anti-Gal Alpha Research Articles

Normal human serum contains high levels of anti‐Galα1‐4GlcNAc antibodies

Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF‐transgenic porcine hearts

Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs. Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes. Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv. DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.

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

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. 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. 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. 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.

In vitro and in vivo investigation of a novel monoclonal antibody to plasma cells (W5 mAb).

Natural antibodies (Abs), predominantly anti-Gal alpha 1-3Gal (Gal) Abs, in non-human primates and human beings present a major hurdle to successful pig-to-primate xenotransplantation. Attempts to inhibit anti-Gal Ab production in naïve baboons using non-specific immunosuppressive or B cell-specific reagents have failed. A new rat monoclonal antibody (W5 mAb) has been generated, which binds to all B cells, including memory cells, and to the majority of plasma cells, but not to T cells. It has been tested in vitro and in vivo. By immunoprecipitation, W5 mAb bound a human leukocyte antigen class II (HLA-DR) determinant. Sorting splenic or bone marrow W5+ cells resulted in a highly enriched anti-Gal Ab and total immunoglobulin (Ig)-secretory population. In vivo studies in baboons demonstrated that W5 mAb was safe but, despite the concomitant administration of an anti-CD154 mAb to inhibit sensitization, anti-rat Abs were detected within 10 days and inhibited the effect of the W5 mAb. High levels of W5 mAb were able to completely deplete B cells in the blood, but not in lymphoid tissues. Enzyme-linked spot-forming assay (ELISPOT) demonstrated that only 50 to 60% of secreting cells (SC) were depleted in the bone marrow. No reduction in the serum levels of anti-Gal Ab was observed. W5 mAb did not cause complete inhibition of anti-Gal Ab production, probably as a result of its inability to completely deplete B and plasma cells from all lymphoid compartments.

Initial investigation of the potential of modified porcine erythrocytes for transfusion in primates.

There is a shortage of human blood for transfusion. The possibility of using alpha-galactosidase-treated pig red blood cells (pRBCs) for transfusion into humans has been investigated. pRBCs were treated in vitro with alpha-galactosidase. In vitro binding of antibodies (Abs) in baboon or human sera to untreated/treated pRBCs was assessed by flow cytometry and serum cytotoxicity. In vivo clearance rates of (1) autologous baboon red blood cells (RBCs), (2) unmodified pRBCs, and (3) alpha-galactosidase-treated pRBCs were measured after transfusion into baboons receiving either no treatment or depletion of complement +/- depletion of anti-Gal alpha 1-3Gal (Gal) Ab or of macrophage phagocytes. In vitro binding of baboon or human Abs to treated pRBCs was absent or minimal compared with untreated pRBCs, and serum cytotoxicity was completely inhibited. In vivo autologous baboon RBCs survived for >16 days and unmodified pRBCs for <15 min in an untreated baboon. Treated pRBCs survived for 2 h in an untreated baboon, for 24 h in a complement-depleted baboon, and for 72 h when the baboon was depleted of both complement and anti-Gal Ab, or of complement and macrophage phagocytes. All baboons, however, became sensitized to Gal antigens. Failure to prolong the in vivo survival of treated pRBCs could be due to inadequate removal of Gal epitopes because sensitization to Gal developed, or could imply other, as yet unidentified, causes for RBC destruction. To fully assess the potential of pRBC transfusion in humans, more complete alpha-galactosidase treatment of pRBCs will be required.

Anti-Gal alpha 1-3Gal IgM and IgG antibody levels in sera of humans and old world non-human primates.

Organs transplanted from pig to primate are rejected within minutes or hours by an antibody-dependent, complement-mediated mechanism [hyperacute rejection (HAR)]. Even after depletion of anti-Gal alpha 1-3Gal (Gal) antibody (Ab), for example by extracorporeal immunoadsorption, return of natural Ab is believed to be a major factor in the initiation of acute humoral xenograft rejection. Various non-human primates are used as recipients of pig organs in experimental discordant xenotransplantation (XTx) models. However, anti-Gal IgM and IgG levels in non-human primates may differ from those in humans. Serum levels of anti-Gal IgM and IgG were measured by enzyme-linked immunosorbent assay (ELISA) in humans (n=14), chimpanzees (n=8), baboons (n=214), cynomolgus monkeys (n=29), rhesus monkeys (n=23) and Japanese monkeys (n=6). The mean level of anti-Gal IgM was significantly higher in chimpanzees than in other groups, while in rhesus monkeys it was significantly lower than in other groups, except baboons and Japanese monkeys. The mean human anti-Gal IgG level was higher than in other groups and this difference reached statistical significance except with regard to chimpanzees. The mean anti-Gal IgG level in baboons was significantly lower than that in humans, chimpanzees and cynomolgus monkeys. The measured differences in anti-Gal IgM and IgG levels may affect the kinetics of Ab removal and rate of return in different species, and thus may have relevance for translating work in non-human primate models to the clinical setting.

Tolerization of Gal alpha 1,3Gal-reactive B cells in pre-sensitized alpha 1,3-galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism.

Using a alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) to deficient (GalT(-/-)) mouse bone marrow transplantation model, we have previously demonstrated that a non-myeloablative conditioning regimen is capable of permitting induction of allogeneic and xenogeneic mixed chimerism. Chimerism is associated with the rapid and lasting tolerization of anti-Gal alpha 1,3Gal (Gal) natural antibody (Ab)-producing B cells. However, one limitation of this model is that anti-Gal natural Ab levels are lower in GalT(-/-) mice than in humans and other primates. To overcome this limitation, we have now investigated the possibility of inducing such tolerance in GalT(-/-) mice that produce much higher levels of anti-Gal Abs due to presensitization with Gal-bearing xenogeneic cells. B6 GalT(-/-) mice that were pre-sensitized with rabbit red blood cells received non-myeloablative conditioning with depleting anti-CD4 and CD8 mAbs, 3Gy whole body and 7Gy thymic irradiation, and infusion of BALB/c GalT(+/+) bone marrow cells (BMC). Although engraftment of standard marrow doses was inhibited by the presensitization, long-lasting mixed chimerism could be induced in recipients of a high dose [160 x 10(6)] of allogeneic wild-type BMC. Achievement of persistent chimerism was associated with high levels of anti-Gal IgG(1) pretransplant, suggesting an inhibitory effect of non-complement-fixing IgG(1) Ab on anti-Gal-mediated marrow rejection. Induction of mixed chimerism was associated with a rapid disappearance of serum anti-Gal and tolerization of anti-Gal Ab-producing cells. B cells with anti-Gal receptors became undetectable in mixed chimeras. Mixed chimeras accepted subsequently transplanted donor-type GalT(+/+) hearts (> 140 days), whereas rapid (within 2 days) rejection of GalT(+/+) hearts occurred in conditioned control GalT(-/-) mice. In conclusion, when a high dose of GalT(+/+) BMC was administered to pre-sensitized GalT(-/-) mice, chimerism and tolerance were achieved. The absence of B cells with receptors recognizing Gal in mixed chimeras suggests a role for clonal deletion/receptor editing in the maintenance of B cell tolerance.

A COMPARISON OF FETAL AND ADULT PORCINE ISLETS WITH REGARD TO Gal?? (1,3)Gal EXPRESSION AND THE ROLE OF HUMAN IMMUNOGLOBULINS AND COMPLEMENT IN ISLET CELL CYTOTOXICITY1

It is still debated whether fetal or adult porcine islets should be the preferred choice for future clinical islet xenotransplantation. Each type of islet preparation has advantages and disadvantages compared with the other. Here we present a direct comparison between fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression, immunoglobulin and complement binding, and cytotoxicity after exposure to fresh human serum. Islet single cell suspensions were prepared from adult and fetal islets by trypsin digestion. Fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (BS-IB4) and affinity-purified chicken anti-Gal alpha(1,3)Gal antibody was used to detect Gal alpha(1,3)Gal expression. Immunoglobulin and complement binding to the islet cells and cytotoxicity for islet cells was compared after incubation with fresh and heat-inactivated human sera and with an immune serum from a diabetic patient who received a fetal porcine islet transplant. Furthermore, two pools of human AB sera were depleted of porcine endothelial cell cytotoxic human anti-Gal alpha(1,3)Gal antibodies by absorption and were used to analyze the effect of Gal alpha(1,3)Gal antibody removal on islet cell cytotoxicity. Fetal islet cells readily bound both BS-IB4 and the chicken anti-Gal alpha(1,3)Gal antibody. None of 10 adult porcine islet preparations were stained by BS-IB4. In comparison, IgY anti-Gal Ab binding was detected in two of eight adult islet isolations, whereas the other six preparations showed marginal/no binding. After incubation of fetal islet cells with fresh human serum, C3c binding was strongly positive and IgM binding variable, with occasional binding of IgG and no detectable binding of IgA. Adult islet cells were also strongly positive for C3c but did not bind detectable amounts of IgM, IgG, or IgA. Immune sera from a patient who had received fetal porcine islets showed the presence of induced antibodies that bound to fetal islet cells and to porcine peripheral blood lymphocytes, whereas binding to adult islet cells was barely detectable. Fresh human sera showed a high and similar level of complement-mediated lytic activity for both adult islet cells (78+/-22%) and fetal islet cells (75+/-16%). Cytotoxicity for fetal islet cells and peripheral blood lymphocytes was significantly reduced when the corresponding sera were depleted of anti-Gal antibodies before use (P=0.002 and P=0.003, respectively). In contrast, no difference in cytotoxicity for adult islet cells was detected when anti-Gal-depleted human sera were used. Gal alpha(1,3)Gal expression is occasionally detectable on adult porcine islet cells, but not as readily and at a lower level, compared with fetal islet cells. Thus, as porcine fetal islets mature to adult islets, the expression of the Gal alpha(1,3)Gal epitope gradually diminishes. Consequently, cytotoxic anti-Gal alpha(1,3)Gal antibodies in human serum play an important role in the lysis of fetal but not adult porcine islet cells.

Definition and characterization of chicken Gal alpha(1,3)Gal antibodies.

The Gal alpha(1,3)Gal epitope is of interest as, in pig-to-primate xenotransplantation, it is the major target of naturally occurring human IgM and IgG antibodies, leading to hyperacute rejection. Human and Old World monkeys make anti-Gal alpha(1,3)Gal antibodies as they lack a functional gene and do not express Gal alpha(1,3)Gal. Interestingly, the cultured fibroblasts of some other species, such as chickens, have been reported also not to express Gal alpha(1,3)Gal--if this is true for other tissues, and chickens do not express Gal alpha(1,3)Gal antigen, then they would have anti-Gal antibodies--which could have diagnostic and therapeutic value, particularly as chicken antibodies do not fix mammalian complement. Standard serological methods were used to characterize the antibodies. Several baboons received pig kidney xenografts that had been perfused with hyperimmune chicken anti-Gal antibodies. We now demonstrate that chickens do not express Gal alpha(1,3)Gal on their red cells, leukocytes, or tissues, and that their serum contains large amounts of anti-Gal alpha(1,3)Gal antibodies. In addition, chickens could be immunized to produce high-titer, high-avidity antibodies (9.5x10(9) M(-1))--an avidity considerably greater than that of the Gal alpha(1,3)Gal binding lectin IB4 (2.9x10(8) M(-1)) or Gal antibodies in human serum (2.2x10(5) M(-1)). Chicken antibodies, obtained from both normal and immunized chickens, could block the in vitro cytolysis of pig endothelial cells or lymphocytes by human or baboon antibodies. However, such antibodies tested in vivo in pig-to-baboon xenotransplantation failed to block hyperacute rejection and, indeed, may have accelerated this.

Immunochemical properties of anti-Gal alpha 1-3Gal antibodies after sensitization with xenogeneic tissues.

In antigen-driven immune responses to proteins, antibodies of low avidity and limited complement fixing capacity undergo affinity maturation to yield antibodies of higher avidity which fix complement to a greater extent. The products of antigen-driven responses to carbohydrates are less defined. To investigate the evolution of natural antibodies against carbohydrates following sensitization, we studied natural antibodies specific for Gal alpha 1-3Gal in patients sensitized to that antigen as a result of perfusion of their blood through porcine livers for the treatment of hepatic failure. The natural antibodies against Gal alpha 1-3Gal, which occur in all unsensitized individuals, were predominantly IgM and IgG2, with average functional avidities of 5 x 10(-9) and 2 x 10(-8) M, respectively. After sensitization, the classes of anti-Gal alpha 1-3Gal included IgM, IgG2, and IgG1, and the average functional avidities of IgM and IgG were 3 x 10(-9) and 2 x 10(-9) M, respectively. The activation of complement by anti-Gal alpha 1-3Gal per microgram of Ab, measured by the fixation of C3bi on porcine cells, increased after sensitization owing to changes in subclass and avidity. Deposition of C3bi correlated with the concentrations of IgG1 and IgM but not IgG2 against Gal alpha 1-3Gal. Consistent with this finding, purified IgG1, but not IgG2, anti-Gal alpha 1-3Gal fixed complement on porcine cells. These results demonstrate that the properties of anticarbohydrate antibodies evolve after sensitization to increase complement fixation on potential targets. These properties may result from the altered costimulation of the humoral response to Gal alpha 1-3Gal due to sensitization.

A murine model of antibody-mediated hyperacute rejection by galactose-alpha(1,3)galactose antibodies in Gal o/o mice.

In pig-to-primate/human xenografts, hyperacute rejection of primarily vascularized organs usually occurs in 10-60 min and is due to the reaction of the recipients' natural antibodies with antigens expressed on the donor endothelium, the fixation of complement, and ultimately vascular stasis and hemorrhage. Surprisingly, the major target of the natural antibodies is the disaccharide galactose-alpha(1,3)galactose (Gal alpha(1,3)Gal), which is found on many different molecules in pig tissues and reacts with naturally occurring human anti-pig IgM and IgG antibodies. There are a number of strategies to remove/block/alter Gal alpha(1,3)Gal expression in pig tissues, all of which involve the expression of transgenes in pigs. To overcome the difficulty of preclinical studies using primates, we describe a model of hyperacute rejection of heart transplants to Gal o/o mice, which are similar to humans in that they have anti-Gal alpha(1,3)Gal antibodies. Gal o/o mice received skin or heart grafts from Gal+ mice or rats, and additional antibody and complement were provided; hyperacute rejection was monitored by observation and histology. Gal alpha(1,3)Gal+ mouse tissues (skin or heart) are not rejected by Gal o/o mice. This was not unexpected, as mice do not utilize alloantibody/complement systems satisfactorily in experimental transplantation studies. However, with the addition of anti-Gal alpha(1,3)Gal antibody and complement, hyperacute rejection of hearts can occur in 10-20 min; it is mediated by IgM, not IgG, antibodies and leads predominantly to tissue hemorrhage. Gal alpha(1,3)Gal antigen modification by expression of the H transferase cDNA leads to "indefinite" survival (>120 min) and no hyperacute rejection, which shows that this model is suitable for the study of antibody-mediated rejection of relevance to pig-to-human xenografts.

Down-regulation of Gal alpha(1,3)Gal expression by alpha1,2-fucosyltransferase: further characterization of alpha1,2-fucosyltransferase transgenic mice.

In pig-to-primate transplantation, antibody-mediated hyperacute rejection is the consequence of binding of natural antibodies to Gal alpha(1,3)Gal on pig endothelium. The elimination of the Gal alpha(1,3)Gal antigen from pig cells should prevent hyperacute rejection. Using in vitro techniques, we have previously reported that using the alpha1,2-fucosyltransferase gene induces the preferential expression of H substance with a concomitant reduction in the expression of Gal alpha(1,3)Gal. The aim of the present study was to examine the effect of expressing the alpha1,2-fucosyltransferase gene in vivo on Gal alpha(1,3)Gal. Three alpha1,2-fucosyltransferase transgenic lines of mice were produced and characterized serologically and histologically. Immunohistological studies showed heavy staining for H substance in liver, spleen, kidney, and heart, with a reduction in staining for Gal alpha(1,3)Gal. In addition, there was a reduction in the binding of human anti-Gal alpha(1,3)Gal antibody to lymphocytes from alpha1,2-fucosyltransferase transgenic mice and a substantial decrease in complement-mediated cytolysis of alpha1,2-fucosyltransferase transgenic lymphocytes when compared with that obtained with normal mice. The findings have important implications, in that alpha1,2-fucosyltransferase transgenic pigs could be produced as a source for humans. Such pigs should have a reduced expression of Gal alpha(1,3)Gal.

Natural human anti-Gal alpha(1,3)Gal antibodies react with human mucin peptides.

We have recently demonstrated that both antibodies to Gal alpha(1,3)Gal, and the Gal alpha(1,3)Gal binding lectin (IB4), bind a synthetic peptide (DAHWESWL), there being a similar recognition of carbohydrate and peptide structures. We now report that the anti-Gal alpha(1,3)Gal antibodies and IB4 lectin also react with peptides encoded by mucin genes (MUC 1, 3, 4)-sequences known to be rich in serine, threonine and proline. This activity was demonstrated (1) by the ability of mucin derived peptides to block the reaction of anti-Gal alpha(1,3)Gal antibodies and IB4 lectin with a Gal alpha(1,3)Gal+ pig endothelial cell line; the reactions were specific and did not occur with a random peptide containing the same sequences or with other mucin peptides; (2) by the fact that anti-mucin1 antibodies could react with the Gal alpha(1,3)Gal expressed after transfection of COS cells (Gal alpha(1,3)Gal-,Muc1-) with cDNA encoding the pig alpha, 3galactosyltransferase; and (3) that the IB4 lectin and anti-Gal alpha(1,3)Gal antibodies could react with mucin 1 found on the surface of human breast cancer cells. Thus natural occurring anti-Gal alpha(1,3)Gal antibodies found in all human serum can react with self (Muc1) peptides expressed in large amounts on the surface of tumour cells but not on normal cells. The findings are of interest and serve to explain the previously reported findings that human cells can, at times, express Gal alpha(1,3)Gal; such expression is an artefact, the reaction is due to the phenomenon described herein, i.e. that anti-Gal alpha(1,3)Gal antibodies react with mucin peptides.

Specificity of xenoreactive anti-Gal alpha 1-3Gal IgM for alpha-galactosyl ligands.

The transplantation of organs from lower animals such as pigs into humans is prevented by a severe rejection reaction initiated by complement fixing xenoreactive natural antibodies. Most anti-pig xenoreactive natural antibodies in humans are thought to recognize Gal alpha 1-3Gal beta 1-4GlcNAc and are also thought to recognize, albeit less avidly, Gal alpha 1-6Glc. Gal alpha 1-6Glc has been used as a ligand for purification of 'anti-Gal alpha 1-3Gal antibodies' and as a therapeutic or reagent to prevent the binding of these antibodies to porcine organs or cells. We tested the specificity of anti-Gal alpha 1-3Gal IgM for Gal alpha 1-6Glc and related saccharides. Based on inhibition of binding of xenoreactive anti-Gal alpha 1-3Gal IgM to porcine cells by soluble saccharides, anti-Gal alpha 1-3Gal IgM in a human serum was found to consist of a mixture of antibodies which have a similar affinity for Gal alpha 1-3Gal but varying affinities for Gal alpha 1-6Glc and other structures. Twenty to 40% of the anti-Gal alpha 1-3Gal IgM from the population tested did not recognize Gal alpha 1-6Glc. The binding of anti-Gal alpha 1-3Gal IgM to Gal alpha 1-6Glc varied widely from individual to individual, some samples lacking almost entirely anti-Gal alpha 1-3Gal IgM which bound to Gal alpha 1-6Glc.

Identification of porcine endothelial cell membrane antigens recognized by human xenoreactive natural antibodies.

Rejection of an organ transplanted into a phylogenetically disparate recipient may be initiated by the binding of xenoreactive natural Abs of the recipient to Ags expressed on donor endothelium leading to the activation of the C system. Having previously shown that human xenoreactive Abs bind predominantly to N-linked oligosaccharides on porcine endothelial cell glycoproteins, we sought to identify those glycoproteins to gain insight into the pathogenesis of the rejection reaction and to develop improved methods for depleting those Abs. Based on amino-terminal and internal sequencing of the glycoproteins, reactivity with monospecific rabbit Abs, and functional properties, the porcine endothelial cell targets of human natural Abs were shown to be the von Willebrand factor, integrin alpha 1, alpha v, alpha 3/alpha 5, beta 1, and beta 3 chains and a 95-kDa glycoprotein homologous with chick DM-GRASP. Human and baboon Abs directed against those glycoproteins were shown to be absorbed during perfusion of porcine organs and the binding of human IgM to the isolated glycoproteins to initiate the activation of C leading to formation of iC3b neoantigen. The determinants recognized by human xenoreactive Abs had immunodominant alpha-galactosyl residues based on affinity chromatography, reactivity with anti-Gal alpha(1-3)Gal Abs and sensitivity to alpha-galactosidase. The findings show that members of the integrin family and the von Willebrand factor are major targets of xenoreactive Abs and suggest potential mechanisms by which the binding of Abs to endothelial cells might perturb the physiology of those cells and thus cause aberrant functioning of a tissue or organ.

Galα(1,3)Gal, the Major Xenoantigen(s) Recognised in Pigs by Human Natural Antibodies

The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Distribution of Carbohydrates Recognized by the LectinsEuonymus europaeusand Concanavalin A in Monoxenic and Heteroxenic Trypanosomatids

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.