Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection.

The transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galalphal,3Gal, which is synthesized by Galbeta1,4GlcNAc alpha1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of alpha1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking alpha1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of alpha1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses.

Relationship between the secretor status and the expression of ABH blood group antigenic determinants in human intestinal brush-border membrane hydrolases.

At least six hydrolases of the human intestinal brush-border membrane bear ABH blood group antigenic determinants related to the erythrocyte phenotype: the intestinal glycoproteins of blood group A and B subjects express A or B determinants, respectively, while blood group O subjects express the H determinant identified with Ulex europaeus lectin I. These expressions are under the control of the secretor gene: ABH antigens were not detected in the hydrolases of non-secretor subjects.

Identification of two functionally deficient plasma alpha 3-fucosyltransferase (FUT6) alleles.

One Indonesian individual without detectable plasma alpha3-fucosyltransferase activity was identified with three point mutations, 730C>G (L244V), 907C>G (R303G), and 370C>T (P124S), in the coding region of one FUT6 allele. Another individual, expressing weak plasma alpha3-fucosyltransferase activity, had the 907C>G together with the 370C>T mutation, but did not have the 730C>G mutation. PCR-RFLP analyses of complete families confirmed the segregation of these alleles and illustrated the existence and inheritance of the [370C>T; 907C>G] mutated allele in three additional families. Altogether, this allele was found heterozygously in nine Indonesian and two Swedish individuals, all with detectable plasma alpha3-fucosyltransferase activities. The FUT6 allele with the three mutations (370C>T; 730C>G; 907C>G) was identified heterozygously in only two Indonesian individuals, both having the inactivating 739G>A mutation in the other allele and both lacking plasma alpha3-fucosyltransferase activity. Enzyme studies made on transiently transfected COS-7 cells demonstrated that the combination of the 370C>T, 730C>G and 907C>G mutations decreased the V(max) by more than 80%, but caused no obvious change of the apparent K(m) values for GDP-fucose and Gal-N-acetyllactosamine. In comparison, chimeric constructs with the isolated 730C>G or 907C>G mutations decreased the V(max) values by about two thirds and one third, respectively.

The jelly layers and cortex of the unfertilized Xenopus laevis egg: carbohydrate, phospholipid and protein analysis.

Differences in the carbohydrate composition were found in the jellies and the cortex of the unfertilized Xenopus laevis egg using lectins and blood group antibodies. Blood group H trisaccharide was detected in the outer jelly and "A-like" oligosaccharide was found in the inner jelly. The blood group A trisaccharide was detected in the vitelline envelope and the cortex. The plasma membranes were isolated and partially purified by differential centrifugation on sucrose cushion. The phospholipid composition of the membrane was assessed by quantitative two-dimensional thin layer chromatography. The major phospholipids were sphingomyelin (8%), phosphatidylcholine (55%), phosphatidylinositol and phosphatidylserine (7%), and phosphatidylethanolamine (26%). The external application of phospholipase A2 indicated a possible asymmetry of the phospholipids in the membrane such as the acidic phospholipids are preferentially located at the inner leaflet. The membrane protein and glycoprotein pattern was examined by gel electrophoresis using Triton and selective staining. Six major glycoproteins ranging from 250 to 32 kDa, were detected among the Triton-insoluble components.

Cellular expression of H and B antigens in the rat olfactory system during development.

Developmental expression of H and B antigens in the rat olfactory system was studied from the embryonic day 14 up to the postnatal day 30. The H antigen was detected in the olfactory and vomeronasal epithelia as early as fetal day 14, whereas the B antigen first appeared 2 days later. The anti-H reagent reacted strongly with sensory receptors and weakly with supporting cells in both epithelia, whereas the anti-B reagent was specific for olfactory receptors. In the main olfactory epithelium, the H antigen was expressed from fetal day 19 by most of the receptor cells, whereas the B determinant was expressed from fetal day 16 to postnatal day 3 by only a few neuroreceptors mostly located near the epithelial surface. After the postnatal day 3, B positive neurons increased in number from the periphery toward the deeper mucosal layers and they were distributed over 3/4 of the epithelial thickness in 15- and 30-day-old rats. In the main olfactory bulb, a widespread glomerular B staining with variable binding intensity between adjacent glomeruli was already observed at birth. The vomeronasal receptor cells and their axon terminals in the accessory olfactory bulb exhibited a comparable developmental expression of the B antigen. Results suggest that the B antigen could be regarded as a marker of neuronal maturation of both the olfactory and vomeronasal receptor cells; moreover, its first appearance in the receptor cells might be temporally related to the formation of synapses between receptor axons and deutoneurons in the bulb.

Expression of ABH and X (Lex) antigens in various cells.

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.

Fluorescent staining of nuclei and amyloid substance. Two useful properties of p-phenylenediamine.

Mounting immunofluorescent slides in an oxidized solution of p-phenylenediamine in 90% glycerol, resulted in brown fluorescent staining of nuclei. This contrasts well with the fluorescence of fluorescein and rhodamine conjugates and facilitates excellent morphological localization of antigens. The antifading effect of p-phenylenediamine is maintained in this oxidized solution. In addition, amyloid substance gives a bright yellow-orange fluorescence with this medium. This method of staining is more sensitive and less time consuming than the usual methods for amyloid. Alternative techniques to keep the antifading effect without either nuclear or amyloid counterstain (for identification of nuclear antigens), or to show up amyloid deposits without nuclear counterstain (for small amyloid deposits) are described.

Immunohistological characterization of mucin epitopes by pre-treatment of gastro-intestinal sections with periodic acid.

Periodate pretreatment of paraffin sections of ethanol-fixed gastrointestinal mucosae was used to characterize the carbohydrate or peptidic nature of mucin epitopes by immunoperoxidase. Immunoreactivity of monoclonal antibodies (MAbs) against histo-blood group related carbohydrate epitopes such as A, Lea, Lec, Sialosyl Lea, H type 2, I, T, Tn and sialosyl Tn dramatically decreased or even disappeared after periodate pretreatment of deparaffinized sections. In contrast, the immunoreactivity of MAbs against peptide mucin epitopes such as the gastric M1 mucin epitopes was almost unaffected by this treatment. Moreover, periodate treatment revealed cryptic peptide M1 mucin epitopes and the peptide MUSE11 epitope associated with the 20 amino acid tandem repeat (PDTRPAPGSTAPPAHGVTSA). An increase of cross-reactions of anti-human M1 MAbs with gastric epithelium of different vertebrate species was detected with periodate treatment. Our results suggest that this method can be useful for preliminary characterization of the biochemical nature of mucin epitopes (peptidic or saccharidic) and for demasking peptidic tumour markers which are hidden by saccharide molecules in normal tissues.

A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation.

Removal and/or neutralization of preformed anti-pig antibodies in non-human primate blood have been shown to prevent the hyperacute rejection of transplanted pig organs. The purpose of this study was to establish a suitable in vitro method that would allow for screening and comparison of various agents and methods potentially useful in the prevention of hyperacute rejection. The pig kidney cell line (PK15), pig aortic endothelial cell line (AG08472), and a primary culture of endothelial cells explanted from a pig aorta were incubated with either human or baboon sera. Complement-dependent cytotoxic activity of human and baboon sera was determined on all three types of pig cells using a two-color fluorescence assay and compared with the conventional 51Chromium (51Cr)-release assay. The assay was also performed on PK15 cells as a 2-chambered slide assay and compared with a microcytotoxicity assay performed in Terasaki trays. Using the microcytotoxicity assay, a 1-step assay utilizing endogenous complement was compared with a 2-step assay where rabbit complement was added. Of the three types of cells studied, PK15 cells were the most sensitive to cytotoxic injury, followed by AG cells and the primary endothelial culture. Good correlation between the 51Cr-release and the two-color fluorescence method was documented. There was good agreement between the results obtained using the 2-chambered slide method and the microcytotoxicity assay, as there was between the 1- and the 2-step assays. The 1- and 2-step assays provided information on the level and efficacy of endogenous complement. We conclude that the two-color fluorescence assay is suitable for the rapid and inexpensive screening of therapeutic interventions that might be useful in the prevention of hyperacute xenograft rejection, and that PK15 cells are suitable for use in this assay.

Molecular cloning of a putative alpha3-fucosyltransferase from Schistosoma mansoni.

Alpha 3-fucosylation of protein or lipid substrates is an important component of the host/parasite interactions during schistosomiasis. In this process, alpha3-fucosyltransferases (alpha3-FucTs) are considered as key enzymes ensuring both parasite survival and adaptation in their (in)vertebrate hosts. In this paper, we report the molecular cloning of a putative alpha3-FucT from Schistosoma mansoni that we termed SmFucTA. The full-length SmFucTA encodes a typical transmembrane type II protein with a short cytoplasmic domain, a transmembrane segment and a long C-terminal catalytic domain. In this region, the GDP-fucose binding site is well conserved whereas the putative acceptor site displays sequence divergence compared to the corresponding region from vertebrate and invertebrate alpha3-FucTs. Southern blot analysis suggested that SmFucTA is present as several copies or has highly related counterparts in the S. mansoni genome. Northern blot revealed a single SmFucTA transcript at 2 kb in adult worms. Affinity purified antibodies directed against recombinant SmFucTA identified a 50 kDa native protein that localizes to the subtegumental and parenchymal regions of adult worms.

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human.

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.

Influence of the original disease, race, and center on the outcome of kidney transplantation.

The low graft survival rate in black recipients (36 +/- 2% at 1 year) as compared with the graft survival rate in white recipients (48 +/- 1%) might be secondary to a higher incidence of vascular lesions, inducing hypertensive disease, in blacks than in whites. The relative frequency of malignant hypertension in black recipients was six times that of white recipients, and recipients with malignant hypertension had a significant lower graft survival rate (43 +/- 2%) than recipients with glomerulonephritis (54 +/- 1%). In addition, patients with vascular lesions (diabetes, malignant hypertension, and glomerulonephritis) showed significantly lower graft survival rates in black than in white recipients, in contrast to patients with primary tubular or interstitial lesions (polycystic kidneys and pyelonephritis), who showed similar graft survival rates in blacks and whites. Only a small fraction of this racial effect could be traced back to the higher incidence of Lewis-negative phenotypes in black recipients and a similar beneficial effect of transfusions, on graft survival, was observed in both black and white recipients. The effects of graft survival of age (6%), race (9%), and transfusions (18%) were significant in good (A) and poor (B) centers. No overlap between A and B centers was observed for any of these three parameters when analyzed separately. However, when the cumulative effects of these three risk parameters were analyzed together a partial overlap appeared, i.e., higher graft survival rates were observed in low-risk recipients that received transplants in B centers than in high-risk recipients that received transplants in A centers. Consequently, the selection of the recipient may play a role in the overall results of different transplantation units, leading to their classification into A or B centers, but cannot explain all of the differences between A and B centers.

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-alpha Gal isolectin B4 from Griffonia simplicifolia 1 and with human natural anti-alpha Gal antibodies specifically purified by affinity chromatography on synthetic oligosaccharides containing the terminal nonreducing alpha Gal1-->3 beta Gal-R disaccharide. This antigenic activity is destroyed by treatment of pig tissues with alpha-galactosidase. The localization of this linear-B epitope on vascular endothelium and its reactivity with natural human anti-alpha Gal 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 alpha NeuAc2-->3 beta Gal1-->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.

Combined effects of HLA matching and age in renal transplantation.

Recipient age and matching for the HLA-A and B antigens each influence the graft survival rate of cadaver kidney transplants by approximately 10%. These two factors are cumulative and have a combined effect of nearly 20%. Thus, other factors that have a relatively weak influence on graft success may have a combined effect that makes their clinical consideration highly relevant.

The Lewis system and kidney transplantation.

A significant effect of Lewis antigens on cadaver kidney graft survival was found in 1,300 North American transplants. Lewis-negative recipients had a graft survival rate that was 8% lower than that of Lewis-positive recipients (P = 0.05). This effect of Lewis antigens was enhanced in patients at a high failure risk as determined by age, race, or transplant center. In patients older than 30 years, the effect of Lewis was 14% (P = 0.07), in non-Caucasians 12% (P = 0.07), in all grafts performed at centers with less than 50% overall 1-year graft survival 12% (P = 0.03), and in non-Caucasians that received transplants in centers with less than 50% overall graft survival it was 18% (p = 0.01). These data confirm previous results on the role of Lewis as a histocompatibility system in renal transplantation; furthermore, they demonstrate that the influence of Lewis is larger in patients at high risk.

Cellular expression and genetic control of ABH antigens in primary sensory neurons of marmoset, baboon and man.

ABH antigens have been demonstrated in the posterior root ganglia (PRG) of 3 primate species (marmoset, baboon and man). Their expression corresponded to the ABO phenotype of the individual and was independent of the secretor gene. In marmosets more cells were positive for H (33 +/- 9%) than for A (19 +/- 6%). In baboons A or B antigens were more easily detected (66 +/- 9%) than the H antigens (48 +/- 5%). In humans more than two-thirds of PRG cells were positive for H but only a small proportion of these were positive for A or B. The ABH antigens were found mainly in the small and intermediate-size neurons whose central processes project to lamina II of the spinal cord posterior horn. Unipolar neurons of the Gasserian ganglion, neurons of the mesencephalic nucleus of the trigeminal nerve and of some visceral ganglia have also been shown to express these antigens which are also present in the fibre layer and glomeruli of the olfactory bulbs.

Genetic control of the humoral immune response to xenografts. I. Functional characterization of rat monoclonal antibodies to hamster heart xenografts.

The rejection of cardiac xenografts in the hamster-to-rat combination is characterized by the production of IgM antibodies that result in the rapid loss of the graft. We have recently produced rat monoclonal antibodies (mAb) to hamster heart xenografts in an attempt to develop reagents for use in identifying the target antigens for this reaction and to study the nature of the genetic control of the humoral response. The monoclonals were created by the fusion of myeloma cells with splenic lymphocytes from LEW rat recipients of hamster cardiac xenografts. The hybridomas were screened for antibody production, reactivity to hamster cell surface antigens, and the ability to mediate hyperacute rejection of hamster heart xenografts. A panel of monoclonal antibodies has been identified that are capable of inducing hyperacute rejection. All of these mAbs are IgM and bind strongly to hamster vascular endothelium. None of the mAbs were lymphocytotoxic or bound to hamster lymphocytes or erythrocytes. Immunopathologic studies demonstrated that these mAbs react specifically with hamster vascular endothelium and mediate a complement-dependent humoral reaction leading to the destruction of the cardiac xenografts. One of the mAbs (designated as HAR-1) has been characterized in detail. HAR-1 detects antigens distributed in the vascular endothelium, epithelium of bronchi in the lung, small intestine, tubules of kidney, and selective components of lymphoid organs--e.g., the stromal cells of the spleen and thymic medullary epithelium. Western blot analysis of hamster heart proteins with HAR-1 showed multiple bands with two major bands migrating at 80 kDa and 48 kDa. Absorption of the HAR-1 antibody with 48 individual carbohydrate molecules demonstrated that the strongest reactivity of the antibody is with a sialyl-Lea carbohydrate antigen.

Use of embryonic human trachea grown in nude mice to patch-repair congenital tracheal stenosis.

BACKGROUND: Long congenital tracheal stenosis is a life-threatening condition, and the available surgical treatments do not give satisfactory long-term results. METHODS: Human embryonic tracheas were implanted in the abdominal cavities of nude mice until their differentiation was completed. These differentiated tracheas were used to patch-repair surgically induced tracheal stenosis in piglets. The human, mouse, or pig origin, of all the cells in the two successive xenotransplants in the nude mouse and the pig, was determined on tissue sections by in situ hybridization with species-specific DNA probes. RESULTS: The transplanted pigs thrived and reached normal adulthood, irrespective of the administration of immunosuppressive treatment. The human tracheal tissue developed in nude mice conserved human structures, with the exception of feeding capillaries, which were of mouse origin. The tracheal patch in the adult healthy pigs comprised only pig cells organized into a fibrous scar, which was covered by normal pig epithelium. CONCLUSIONS: Results suggest that human embryonic trachea grown in nude mice can be successfully used as patch tracheoplasty for long congenital tracheal stenosis without conventional immunosuppression.