Target antigen specificity of a monoclonal antibody (HAR-1) that causes the rejection of hamster heart xenografts in rats.
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Animals↗
Biomedical subjects
R Oriol
Publications and source records attributed to R Oriol.
Evidence that intravenously administered alpha-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.
Animals↗
Molecular basis for plasma alpha(1,3)-fucosyltransferase gene deficiency (FUT6).
While most humans express an alpha(1,3)-fucosyltransferase in plasma, 9% of individuals on the isle of Java (Indonesia) do not express this enzyme. Ninety-five percent of these plasma alpha(1,3)-fucosyltransferase-deficient individuals have Lewis negative phenotype on red cells, suggesting strong linkage disequilibrium between these two traits. To define the molecular basis for this plasma deficiency and to determine which of two candidate human alpha(1,3)-fucosyltransferase genes encode this enzyme (FUT5 and FUT6), we cloned and analyzed alleles at these two loci from an Indonesian individual deficient in plasma alpha(1,3)-fucosyltransferase activity. Single base pair changes were identified in the coding region of each gene, relative to previously published wild type alleles. These changes in turn yield three codon changes in FUT5 and three in FUT6. The codon changes in the FUT5 gene do not yield detectable diminutions in alpha(1,3)-fucosyltransferase activity when tested by expression in transfected COS-1 cells, and none of the FUT5 alleles co-segregate with plasma alpha(1,3)-fucosyltransferase deficiency in Indonesian pedigrees. By contrast, two of the codon changes in the FUT6 alleles inactivate this gene when tested by expression in transfected COS-1 cells. One of these inactivating changes is a missense mutation (Glu-247-->Lys) within the enzyme's catalytic domain. The other inactivating mutation represents a nonsense mutation (Tyr-315-->stop) that truncates the COOH terminus of the enzyme by 45 amino acids. The Glu-247-->Lys missense mutation is present in double dose in the nine plasma alpha(1,3)-fucosyltransferase-deficient individuals tested, whereas the nonsense mutation at tyrosine 315 is present in double dose in just one of these persons. These results demonstrate that the alpha(1,3)-fucosyltransferase activity in human plasma is encoded by the FUT6 gene and that the missense mutation within codon 247 of this gene is responsible for deficiency of this activity in these Indonesian families.
Alleles↗
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 alpha-galactosyl (alpha Gal) 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 alpha Gal 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 alpha Gal based did not significantly protect PK15 cells. Of the alpha Gal-based carbohydrates, only B disaccharide protected PK15 cells from both human and baboon serum (76% and 93% protection, respectively, at 1 mg/ml). Three alpha Gal 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-alpha Gal antibody-depleted serum caused < 10% pig cell death, whereas the antibodies eluted from the alpha Gal columns caused > 70% pig cell death. In conclusion, this study provides further evidence that (1) alpha Gal structures are the targets for human and baboon anti-pig antibodies, and (2) there may be a therapeutic role for the infusion of specific alpha Gal carbohydrates, or for antibody removal using alpha Gal immunoaffinity columns, in order to prevent hyperacute rejection of pig organs in man.
Animals↗
H and B human blood-group antigen expression in cochlear hair cells is modulated by thyroxine.
The presence of human blood-group antigens in developing and adult hypothyroid rat cochleas was analyzed using antibodies directed against antigens H and B. During postnatal development, hypothyroid rat cochleas exhibited a highly selective expression of both B and H antigens, mainly at the hair cell level. Labeling for antigen B was found throughout the hair cells, whereas the antibody directed against antigen H selectively labeled the apical part of these cells. These immunostaining patterns were similar to those found in normal (euthyroid) rat cochleas, but antigenic expression periods were clearly prolonged. Thus, whereas in normal rat cochleas, the B and H antigenic expression disappears from postnatal day (PD) 9 on, in cochleas of hypothyroid rats the reactivity was intense until PD15; it decreased from this developmental stage, and was negative or only faintly positive at PD30. Therefore, in congenital hypothyroidism, hair cell immunoreactivity is present at developmental stages that are negative in normal rat cochleas. These results suggest that human blood-group antigen expression on the developing cochlear hair cells of rats is modulated by thyroxine and that thyroxine is necessary for the temporal expression pattern and secretion of normal glycoproteins.
ABO Blood-Group System↗
Monomorphic and polymorphic carbohydrate antigens on pig tissues: implications for organ xenotransplantation in the pig-to-human model.
The existence of the alpha Gal 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-alpha Gal 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 phenotypes-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 0 or B blood group recipients. Once the problem of the initial hyperacute vascular rejection directed against the alpha Gal 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.
ABO Blood-Group System↗
Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes.
Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b-) ABH nonsecretor who secreted Lewis substances; a Le(a+b-) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b-) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le(a) and Le(b) co-expressed. The copresence of Le(a) and Le(b) in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le(a) or Le(b). These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.
Aldehydes↗
Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a-b-) nonsecretor phenotypes.
Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a-b-) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Lea and Leb glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Lea, Leb and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a-b-) nonsecretor and Le(a+b-) samples. In the Le(a-b-) nonsecretor Lea and Leb activity was absent and type 1 precursor was present in brush border, while Leb activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Lea and Leb glycolipids were identified in this sample. In parallel trace Leb activity could also be detected in the glycolipids of the Le(a+b-) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a-b-) nonsecretor.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Immunofluorescence techniques in the investigation of a transfusion fatality due to ABO incompatibility.
A 5-year-old girl died some minutes after starting to receive a transfusion of blood, without having previously typed her blood group or that of the donor, who were both supposedly of group 0. Blood groups were investigated in pharynx tissues taken 10 days after her death using the immunofluorescence technique and counter-staining with p-phenylenediamine. It was discovered that the girl was of blood group B and that the transfusion was of blood group A, hence a transfusion error as cause of death is proposed. A field of great medicolegal interest is opened by using immunofluorescent techniques in forensic pathology as an efficient complementary test when doing an autopsy on a person who died during a blood transfusion.
ABO Blood-Group System↗
Molecular genetics of alpha-L-fucosyltransferase genes (H, Se, Le, FUT4, FUT5 and FUT6).
Six human alpha-L-fucosyltransferase genes have been registered in the GDB as FUT1 to FUT6 according to the chronology of their description. FUT1 and FUT2 encode the alpha(1,2)fucosyltransferases H and Se respectively. The FUT2 gene has not been cloned, but it is expected to be closely linked to FUT1 on the long arm of chromosome 19. FUT3, FUT4, FUT5 and FUT6 encode different alpha(1,3)fucosyltransferases which share between 60 and 90% homology with each other, but none with FUT1. Missense and nonsense point mutations have been found to inactivate the cognate enzymes of FUT1, FUT3 and FUT6. FUT3 and FUT6 are closely linked on the short arm of chromosome 19 and encode the Lewis and plasma enzymes respectively. The FUT5 gene has been cloned and sequenced, but its tissue expression has not been defined as yet. FUT4 has been mapped to 11q21 and encodes a monomorphic myeloid enzyme. All but FUT4 are genetically polymorphic. The deficient alleles of FUT1 and FUT6 have a very low incidence and they have been found mainly around the Indian Ocean. A myeloid enzyme is present in 5 to 10 week old human embryos and is later progressively replaced by different patterns of adult fucosyltransferase enzymes in all tissues, except in leukocytes and brain which continue to express a FUT4 like enzyme in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)
Fucosyltransferases↗
Detection and characterization of Lewis antigens in plasma of Lewis-negative individuals. Evidence of chain extension as a result of reduced fucosyltransferase competition.
Nonacid plasma glycolipids from Lewis-negative individuals of nonsecretor, partial-secretor and secretor phenotypes were prepared and separated by thin-layer chromatography and immunostained with radiolabelled Lewis antibodies. Lewis-positive plasma and intestinal epithelial cell glycolipids from Caucasians representing the four recognized Lewis and secretor combined phenotypes were used as controls. By presenting these purified total glycolipids in a cell-free environment to Lewis antibodies we were able to demonstrate the presence of small amounts of Lewis antigens in Lewis-negative individuals. It is shown that lactotetraosylceramide and extended precursor glycolipids are present in all Le(a-b-) nonsecretors. Le(a) was detected in 1 of the 3 Le(a-b-) nonsecretor plasmas and in the intestinal sample of the same phenotype. Lactotetraosylceramide was absent but H type 1 and Le(b) were both present in all group O Le(a-b-) secretors, and extended H type 1 reactive structures were also found in the partial secretor. These results clearly demonstrate that although the Lewis-negative phenotype exists at the serological level, this phenotype is not an 'all-or-nothing' phenomenon at the chemical level. We also show that in the presence of reduced fucosyltransferase activity, increased elongation of the precursor chain occurs, which allows us to postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.
ABO Blood-Group System↗
Oligosaccharides and discordant xenotransplantation.
The initiating factor in the hyperacute rejection of pig organs by human or non-human primates is believed to be related to the presence of preformed "natural" antibodies in the host. In 1991, we demonstrated that human anti-pig antibodies were IgG, IgM and IgA and bound most strongly to oligosaccharides with an alpha galactose (alpha Gal) terminal residue. These included (i) alpha Gal-R (alpha galactose), (ii) alpha Gall-3 beta Gal-R (B disaccharide), (iii) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide) and (iv) alpha Gall-3 beta Gall-4 beta Glc-R (linear B type 6 trisaccharide) where R is (CH2) 8COOCH3. In vitro studies using both the chromium release assay and a live/dead staining technique demonstrated that the cytotoxicity of human sera towards pig cells can be significantly reduced or abolished by immunoadsorption of the serum with immunoaffinity columns of an alpha Gal structure, particularly those with an alpha 1-3 linkage, and not by a large selection of other carbohydrates. Similarly, human anti-pig antibodies can be largely inhibited or "neutralized" by the addition of an alpha 1-3Gal di- or trisaccharide to the serum. Staining of pig vascular endothelium utilizing a panel of carbohydrate-specific lectins and immunoaffinity antibodies demonstrated the presence of three different carbohydrate epitopes, namely (i) alpha Gall-3 beta Gall-4 beta GlcNAc-R (linear B type 2 trisaccharide (ii) alpha NeuAc2-3 beta Gall-4 beta GlcNAc-R (sialyl-N-acetyllactosamine), and (iii) beta Gall-4 beta GlcNAc-R (N-acetyllactosamine). We have investigated organs from several breeds of pig and have concluded that the alpha Gal epitope is either monomorphic or at least has a high incidence in porcine species, since we have not found any pig negative for this antigen. Human vascular endothelial cells have at their surface the same lactosamine-ended precursor and sialylated chains as pigs, but instead of terminal alpha Gal they express the fucosylated polymorphic ABH histo-blood group epitopes. As we have found no evidence that human or baboon plasma contain antibodies directed against sialic acid or lactosamine, and as human tissues contain both of these carbohydrates, it seems unlikely that either of these epitopes plays a role in the vascular rejection that takes place when pig organs are transplanted into primates. Unfortunately, the alpha Gal disaccharide and trisaccharides were not available to us in the large quantities required for extracorporeal immunoadsorption or continuous intravenous infusion in adult baboons.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The gene encoding myeloid alpha-3-fucosyl-transferase (FUT4) is located between D1 1S388 and D11S919 on 11q21.
The last step in the biosynthesis of Le(x) antigen, the addition of a fucose to precursor polysaccharides, can be catalyzed by different alpha-3-fucosyltransferases. We localized the gene (FUT4) encoding myeloid alpha-3-fucosyltransferase by PCR assay using panels of somatic cell and radiation hybrids which retain different rearrangements of chromosome 11. FUT4 was assigned to chromosome band 11q21 between D11S388 and D11S919.
Animals↗
Expression of mucin peptide and blood group ABH- and Lewis-related carbohydrate antigens in normal human conjunctiva.
PURPOSE: The immunohistochemical characterization of mucin peptide antigens and identification of blood group ABH- and Lewis-related carbohydrate antigens expressed in epithelium of normal human conjunctiva. METHODS: Immunoperoxidase characterization of conjunctival glycoproteins was performed using antibodies against peptide and saccharide moieties of gastrointestinal mucins on conjunctival biopsy specimens from 89 healthy individuals of known ABO and Lewis red cell phenotypes. RESULTS: Ocular mucins had epitopes in common with the peptidic core of gastric mucin (M1 epitopes), but also contained sialylated saccharide chains like those of intestinal mucins. Anti-M1 gastric mucins, anti-type 1 precursor, and anti-T antibodies strongly stained the cytoplasm of goblet cells. Anti-Lea, anti-NeuAc-Lea, and anti-Leb antibodies stained both epithelial and goblet cells of Lewis-positive individuals only. Anti-H type 1, anti-H type 2, anti-A, and anti-B stained epithelial cells of ABH secretors. In addition, A-like epitopes independent of secretor phenotype were found in epithelial and goblet cells of all donors. CONCLUSIONS: The conjunctival mucins cross-react with antibodies specific for digestive mucins and for blood group-related carbohydrate epitopes. The distributions of Lewis and secretor phenotypes in conjunctiva are: 19% Lewis-positive nonsecretors, 71% Lewis-positive secretors, and 10% Lewis-negative secretors. Lea and Leb antigens are present on epithelial cells of Lewis-positive nonsecretors and Lewis-positive secretors, respectively, as expected for antigens under control of Lewis and secretor genes. The secretor alpha-2-fucosyltransferase is not expressed on goblet cells, as described for the goblet cells of the distal rectal colonic mucosae. ABH antigens synthesized by the products of A1, A2, B, and Se genes and modulated by the presence of the product of the Lewis gene are found in epithelial cells. In addition, A-related epitopes independent of the A1-A2 subtype, secretor, and Lewis phenotypes also are present in both epithelial and goblet cells.
ABO Blood-Group System↗
Clinical potential of xenotransplantation.
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Animals↗
In vitro model for hyperacute rejection of xenogeneic cells.
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Acute Disease↗
Immunoadsorption of natural antibodies from human serum by affinity chromatography using specific carbohydrates protects pig cells from cytotoxic destruction.
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Animals↗
Carbohydrate antigens of vascular endothelium and other pig tissues reacting with human natural antibodies.
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Animals↗