Department of Cardiology, University Hospital, CH-3010 Bern, Switzerland, 2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871, GSP7, V-437 Moscow, Russia, 3INSERM U504 and University of Paris South XI, F94807 Villejuif Cedex, France, 4Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913, B-334 Moscow, Russia, 5Department of Nephrology, Leiden University Medical Center, NL-2300 RC Leiden, The Netherlands, and 6Department of Medical Chemistry, Vrije Universiteit, NL-1081 BT Amsterdam, The Netherlands
Received on April 19, 1999; revised on July 12, 1999; accepted on July 22, 1999.
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Abstract |
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Key words: xenotransplantation/xenoreactive antibodies/oligosaccharides/glycoconjugates/chemico-enzymatic synthesis/1
3-galactosyltransferase
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Introduction |
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It was recently demonstrated that organs derived from pigs transgenic for human complement regulatory proteins, such as decay accelerating factor (DAF), CD59, or membrane co-factor protein (MCP), are protected against hyperacute rejection when transplanted into baboons (McCurry et al., 1995; White and Yannoutsos 1996). However, as the terminal Gal
1
3Gal epitope is still present on the endothelium of such organs, anti-
Gal antibodies are binding nonetheless, leading to either hyperacute, acute, or delayed xenograft rejections, which may resemble the rejections seen in ABO-incompatible transplantations (Kobayashi et al., 1997
; Palmetshofer et al., 1998a,b). Two principally different strategies to prevent anti-
Gal binding to the endothelial cells have therefore been followed by various research groups. The first is the genetic manipulation of donor pigs in order to block expression of the Gal
1
3Gal epitope. Mouse experiments showed that both the transgenic expression of human
1
2fucosyltransferase (H-transferase) and the inactivation of the
1
3galactosyltransferase gene by homologous recombination successfully prevented the appearance of the Gal
1
3Gal epitope (Sandrin et al., 1995
; Chen et al., 1996
; Sharma et al., 1996
; Osman et al., 1997
; McKenzie et al., 1998
). The second possibility is the use of synthetic antigens either as immunoabsorption substances to remove anti-
Gal from the recipients circulation or as soluble substances to inhibit the binding of anti-
Gal to the
galactosyl epitope on the cell surface. The use of immunoabsorption substances was shown to be successful in the clinical setting of ABO-incompatible transplantation (Bensinger et al., 1981
; Aeschbacher et al., 1987
; Mendez et al., 1992
) and also in experimental xenotransplantation models (Rieben et al., 1995
; Taniguchi et al., 1996
; Kozlowski et al., 1998
; Xu et al., 1998
). Infusion of soluble oligosaccharides to block anti-
Gal binding was shown to effectively delay rejection in ABO-incompatible transplantation (Cooper et al., 1993
) and in anti-
Gal dependent xenotransplantation models as well (Simon et al., 1998
).
Human anti-Gal that bind to pig endothelial cells or the pig kidney cell line PK15, which abundantly expresses the
-galactosyl epitope, were shown to preferentially react with more complex structures than the Gal
1
3Gal disaccharide (Galili and Matta 1996; Neethling et al., 1996
). In fact, as compared with the Gal
1
3Gal disaccharide the trisaccharide Gal
1
3Galß1
4GlcNAc was shown to have an up to 10-fold higher inhibitory effect on anti-
Gal mediated cytotoxicity against PK15 cells in vitro (Neethling et al., 1996
). The experiments presented here were therefore designed to extend the search for optimal synthetic oligosaccharide inhibitors of human anti-
Gal, including oligo- and polymeric variants thereof. As the difficulties and also the costs for direct chemical synthesis of oligosaccharides increase considerably with increasing chain length, a combined chemico-enzymatic strategy for oligosaccharide production was developed and used for the production of one of the substances analyzed in this report.
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Results |
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Immunoabsorption of human serum on PAA-Bdi Sepharose: effects on titers of anti-Gal as detected by ELISA and PK15-cytotoxicity test
Human serum was absorbed over a column of PAA-Bdi Sepharose, and the reduction of both anti-Gal and anti-A trisaccharide antibody titers monitored isotype-specifically by ELISA (Figure 2) and by PK15 cytotoxicity test (Figure 3). PAA-Bdi Sepharose absorbed 96% (IgG) to 99% (IgM) of the anti-
Gal antibody as assessed by ELISA with PAA-Bdi (structure: Figure 1d) as coating antigen, whereas the same column absorbed only 23% of anti-A IgG and 20% of anti-A IgM (assay with PAA-Atri as coating antigen).
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Isotype-specific differences in inhibition of anti-Gal binding by oligosaccharides and glycoconjugates with terminal Gal
1
3Gal
The inhibitory effects of different oligosaccharides and glycoconjugates on human anti-Gal binding to PAA-Bdi coated microtiter plates was analyzed isotype-specifically by ELISA. Inhibition experiments were performed with the disaccharide Gal
1
3Gal, the trisaccharides Gal
1
3Galß1
4GlcNAc and Gal
1
3Galß1
3GlcNAc, and the pentasaccharide Gal
1
3Galß1
4GlcNAcß1
3Galß1
4Glc. As shown in Figure 4, binding of anti-
Gal IgG (Figure 4, lower panel) was inhibited more easily than binding of the respective IgM isotype (Figure 4, upper panel). For both isotypes, the pentasaccharide antigen was the most effective inhibitor, followed by the tri and disaccharides. The two different trisaccharide isomers with ß1
4 and ß1
3 linkages to GlcNAc were equally active as inhibitors of both anti-
Gal IgM and IgG binding (Figure 4, data shown only for the ß1
4 linked trisaccharide).
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Discussion |
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The chemico-enzymatically produced Gal1
3Galß1
3GlcNAc was compared with its physiological ß1
4 linked counterpart, Gal
1
3Galß1
4GlcNAc, as well as the disaccharide Gal
1
3Gal and the pentasaccharide Gal
1
3Galß1
4GlcNAcß1
3Galß1
4Glc for inhibitory activity on anti-
Gal binding in ELISA and PK15 cytotoxicity test. Our results confirm previous observations (Neethling et al., 1996
; Parker et al., 1996
; Simon et al., 1998
) that the tri and pentasaccharide structures are better inhibitors of human anti-
Gal binding than the disaccharide. In fact, the pentasaccharide, which was originally identified as the main
Gal-bearing oligosaccharide species in pig kidney endothelium glycolipids by the group of Samuelsson (Holgersson et al., 1992
; Samuelsson et al., 1994
), had the highest inhibitory capacity of all tested monomeric oligosaccharides. Interestingly, the ß1
3 and ß1
4 linked trisaccharides showed no difference in inhibitory strength, neither in the ELISA system nor in the PK15 cytotoxicity test. Because the ß1
3 linked trisaccharide is easier and therefore cheaper to synthesize by organic synthesis, this substance might be a valuable alternative for future (pre)clinical xenotransplantation experiments which will require relatively large amounts of oligosaccharides as anti-
Gal blockers.
An important part of this study was devoted to a comparison of the isotype-specific inhibitory effects of mono, oligo, and polymeric variants of oligosaccharides. In general, monomeric antigens, including the tri and pentasaccharide, were poor inhibitors of anti-Gal IgM binding, with only zero (disaccharide) to 10% (pentasaccharide) inhibition at the highest tested concentration. Whereas the di, tetra, and octameric constructs were better inhibitors of anti-
Gal IgG than the monomeric disaccharide this was not the case for inhibition of anti-
Gal IgM, nor the PK15-specific cytotoxicity. However, an approximately 1000-fold enhancement of inhibitory activity, based on calculation per
Gal residue, was achieved by using the flexible, hydrophilic PAA-Bdi polymer, which contains ~40 Gal
1
3Gal epitopes per molecule. The anti-
Gal inhibition by PAA-Bdi was specific as assessed by a PAA-Hdi, which did not inhibit PK15 cytotoxicity up to a concentration of 1000 µM, and by the use of Sepharose-bound PAA-Bdi, which efficiently absorbed anti-
Gal from human serum, but not antibodies against the blood group A trisaccharide.
This study was not designed to allow an exact calculation of the number of anti-Gal IgM or IgG molecules that can be bound by the tested substances, nor of their affinities for human anti-
Gal antibodies. However, it can be assumed that the oligomeric constructs of Gal
1
3Gal used in this studywhile being good inhibitors of anti-
Gal IgGwere not able to efficiently block enough of the 10 binding sites of an anti-
Gal IgM molecule to prevent its binding to either PAA-Bdi coated ELISA plates or PK15 cells. In contrast to the relatively compact and rigid oligomers, the PAA-conjugate seems to be large and/or flexible enough to be a highly efficient inhibitor of anti-
Gal IgM as well as PK15 cytotoxicity. Larger oligomeric structures, up to 64-mer dendrimers of Gal
1
3Gal, are currently under investigation and preliminary experiments showed an enhancement of anti-
Gal IgM binding properties with increasing size; these results will be published elsewhere.
The use of PAA-Bdi as a soluble substance for infusion into a patient might be problematic because of the possibility of immune complex formation and will therefore need careful evaluation. However, the excellent anti-Gal IgM- and IgG-binding properties of PAA-Bdi were also retained when it was used coupled to Sepharose as an immunoabsorption material, whereas direct coupling of Gal
1
3Gal to Sepharose via a C3- or C9 spacer produced less efficient immunoabsorption substances (results not shown). In fact, Sepharose-based immunoabsorption substances have a long tradition in clinical application and Sepharose coated with a PAA-conjugate of the blood group B trisaccharide was recently used at our clinic to treat a patient of blood group O who accidentally received a blood group B heart transplant. The treatment proved to be safe and efficient and the patient is still alive and well more than 2 years after transplantation (Mohacsi et al., 1998
). Similarly, PAA-Bdi Sepharose might be used in the future to remove anti-
Gal antibodies prior to pig-to-human xenotransplantation. In view of the results presented here it is likely that the ligand (Bdi) can be further optimized: tri or pentasaccharides, or a mixture thereof as proposed by others (McKane et al., 1998
), conjugated to PAA might be superior to PAA-Bdi. In addition, organic chemical synthesis of the ligand may be facilitated by use of the Gal
1
3Galß1
3GlcNAc isomer. In conclusion, immunoabsorption on PAA-Bdi Sepharose derivatives for now seems to be the most promising approach for medical application of oligosaccharides with terminal Gal
1
3Gal in xenotransplantation in the near future.
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Materials and methods |
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Chemical reagents, human sera, and antibodies
Recombinant bovine UDP-Gal:Galß14GlcNAc
1
3-galactosyltransferase was produced in an insect cell culture system as described earlier (Joziasse et al., 1989
, 1990), and purified by affinity chromatography on UDP-Sepharose. Human sera were obtained from healthy volunteer donors and selected for high titers of anti-
Gal and a pronounced cytotoxic activity against PK15 cells. Monoclonal antibodies against human IgM or IgG (clones HB57 and HB43) were obtained from the American Type Culture Collection (ATCC), Manassas, VA. Biotinylated goat anti-mouse IgG1 antibody was from Southern Biotechnology Associates, Birmingham, AL, and streptavidin conjugated to alkaline phosphatase from Amersham Life Science (Amersham Pharmacia Biotech, Bucks, UK). All other chemicals were from Merck (E. Merck, Darmstadt, Germany) or Sigma (Sigma Chemical Co., St. Louis, MO).
Synthesis of Gal1
3Galß1
3GlcNAc
The trisaccharide Gal1
3Galß1
3GlcNAc was produced by chemico-enzymatic synthesis. The disaccharide Galß1
3GlcNAc was chemically synthesized by Bovin et al. (Bovin and Khorlin, 1984
), and
1
3-galactosylated by use of recombinant bovine
1
3-galactosyltransferase in a final reaction volume of 2.50 ml containing: 5.0 µmol Galß1
3GlcNAc, 0.25 mmol Tris-maleic acid buffer pH 6.8, 10 µmol ATP, 50 µmol
galactonolacton, 100 µg bovine serum albumin, 20 µmol MnCl2, 7.5 µmol UDPGal, and 150 mU bovine
1
3-galactosyltransferase. After incubation at 37°C for 16 h the reaction was stopped on ice. The mixture was chromatographed on a column (bed volume 3 ml) of Dowex 1-X8 (Cl), the flow-through collected and lyophilized. The dry residue was dissolved in 1.0 ml 50 mM ammonium acetate buffer at pH 5.2, and purified by gel filtration on a calibrated column of Bio-Gel P-4 (100200 mesh, 1.6 x 200 cm), equilibrated and run in 50 mM ammonium acetate buffer pH 5.2. Fractions of 3.6 ml were collected, and the elution position of the product was determined based on orcinol assay. Fractions containing hexose, eluting in the trisaccharide region, were collected and lyophilized.
The total amount of product was assayed by the phenol-sulfuric acid hexose assay, and on the basis of the detector response upon HPLC analysis (Lichrosorb-NH2 column, elution with acetonitrile/buffer 80/20 at a flow rate of 0.2 ml/min; detection based on UV absorption at 195 nm). Part of the product was analyzed by 400 MHz 1H-NMR spectroscopy as described earlier (Joziasse et al., 1990).
Immunoabsorption of human serum on PAA-Bdi Sepharose
Polypropylene chromatography columns (Poly-Prep, Bio-Rad Laboratories, Hercules, CA) were packed with 2 ml of PAA-Bdi Sepharose and rinsed with PBS. Nine milliliters of human serum were absorbed over the column and the amount of anti-Gal as well as anti-blood group A trisaccharide antibodies (as a control) measured by ELISA before and after absorption. For the ELISAs, microtiter plates were coated with PAA-Bdi and Atri, respectively, and the sera were diluted 1:40 in PBS-BSA-Tween. Detection of bound antibodies was performed analogously to the oligosaccharide-inhibition test described below. Serum samples before and after absorption were also analyzed in the cytotoxicity test with PK15 cells.
Inhibition of anti-Gal antibody binding by different oligosaccharides and glycoconjugates: isotype-specific detection by ELISA
The degree of inhibition of human anti-Gal binding by the different oligosaccharide inhibitors was analyzed isotype-specifically by ELISA (Rieben et al., 1995
). Human serum was diluted 1:400 in phosphate-buffered saline pH 7.4 (PBS) containing 1% bovine serum albumin (BSA) and 5% Tween 20 (PBS-BSA-Tween). A serial dilution of the oligosaccharide or glycoconjugate to be tested was added to the serum and the mixture incubated for 120 min at 37°C or overnight at 4°C. Polystyrene microtiter plates (NUNC MaxiSorp, NUNC A/S, Roskilde, Denmark) were coated overnight at 4°C with 5 µg/ml of PAA-Bdi in 0.1 M carbonate buffer pH 9.6 and then washed with PBS containing 0.02% Tween 20. The serum-oligosaccharide mixture was added to the coated wells and incubated for 90 min at 37°C. After washing, the bound human antibodies were revealed isotype-specifically with monoclonal antibodies against IgM (HB 57) or IgG (HB 43), followed by biotinylated goat anti-mouse IgG1, streptavidinalkaline phosphatase conjugate, and 4-nitrophenyl phosphate substrate. The development of yellow color was measured with a microplate reader at 405 nm and the data analyzed in Microsoft Excel; values for 50% inhibition (IC50) were calculated by log-logit curve fitting.
Culture of PK15 cells
The PK15 cell line (order no. CCL 33) was obtained from ATCC. Cells were grown in Dulbeccos Modified Eagle Medium (DMEM, Life Technologies Inc., Rockville, MD) with addition of 10% fetal bovine serum (FCS, Life Technologies) and 200 IU/ml of penicillin/streptomycin (Pen/Strep, Life Technologies); DMEM++. Cells were grown in 75 cm2 polystyrene culture flasks (Becton Dickinson Franklin Lakes, NJ) until they were used in the cytotoxicity assay (see below).
Inhibition of anti-Gal antibody-mediated cytotoxicity by different oligosaccharides and glycoconjugates: quantitation by nonradioactive cytotoxicity assay with PK15 cells
The cytotoxicity test was performed analogously to the one described by Neethling and Cooper (Neethling et al., 1999). PK15 cells were seeded at ~150,000/ml in 10 µl DMEM++ into 60 well Terasaki plates (Robbins Scientific, Sunnyvale, CA) and incubated for 2448 h. The oligosaccharide or glycoconjugate to be tested was serially diluted in human serum and the mixture incubated over night at 4°C. Immediately before use in the assay 10% rabbit serum (Sigma) was added as additional complement source and the mixture then incubated for 10 min in the Terasaki plates with the PK15 cells. The plates were washed and the amount of cytotoxicity was revealed with a two-color fluorescent live/dead stain (calcein AM/ethidium homodimer 1, Molecular Probes Europe BV, Leiden, The Netherlands).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Auchincloss,H. and Sachs,D.H. (1998) Xenogeneic transplantation. Annu. Rev. Immunol., 16, 433470.[ISI][Medline]
Bensinger,W.I., Baker,D.A., Buckner,C.D., Clift,R.A. and Thomas,E.D. (1981) In vitro and in vivo removal of anti-A erythrocytic antibody by adsorption to a synthetic immunoadsorbent. Transfusion, 21, 335342.[ISI][Medline]
Blanken,W.M. and Van den Eijnden,D.H. (1985) Biosynthesis of terminal Gal1
3Galß1
4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide
1
3-galactosyltransferase from calf thymus. J. Biol. Chem., 260, 1292712934.
Bovin,N.V. and Khorlin,A. (1984) Synthesis of determinant oligosaccharides of the ABH (type 1) blood group antigen and Leb tetrasaccharide from a common precursor. Bioorg. Khim., 10, 853860.[ISI][Medline]
Bovin,N.V., Korchagina,E.Y., Zemlyanukhina,T.V., Byramova,N.E., Galanina,O.E., Zemlyakov,A.E., Ivanov,A.E., Zubov,V.P. and Mochalova,L.V. (1993) Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides. Glycoconjugate J., 10, 142151.[ISI][Medline]
Chen,C.G., Fisicaro,N., Shinkel,T.A., Aitken,V., Katerelos,M., van Denderen,B.J.W., Tange,M.J., Crawford,R.J., Robins,A.J., Pearse,M.J. and dApice,A.J.F. (1996) Reduction in Gal1
3Gal epitope expression in transgenic mice expressing human H-transferase. Xenotransplantation, 3, 6975.[ISI]
Collins,B.H., Cotterell,A.H., McCurry,K.R., Alvarado,C.G., Magee,J.C., Parker,W. and Platt,J.L. (1995) Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection. J. Immunol., 154, 55005510.
Cooper,D.K., Human,P.A., Lexer,G., Rose,A.G., Rees,J., Keraan,M. and Du Toit,E. (1988) Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J. Heart Transplant., 7, 238246.[ISI][Medline]
Cooper,D.K.C., Ye,Y., Niekrasz,M., Kehoe,M., Martin,M., Neethling,F.A., Kosanke,S., Debault,L.E., Worsley,G., Zuhdi,N., Oriol,R. and Romano,E. (1993) Specific intravenous carbohydrate therapya new concept in inhibiting antibody-mediated rejection: experience with ABO-incompatible cardiac allografting in the baboon. Transplantation, 56, 769777.[ISI][Medline]
Cooper,D.K., Koren,E. and Oriol,R. (1994) Oligosaccharides and discordant xenotransplantation. Immunol. Rev., 141, 3158.[ISI][Medline]
Dalmasso,A.P., Platt,J.L. and Bach,F.H. (1991) Reaction of complement with endothelial cells in a model of xenotransplantation. Clin. Exp. Immunol., 86 (Suppl. 1), 3135.
de Vries,T., Palcic,M.P., Schoenmakers,P.S., van den Eijnden,D.H. and Joziasse,D.H. (1997) Acceptor specificity of GDP-Fuc:Galß14GlcNAcR
3-fucosyltransferase VI (FucT VI) expressed in insect cells as soluble, secreted enzyme. Glycobiology, 7, 921927.[Abstract]
Galili,U. and Matta,K.L. (1996) Inhibition of anti-Gal IgG binding to porcine endothelial cells by synthetic oligosaccharides. Transplantation, 62, 256262.[ISI][Medline]
Galili,U., Rachmilewitz,E.A., Peleg,A. and Flechner,I. (1984) A unique natural human IgG antibody with anti -galactosyl specificity. J. Exp. Med., 160, 15191531.[Abstract]
Good,A.H., Cooper,D.K.C., Malcolm,A.J., Ippolito,R.M., Koren,E., Neethling,F.A., Ye,Y., Zuhdi,N. and Lamontagne,L.R. (1992) Identification of carbohydrate structures that bind human antiporcine antibodiesimplications for discordant xenografting in humans. Transplant Proc., 24, 559562.[ISI][Medline]
Hammer,C., Linke,R., Wagner,F. and Diefenbeck,M. (1998) Organs from animals for man. Int. Arch. Allergy Immunol., 116, 521.[ISI][Medline]
Hokke,C.H., van der Ven,J.G., Kamerling,J.P. and Vliegenthart,J.F. (1993) Action of rat liver Galß14GlcNAc
2
6-sialyltransferase on Manß1
4GlcNAcß-OMe, GalNAcß1
4GlcNAcß-OMe, Glcß1
4GlcNAcß-OMe and GlcNAcß1
4GlcNAcß-OMe as synthetic substrates. Glycoconj. J., 10, 8290.[ISI][Medline]
Holgersson,J., Cairns,T.D.H., Karlsson,E.C., Backer,A.E., Breimer,M.E., Taube,D.H., Welsh,K.I. and Samuelsson,B.E. (1992) Carbohydrate specificity of human immunoglobulin-M antibodies with pig lymphocytotoxic activity. Transplant Proc., 24, 605608.[ISI][Medline]
Joziasse,D.H., Shaper,J.H., van den Eijnden,D.H., van Tunen,A.J. and Shaper,N.L. (1989) Bovine 1
3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. J. Biol. Chem., 264, 1429014297.
Joziasse,D.H., Shaper,N.L., Salyer,L.S., van den Eijnden,D.H., van der Spoel,A.C. and Shaper,J.H. (1990) 1
3-Galactosyltransferase: the use of recombinant enzyme for the synthesis of a-galactosylated glycoconjugates. Eur. J. Biochem., 191, 7583.[Abstract]
Kobayashi,T., Taniguchi,S., Neethling,F.A., Rose,A.G., Hancock,W.W., Ye,Y., Niekrasz,M., Kosanke,S., Wright,L.J., White,D.J.G. and Cooper,D.K.C. (1997) Delayed xenograft resection of pig-to-baboon cardiac transplants after cobra venom factor therapy. Transplantation, 64, 12551261.[ISI][Medline]
Kozlowski,T., Ierino,F.L., Lambrigts,D., Foley,A. andrews,D., Awwad,M., Monroy,R., Cosimi,A.B., Cooper,D.K.C. and Sachs,D.H. (1998) Depletion of anti Gal1
3Gal antibody in baboons by specific
Gal immunoaffinity columns. Xenotransplantation, 5, 122131.[ISI][Medline]
McCurry,K.R., Kooyman,D.L., Alvarado,C.G., Cotterel,A.H., Martin,M.J., Logan,J.S. and Platt,J.L. (1995) Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury. Nature Med., 1, 423427.[ISI][Medline]
McKane,W., Lee,J., Preston,R., Hacking,A., Simpson,P., Lynds,S., Goldberg,L., Cairns,T. and Taube,D. (1998) Polymorphism in the human anti-pig natural antibody repertoireimplications for antigen-specific immunoadsorption. Transplantation, 66, 626633.[ISI][Medline]
McKenzie,I.F.C., Li,Y.Q., Patton,K., Thall,A.D. and Sandrin,M.S. (1998) A murine model of antibody-mediated hyperacute rejection by Gal1
3Gal antibodies in Gal o/o mice. Transplantation, 66, 754763.[ISI][Medline]
Mendez,R., Sakhrani,L., Aswad,S., Minasian,R., Obispo,E. and Mendez,R.G. (1992) Successful living-related ABO incompatible renal transplant using the BIOSYNSORB immunoadsorption column. Transplant Proc., 24, 17381740.[ISI][Medline]
Mohacsi,P.J., Carrel,T., Tschanz,H.U., Nydegger,U.E., Wuillemin,W.E. and Rieben,R. (1998) Rescuing of an accidentally transplanted ABO-incompatible cardiac allograft by combined use of specific immunoabsorption and anti-complement treatment (abstract). 3rd International Conference on new Trends in Clinical and Experimental Immunosuppression, Geneva.
Neethling,F.A., Joziasse,D., Bovin,N., Cooper,D.K. and Oriol,R. (1996) The reducing end of Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera. Transpl. Int., 9, 98101.[ISI][Medline]
Neethling,F.A., Koscec,M., Oriol,R., Cooper,D.K.C. and Koren,E. (1999) A reliable, rapid and inexpensive two-color fluorescence assay to monitor serum cytotoxicity in xenotransplantation. J. Immunol. Methods, 222, 3144.[ISI][Medline]
Oriol,R., Ye,Y., Koren,E. and Cooper,D. (1993) Carbohydrate antigens of pig tissues reacting with human natural antibodies as potential targets for hyperacute vascular rejection in pig-to-man organ xenotransplantation. Transplantation, 56, 14331442.[ISI][Medline]
Oriol,R., Candelier,J.J., Taniguchi,S., Balanzino,L., Peters,L., Niekrasz,M., Hammer,C. and Cooper,D.K.C. (1999) Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research. Xenotransplantation, 6, 7889.
Osman,N., McKenzie,I.F.C., Ostenried,K., Ioannou,Y.A., Desnick,R.J. and Sandrin,M.S. (1997) Combined transgenic expression of -galactosidase and
1
2-fucosyltransferase leads to optimal reduction in the major xenoepitope Gal
1
3Gal. Proc. Natl. Acad. Sci. USA, 94, 1467714682.
Palmetshofer,A., Galili,U., Dalmasso,A.P., Robson,S.C. and Bach,F.H. (1998a) -Galactosyl epitope-mediated activation of porcine aortic endothelial cells: type I activation. Transplantation, 65, 844853.[ISI][Medline]
Palmetshofer,A., Galili,U., Dalmasso,A.P., Robson,S.C. and Bach,F.H. (1998b) -Galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation. Transplantation, 65, 971978.[ISI][Medline]
Parker,W., Lateef,J., Everett,M.L. and Platt,J.L. (1996) Specificity of xenoreactive anti-Gal 13Gal IgM for
-galactosyl ligands. Glycobiology, 6, 499506.[ISI]
Platt,J.L., Fischel,R.J., Matas,A.J., Reif,S.A., Bolman,R.M. and Bach,F.H. (1991) Immunopathology of hyperacute xenograft rejection in a swine-to-primate model. Transplantation, 52, 214220.[ISI][Medline]
Rieben,R., von Allmen,E., Korchagina,E.Y., Nydegger,U.E., Neethling,F.A., Kujundzic,M., Koren,E., Bovin,N.V. and Cooper,D.K.C. (1995) Detection, immunoabsorption and inhibition of cytotoxic activity of anti-Gal antibodies using newly developed substances with synthetic Gal
1
3Gal disaccharide epitopes. Xenotransplantation, 2, 98106.
Samuelsson,B.E., Rydberg,L., Breimer,M.E., Backer,A., Gustavsson,M., Holgersson,J., Karlsson,E., Uyterwaal,A.C., Cairns,T. and Welsh,K. (1994) Natural antibodies and human xenotransplantation. Immunol. Rev., 141, 151168.[ISI][Medline]
Sandrin,M.S. and McKenzie,I.F. (1994) Gal1
3Gal, the major xenoantigen (s) recognised in pigs by human natural antibodies. Immunol. Rev., 141, 169190.[ISI][Medline]
Sandrin,M.S., Fodor,W.L., Mouhtouris,E., Osman,N., Cohney,S., Rollins,S.A., Guilmette,E.R., Setter,E., Squinto,S.P. and McKenzie,I.F. (1995) Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis. Nature Med., 1, 12611267.[ISI][Medline]
Sharma,A., Okabe,J., Birch,P., McClellan,S.B., Martin,M.J., Platt,J.L. and Logan,J.S. (1996) Reduction in the level of Gal1
3Gal in transgenic mice and pigs by the expression of an
1
2-fucosyltransferase. Proc. Natl Acad. Sci. USA, 93, 71907195.
Simon,P.M., Neethling,F.A., Taniguchi,S., Goode,P.L., Zopf,D., Hancock,W.W. and Cooper,D.K.C. (1998) Intravenous infusion of Gal1
3Gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts. Transplantation, 65, 346353.[ISI][Medline]
Taniguchi,S., Neethling,F.A., Korchagina,E.Y., Bovin,N., Ye,Y., Kobayashi,T., Niekrasz,M., Li,S., Koren,E., Oriol,R. and Cooper,D.K.C. (1996) In vivo immunoadsorption of antipig antibodies in baboons using a specific Gal1
3Gal column. Transplantation, 62, 13791384.[ISI][Medline]
Tsvetkov,D.E., Cheshev,P.E., Tuzikov,A.B., Pazynina,G.V., Bovin,N.V., Rieben,R. and Nifantev,N.E. (1999) Synthesis of neoglycoconjugate dendrimers. Mendeleev Commun., 4750.
White,D.J. and Yannoutsos,N. (1996) Production of pigs transgenic for human DAF to overcome complement-mediated hyperacute xenograft rejection in man. Res. Immunol., 147, 8894.[ISI][Medline]
Xu,Y., Lorf,T., Sablinski,T., Gianello,P., Bailin,M., Monroy,R., Kozlowski,T., Awwad,M., Cooper,D.K.C. and Sachs,D.H. (1998) Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Gal1
3Galß1
4ßGlc-X immunoaffinity column. Transplantation, 65, 172179.[ISI][Medline]
Yang,Q., Wang,Y.Y., Bollinger,R.R. and De Buysscher,E.V. (1992) Discordant complement systems as a factor in hyperacute xenograft rejection. Transplant Proc., 24, 481482.[ISI][Medline]
Ye,Y., Niekrasz,M., Kosanke,S., Welsh,R., Jordan,H.E., Fox,J.C., Edwards,W.C., Maxwell,C. and Cooper,D.K. (1994) The pig as a potential organ donor for man. A study of potentially transferable disease from donor pig to recipient man. Transplantation, 57, 694703.[ISI][Medline]