infusion of oligosaccharides. The cytotoxicity of the serum was then assessed on cultures of pig kidney cells (PK15). Unmodified serum caused approximate 65-100% pig cell destruction. Splenectomy and/or pharmacologic immunosuppression, and infusions of dextran, dextrose or mannitol, did not result in any reduction of cytotoxicity. Infusions of melibiose and/or arabinogalactan, both of which have terminal non-reducing alpha-galactose, however, decreased relative PK15 cell damage significantly in a dose-dependent manner. At high concentrations (< or = 50 g/hr), complete inhibition of cytotoxicity was achieved in 4 of 15 baboons. The extracorporeal immunoadsorption of baboon serum utilizing immunoaffinity columns of melibiose also resulted in a significant reduction (of approximately 80%) in cytotoxic effect. In 1 baboon, melibiose and arabinogalactan infusion delayed vascular rejection of a pig cardiac xenograft from 10 min to about 12 hr, at which time the baboon died from the toxic effects of the carbohydrate infusion. These observations (1) add further support to the role that anti-alpha-galactosyl antibodies play in the hyperacute rejection of pig tissues transplanted into primates, and (2) demonstrate that serum cytotoxicity can be reduced by the intravenous infusion of alpha-galactosyl oligosaccharides or by extracorporeal immunoadsorption using these carbohydrates.

In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal(alpha)1-3Gal column.

The major role of anti-alphaGal antibodies in the hyperacute rejection of pig organs by humans and baboons has been clearly demonstrated. Spacered alpha-galactose disaccharide (Gal(alpha1)-3Gal) hapten was produced by chemical synthesis and covalently attached to a flexible, hydrophilic polymer (PAA), which in turn was covalently coupled to macroporous glass beads, forming an immunoadsorbent that is mechanically and chemically stable and can be sterilized. The extracorporeal immunoadsorption (EIA) of anti-alphaGal antibodies using this column has been investigated in vivo in 3 baboons. In Baboon 1 (which had hyperacutely rejected a pig heart transplant 4 months previously, was not splenectomized, and did not receive any pharmacologic immunosuppression) the levels of anti-alphaGal antibody and antipig IgM and IgG, as well as serum cytotoxicity, fell significantly after each of 3 EIAs but were not eliminated. Serum cytotoxicity, antipig immunoglobulin and anti-alphaGal antibody rose steeply within 24 hr of the final EIA, suggesting that the return of cytotoxicity was associated with anti-alphaGa1 antibody. In Baboons 2 and 3 (which were immunologically naive and splenectomized, and received triple drug immunosuppressive therapy) serum cytotoxicity was totally eliminated and anti-alphaGal antibody and antipig IgM and IgG levels were greatly reduced by courses of EIA. In Baboon 2, cytotoxicity and all antibody levels remained negligible for approximately one week after the final (fourth) daily EIA. In Baboon 3, cytotoxicity and antibody levels were maintained low by intermittent EIA (over a period of 13 days) for almost 3 weeks, although antipig IgM began to rebound 4 days after the final EIA. We conclude that, in an immunosuppressed, splenectomized baboon, repeated EIA using a specific alphaGal disaccharide column will reduce antipig and anti-alphaGal antibody levels and serum cytotoxicity significantly for several days. This reduction in cytotoxicity will almost certainly be sufficient to delay the hyperacute rejection of a transplanted pig organ, but further studies are required to investigate whether it will be sufficient to allow accommodation to develop.

Protection of pig kidney (PK15) cells from the cytotoxic effect of anti-pig antibodies by alpha-galactosyl oligosaccharides.

Anti-pig antibodies in human and baboon serum are believed to be directed against α-galactosyl (αGal) epitopes expressed on various pig cells, including vascular endothelia. We have investigated the effect of human sera on the PK15 pig kidney cell line, which abundantly expresses αGal epitopes. To quantitate cell viability, we have used a staining method that differentiates live cells from dead ones. Various carbohydrates (n=28) were added individually to serum at concentrations of 0.125–50 mg/ml. Unmodified serum caused approximate 100% PK15 cell death within 60 min. Carbohydrates that were not αGal based did not significantly protect PK15 cells. Of the αGal-based carbohydrates, only B disaccharide protected PK15 cells from both human and baboon serum (76% and 93% protection, respectively, at 1 mg/ml). Three αGal oligosaccharides provided approximately 80–90% protection against both human and baboon sera at a concentration of 10 mg/ml. Three other closely related structures protected only against baboon serum (> 80%) at high concentration (50 mg/ml), suggesting a difference in anti-pig antibody affinity between baboon and man. Specific anti-αGal antibody-depleted serum caused < 10% pig cell death, whereas the antibodies eluted from the αGal columns caused > 70% pig cell death. In conclusion, this study provides further evidence that (1) αGal structures are the targets for human and baboon anti-pig antibodies, and (2) there may be a therapeutic role for the infusion of specific αGal carbohydrates, or for antibody removal using αGal immunoaffinity columns, in order to prevent hyperacute rejection of pig organs in man.

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Lupus autoantibodies to native DNA cross-react with the A and D SnRNP polypeptides.

Antibodies to native DNA (nDNA) in sera from patients with systemic lupus erythematosus have been found to frequently correlate with antibodies to the A and D SnRNP proteins measured in Western blot assays. 40 of 54 SLE (74.1%) sera with anti-nDNA bound to A and D proteins, while 9 of 113 sera (8%) without anti-nDNA bound the A and D proteins, P < 10(-8) by Fishers exact test. Antibodies to nDNA correlated closely with anti-A and anti-D in seven of eight patients followed sequentially, r = 0.7865. Nine human polyclonal anti-nDNA populations were isolated from DNA cellulose columns. Seven reacted equally with A and D, and two reacted predominantly with D. Two of three murine monoclonal anti-DNA antibodies isolated from NZB/NZW F1 hybrid mice bound A and D equally in Western blot with a titer > 1/40,000. These reactions were directed to the unfolded A and D proteins measurable in Western blot since these monoclonals (and several of the human anti-nDNA populations) failed to react with native U1RNP in ELISA or in RNA immunoprecipitation experiments. These newly recognized cross reactions of anti-nDNA may amplify the immune response to DNA and be part of the original immunogenic drive.

Detection, immunoabsorption, and inhibition of cytotoxic activity of anti‐αGal antibodies using newly developed substances with synthetic Gal α1–3Gal disaccharide epitopes

Abstract: The presence of naturally occurring anti‐Galα1–3Gal (anti‐αGal) antibodies in human serum is believed to be a major factor in the hyperacute rejection of discordant organ xenografts such as the pig‐to‐human combination. Galα1–3Gal epitopes are expressed on pig tissues and the binding of anti‐αGal leads to endothelial cell activation and complement‐mediated, hyperacute graft rejection. One possible method to overcome this problem is to absorb anti‐αGal antibodies from the plasma of the xenograft recipient using a suitable immunoabsorbent or to interfere with their binding to tissues and thus prevent their cytotoxic activity by the intravenous injection of soluble antigen. We describe here the use of new synthetic antigens containing the Galα1–3Gal disaccharide (Bdi) epitope. Soluble conjugates of the Bdi with polyacrylamide (PAA‐Bdi) were used as coating antigens for an anti‐αGal ELISA as well as for in vitro inhibition of the cytotoxicity of anti‐αGal. An immunoabsorbent consisting of PAA‐Bdi coupled to macroporous glass (Sorbent Bdi) was tested for absorption of anti‐αGal from human serum.

Variability of anti‐αGal antibodies in human serum and their relation to serum cytotoxicity against pig cells

Abstract: One of the major obstacles in pig‐to‐human xenografting is hyperacute rejection (HAR) of pig cells caused by preformed anti‐pig antibodies and complement. In 1991 we suggested that anti‐αGal antibodies play a major role in the HAR of pig cells. Anti‐αGal antibodies recognize terminal α‐galactose‐containing epitopes on glycoproteins and glycolipids. They are present in humans, apes, and Old World monkeys, but not in lower mammals such as pigs. However, pigs, unlike humans, express terminal α‐galactose epitopes on vascular endothelium which represent targets for human anti‐αGal antibodies. Despite increasing recognition that anti‐αGal antibodies are an important factor, many questions related to their precise role in HAR remain to be answered.

Monomorphic and polymorphic carbohydrate antigens on pig tissues: implications for organ xenotransplantation in the pig-to-human model

Abstract The existence of the αCial epitope in 137 pigs belonging to 23 different breeds suggests that this antigen is either monomorphic or occurs at a high incidence in the porcine species. Its histological location at the surface of pig vascular endothelial cells makes it a target for human natural anti‐alGal antibodies and complement, which may be responsible for the hyperacute vascular rejection of transplanted pig organs. The precursor carbohydrate chain (N‐acetyllactosamine) and NeuAc‐substituted epitopes are also exposed at the surface of pig vascular endothelium and were found in all pigs in this study. However, humans also have these two epitopes on vascular endothelium and, consequently, have not made natural antibodies against these carbohydrate antigens. Therefore, these two pig epitopes cannot be the main target of the hyperacute vascular rejection process. Three pig phenotpyes‐A + (51%), A:H + (38%), and A ‐ H ‐ I + (11%) were identified among 37 Large‐white pigs by the presence of polymorphic A, H, and I carbohydrate antigens on the brush border of the surface epithelium of small intestine. These antigens were also present in other exocrine secretions but were not detected on vascular endothelium of the same pigs, suggesting that they are not involved in the hyperacute vascular rejection, although the pig A tissue antigen can induce an immune response in ***O or B blood group recipients. Once the problem of the initial hyperacute vascular rejection directed against the αGal epitope is overcome, typing donor pigs for A, H, and I, as well as for the protein swine leukocyte antigens (SLA) and other pig antigens, may help in elucidating antigens involved in acute or chronic xenograft rejection.