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• W2139676396 abstract "We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future. We have been successful in generating several lines of transgenic mice and pigs that contain the human β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galα1–3Galβ1–4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as α1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future. Galα1–3Galβ1–4GlcNAc-R 3GT, α1,3-galactosyltransferase 2FT, α-1,2-fucosyltransferase 3FT, α1,3-fucosyltransferase 3ST, α2,3-sialyltransferase 6ST, α2,6-sialyltransferase β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III α-3-d-mannoside β-1,4-N-acetylglucosaminyltransferase IV α-6-d-mannoside β-1,6N-acetylglucosaminyltransferase V pig endothelial cell phosphate-buffered saline 2-(N-morpholino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid high performance liquid chromatography normal human serum antibody(ies) monoclonal antibody fluorescein isothiocyanate Griffonia simplicifolia I Hanganutziu-Deicher fluorescence-activated cell sorter lactate dehydrogenase natural killer cell(s) complement hemolytic activity N-glycolylneuraminic acid CMP-N-acetylneuraminic acid The increasing problem of the worldwide shortage of donor organs has led to a revival of interest in xenotransplantation. The expression of complement regulatory proteins, such as membrane cofactor protein (CD46) (1Seya T. Turner J.R. Atkinson J.P. J. Exp. Med. 1986; 163: 837-855Crossref PubMed Scopus (327) Google Scholar), decay accelerating factor (CD55) (2Nicholson-Weller A. Burge J. Fearon D.T. Weller P.F. Austen K.F. J. Immunol. 1982; 129: 184-189PubMed Google Scholar), and CD59 (3Sugita Y. Nakano Y. Tomita M. J. Biochem. 1988; 104: 633-637Crossref PubMed Scopus (218) Google Scholar, 4Okada H. Nagami Y. Takahashi K. Okada N. Hideshima T. Takizawa H. Kondo J. Biochem. Biophys. Res. Commun. 1989; 162: 1553-1559Crossref PubMed Scopus (101) Google Scholar) in transgenic pigs, has been shown to be very effective in protecting against hyperacute rejection in a xenograft (5Rosengard A.M. Cary N.R.B. Langford G.A. Tucker A.W. Wallwork J. White D.J.G. Transplantation. 1995; 59: 1325-1333Crossref PubMed Google Scholar, 6Fodor W.L. Williams B.L. Matis L.A. Madri J.A. Rollins S.A. Knight J.W. Velander W. Squinto S.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11153-11157Crossref PubMed Scopus (306) Google Scholar, 7McCurry K.R. Kooyman D.L. Alvarado C.G. Cotterell A.H. Martin M.J. Logan J.S. Platt J.L. Nat. Med. 1995; 1: 423-427Crossref PubMed Scopus (487) Google Scholar, 8Lambrigts D. Sachs D.H. Cooper D.K. Transplantation. 1998; 66: 547-561Crossref PubMed Scopus (202) Google Scholar). However, since Galili et al. reported that the Galα1–3Galβ1–4 GlcNAc-R (α-Gal)1 is the major antigen to human xenotransplantation in pig, genetic approaches to modify this glycoantigen have been the focus of xenotransplantation studies. This antigen was first described as an internal type 1 chain but was later corrected as a linear type 2 oligosaccharide, the α-Gal that is synthesized by α1,3-galactosyltransferase (α1,3GT) (9Eto T. Ichikawa Y. Nishimura K. Ando S. Yamakawa T. J. Biochem. 1968; 64: 205-213Crossref PubMed Scopus (138) Google Scholar, 10Stellner K. Saito H. Hakomori S. Arch. Biochem. Biophys. 1973; 155: 464-472Crossref PubMed Scopus (673) Google Scholar, 11Uemura K. Yuzawa M. Taketomi T. J. Biochem. 1978; 83: 463-471Crossref PubMed Scopus (69) Google Scholar, 12Egge H. Kordowicz M. Peter-Katalinic Hanfland P. J. Biol. Chem. 1985; 260: 4927-4935Abstract Full Text PDF PubMed Google Scholar, 13Galili U. Rachmilewitz E.A. Peleg A. Flechner I. J. Exp. Med. 1984; 160: 1519-1531Crossref PubMed Scopus (574) Google Scholar). The human sequence, however, has suffered a deletion of a single nucleotide at two separate positions, which disrupts the translational reading frame (14Larsen R.D. Rivera-Marrero C.A. Ernst L.K. Cummings R.D. Low J.B. J. Biol. Chem. 1990; 265: 7055-7061Abstract Full Text PDF PubMed Google Scholar, 15Galili U. Swanson K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7401-7404Crossref PubMed Scopus (268) Google Scholar). As a result, humans produce a natural antibody that comprises as much as 1% of the circulating IgG and which is also found in significant amounts as an IgM antibody (16Galili U. Clark M.R. Shohet S.B. Buehler J. Macher B.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1369-1373Crossref PubMed Scopus (511) Google Scholar). The most reliable approach for the elimination of α-Gal from pig tissue is to disrupt the pig α1,3GT gene via homologus recombination and/or gene transfer. However, gene targeting is not feasible at the present time. Another strategy for down-regulating the α-Gal involves taking advantage of enzymatic competition involving terminal glycosylation between α1,3GT and other glycosyltransferases for the common acceptor substrate in the trans-Golgi stack and network. Several glycosyltransferases, such as α1,2-fucosyltransferase (α1,2FT) (17Larsen R.D. Ernst L.K. Nair R.P. Lowe J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6674-6678Crossref PubMed Scopus (303) Google Scholar, 18Sandrin M.S. Fodor W.L. Mouhtouris E. Osman N. Cohney S. Rollins S.A. Guilmette E.R. Setter E. Squinto S.P. McKenzie I.F.C. Nat. Med. 1995; 1: 1261-1267Crossref PubMed Scopus (282) Google Scholar), α1,3-fucosyltransferase (α1,3FT), α2,3-sialyltransferase (α2,3ST) (19Tanemura M. Miyagawa S. Koyota S. Koma M. Matsuda H. Tsuji S. Shirakura R. Taniguchi N. J. Biol. Chem. 1998; 273: 16421-16425Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and α2,6-sialyltransferase (α2,6ST) represent possibilities (20Koma M. Miyagawa S. Honke K. Ikeda Y. Koyota S. Miyoshi S. Matsuda H. Tsuji S. Shirakura R. Taniguchi N. Glycobiology. 2000; 10: 745-751Crossref PubMed Scopus (25) Google Scholar). The strategy we present in this paper involves controlling sugar chain biosynthesis using the β-d-mannoside β-1,4-N-acetylglucosaminyltransferase III (GnT-III) (21Nishikawa A. Ihara Y. Hatakeyama M. Kangawa K. Taniguchi N. J. Biol. Chem. 1992; 267: 18199-18204Abstract Full Text PDF PubMed Google Scholar,22Ihara Y. Nishikawa A. Tohma T. Soejima H. Niikawa N. Taniguchi N. J. Biochem. 1993; 113: 692-698Crossref PubMed Scopus (102) Google Scholar), which leads to a remodeling of the total antigenicity of the cell surface (23Tanemura M. Miyagawa S. Ihara Y. Matsuda H. Shirakura R. Taniguchi N. Biochem. Biophys. Res. Commun. 1997; 235: 359-364Crossref PubMed Scopus (42) Google Scholar). The mechanism by which the introduction of the GnT-III gene significantly suppresses xenoantigens is not fully understood, but its suppression could, in part, be caused by the inhibition of further branching of the carbohydrate moieties and/or a lack of maturation in processing; that is, once a bisecting GlcNAc residue is added to the core mannose by GnT-III, competitive enzymes, including α-3-d-mannoside β-1,4-N-acetylglucosaminyltransferase IV (GnT-IV) and α-6-d-mannoside β-1,6 N-acetylglucosaminyltransferase V (GnT-V), are prevented from introducing any further tri-structures in the Golgi stack (24Taniguchi N. Yoshimura M. Miyoshi E. Ihara Y. Nishikawa A. Fujii S. Glycobiology. 1996; 6: 691-694Crossref PubMed Scopus (55) Google Scholar). Our previous structural analysis of N-linked sugars of the pig endothelial cell (PEC) transfectant with GnT-III revealed that the complex type oligosaccharides with bi-, tri-, and tetraantennary structures, which contained α-Gal, decreased markedly with a parallel increase in bisected structures that contained no α-galactosyl residues (24Taniguchi N. Yoshimura M. Miyoshi E. Ihara Y. Nishikawa A. Fujii S. Glycobiology. 1996; 6: 691-694Crossref PubMed Scopus (55) Google Scholar, 25Koyota S. Ikeda Y. Miyagawa S. Ihara H. Koma M. Honke K. Shirakura R. Taniguchi N. J. Biol. Chem. 2001; 276: 32867-32874Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the present study, transgenic mouse and pig lines carrying GnT-III were produced, and the expression levels of GnT-III, as well as changes in antigenicity in the various tissues, were analyzed. A cDNA of human GnT-III was subcloned into the pCX vector; a β-actin promoter and a cytomegalovirus enhancer (26Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4583) Google Scholar). The plasmid was separately transformed into Escherichia coli C600 and then amplified using standard techniques. B6C3F1 female mice were induced to superovulate and then crossed with B6C3F1 males. Microinjection and embryo transfer were performed by standard methods to generate transgenic mice. The DNA fragments for microinjection were prepared by digesting the plasmids with SalI and HindIII to remove the vector sequences. DNA fragments were microinjected into mouse ova (C57BL/6 ×C3H), resulting in transgenic mice. Genomic DNA from the tail tips of newborn mice was analyzed by polymerase chain reaction and Southern blots to identify the produced transgenic animals. Mice carrying these pCX-GnT-III plasmids were crossed with B6 to obtain offspring. The pCX-GnT-III gene was also used to produce transgenic pigs. Prepubertal cross-bred gilts (Large White/LandraceXDuroc) were used as embryo donors and recipients. Methods used in the superovulation for gilts have been presented previously (27Murakami H. Nagashima H. Takahagi Y. Fujimura T. Miyagawa S. Okabe M. Seya T. Shigehisa T. Taniguchi N. Shirakura R. Kinoshita T. Transplant. Proc. 2000; 32: 2505-2506Crossref PubMed Scopus (15) Google Scholar). Embryo donors were artificially inseminated, and embryos were collected 50–54 h after human chorionic gonadotropin injection. Embryos were centrifuged at 12,000 × g for 8 min to visualize the pronuclei and microinjected with several thousand copies of the hybrid gene. Microinjected embryos were then transferred to unmated synchronized recipients or the embryo donors (donor-recipients). Transgenic pigs were identified by polymerase chain reaction and/or Southern blot analysis with genomic DNA extracted from the tail tips of the newborn pigs. Founder transgenic pigs were bred with nontransgenic boars or gilts to obtain the second generation. For the assay of enzyme activity, tissues were sonicated and lysed in PBS. The enzyme activities of GnT-III, GnT-IV (28Nishikawa A. Gu J. Fuji S. Taniguchi N. Biochim. Biophys. Acta. 1990; 1035: 313-318Crossref PubMed Scopus (113) Google Scholar), and GnT-V (29Gu J. Nishikawa A. Tsuruoka N. Ohno M. Yamaguchi N. Kangawa K. Taniguchi N. J. Biochem. 1993; 113: 614-619Crossref PubMed Scopus (137) Google Scholar) were determined using the pyridylaminated biantennary sugar chain GlcNAcβ1–2Manα1–6 (GlcNAcβ1–2Manα1–3)Manβ1–4GlcNAcβ1–4GlcNAc-PA as a substrate (30Nishikawa A. Fujii S. Sugiyama T. Taniguchi N. Anal. Biochem. 1988; 170: 349-354Crossref PubMed Scopus (59) Google Scholar, 31Hase S. Ibuki T. Ikenaka T. J. Biochem. (Tokyo). 1984; 95: 197-203Crossref PubMed Scopus (390) Google Scholar). The reaction buffer for the GnT-III assay consisted of 125 mm MES buffer, pH 6.25, containing 40 mmUDP-GlcNAc, 20 mm MnCl2, 400 mmGlcNAc, and 1% Triton X-100. The reaction mixture for GnT-IV contained 250 mm MOPS buffer, pH 7.3, 80 mm UDP-GlcNAc, 15 mm MnCl2, 400 mm GlcNAc, and 1.0% (W/V) Triton X-100. Assayed GnT-V activity employed, pH 6.25, 250 mm MES buffer, containing 80 mm UDP-GlcNAc, 20 mm EDTA, 400 mm GlcNAc, and 1.0% (W/V) Triton X-100. It should be noted that Mn2+ is not essential for GnT-V activity. 20 mm EDTA, contained in the reaction mixture, completely inhibited GnT-III activity. To 25 µl of these solutions 10 µl of 100 µm substrate was added followed by 15 µl of cell lysate. The assay mixture was then incubated at 37 °C for 3 h (28Nishikawa A. Gu J. Fuji S. Taniguchi N. Biochim. Biophys. Acta. 1990; 1035: 313-318Crossref PubMed Scopus (113) Google Scholar). The acceptor substrate, pyridylaminated lacto-N-neotetraose (Galβ1–4GlcNAcβ1–3Galβ1–4Glc-PA) at a final concentration of 10 µm was employed in the α1,3GT activity assays. Lacto-N-neotetraose was purchased from Seikagaku Kogyo (Tokyo, Japan) and pyridylaminated according to the method of Kondoet al. (32Kondo A. Suzuki J. Kuraya N. Hase S. Kato I. Ikenaka T. Agric. Biol. Chem. 1990; 54: 2169-2170Crossref PubMed Scopus (195) Google Scholar). α1,3GT activity was assayed in a reaction mixture containing 10 µm HEPES, pH 7.2, 20 mmUDP-galactose, 10 mm MnCl2, 33 mmNaCl, and 3 mm KCl. 10 µl of 50 µmsubstrate and 15 µl of cell lysate were added to this mixture, which was then incubated at 37 °C for 3 h (19Tanemura M. Miyagawa S. Koyota S. Koma M. Matsuda H. Tsuji S. Shirakura R. Taniguchi N. J. Biol. Chem. 1998; 273: 16421-16425Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The enzyme reactions were quenched by boiling for 5 min. The samples were then centrifuged at 12,000 × g for 5 min, and an aliquot of each supernatant was subjected to HPLC analysis, using a TSK-gel ODS-80TM column (4.6 × 250 mm). The reaction products were eluted with 20 mm acetate buffer, pH 4.0, containingn-butyl alcohol at a flow rate of 1.0 ml/min at 55 °C and were monitored with a fluorescence spectrophotometer (Shimadzu model RF-10AXL, Tokyo) using excitation and emission wavelengths of 320 and 400 nm, respectively. The specific activity of the enzyme is expressed as mol of product produced per h of incubation per mg of protein. Protein concentrations were determined with a BCA protein assay kit (Pierce, Rockford, IL), using bovine serum albumin as a standard. Various organs were excised from transgenic mice and pigs. A portion of each organ was fixed with 4% paraformaldehyde and Dulbecco's PBS for 30 min. The fixed sections were incubated with blocking solution (2% bovine serum albumin and Dulbecco's PBS) for 1 h and then reacted with normal human pooled serum (NHS) of blood type O or a mouse mAb anti-α-Gal, M86 (a generous gift from Dr. U. Galili) (33Galili U. LaTemple D.C. Radic M.Z. Transplantation. 1998; 65: 1129-1132Crossref PubMed Scopus (102) Google Scholar). After removal of excess antibody, the sections were reacted with FITC-conjugated goat anti-human Ig (Cappel, West Chester, PA), or FITC-conjugated rabbit anti-mouse IgM (Cappel ICN, Aurora, OH), respectively. Each section was also reacted FITC-conjugated Griffonia simplicifolia I (GS-IB4) lectin, which binds the α-Gal (Honen, Tokyo). Double staining of pancreas islets was also carried out using anti-GnT-III mAb (Fujirevio, Tokyo) and anti-pig insulin polyclonal Ab (DAKO Japan, Kyoto), and subsequently stained with FITC-conjugated anti-mouse Ig (Cappel ICN) and Alexa Fluor 594-labeled goat anti-guinea pig IgG secondary antibody (Molecular Probes Europe BV, Leiden, The Netherlands), respectively. For the detection of monkey C3 and C5b-9 deposition, mouse anti-human C3 mAb and mouse anti-human C5b-9 mAb (DAKO Japan) were used as the first antibody and subsequently stained with FITC-conjugated rabbit anti-mouse IgG (Cappel ICN). The slides were viewed by means of a Zeiss Axioplan 2 universal microscope (Jena, Germany). The PEC from transgenic pigs with or without human GnT-III was removed from the aorta and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum withl-glutamine (Life Technologies, Inc., Rockville, MD) and penicillin/streptomycin (Meiji, Tokyo) (34Miyagawa S. Shirakura R. Iwata K. Nakata S. Matsumiya G. Izutani H. Matsuda H. Terado A. Matsumoto M. Nagasawa S. Seya T. Transplantation. 1994; 58: 834-840Crossref PubMed Scopus (119) Google Scholar). The PECs were incubated with various dilutions of NHS at 4 °C for 1 h, washed, and then incubated with 1.25 µg of FITC-conjugated anti-human Ig (Cappel) as a second antibody for 1 h at 4 °C. The cell surface carbohydrate epitopes were also examined with an FITC-conjugated GS-IB4 lectin (Honen) and chicken anti-Hanganutziu-Deicher (H-D) antigen polyclonal Ab (a gift from Dr. N. Wakamiya, Osaka University, Osaka, Japan) and FITC-conjugated rabbit anti-chicken IgG (Cappel). The stained cells were analyzed with a FACS Calibur flow cytometer (Nippon Becton Dickinson, Tokyo). This assay was performed according to the manufacture's recommended protocol, using an MTX LDH kit (Kyokuto, Tokyo). The PEC from transgenic pigs was plated at 2 × 104 cells/well in flat bottomed gelatin-coated 96-well trays 1 day prior to assay. Fifteen hours after plating the cells, the wells were washed twice with serum-free Dulbecco's modified Eagle's medium to remove the LDH, which is present in fetal calf serum, and incubated with several concentrations of NHS that had been diluted with Dulbecco's modified Eagle's medium. The plates were incubated for 2 h at 37 °C and the released LDH was then measured. The percent cytotoxicity was calculated using Equation 1 Cytotoxicity=(E−N−S)/(M−N−S)×100Equation 1 where E is the experimentally observed release of LDH activity from the target PEC, N the LDH activity in each concentration of NHS, S the spontaneous release of LDH activity from target PEC incubated in the absence of NHS, and M the maximal release of LDH activity, as determined by sonication. The spontaneous release of LDH activity from PEC was less than 5%, compared with the maximal release obtained by sonication (34Miyagawa S. Shirakura R. Iwata K. Nakata S. Matsumiya G. Izutani H. Matsuda H. Terado A. Matsumoto M. Nagasawa S. Seya T. Transplantation. 1994; 58: 834-840Crossref PubMed Scopus (119) Google Scholar). The PEC from transgenic pigs were plated at 2 × 104 cells/well in a flat bottomed gelatin-coated 96-well plate. Fifteen hours after plating the cells, the plates were incubated with effector cells, an NK-like cell line, YT cells, which were kindly provided by Drs. Junji Yodoi and Keisuke Teshigawara (University of Kyoto) (35Yodoi J. Teshigawara K. Nikaido T. Fukui K. Noma T. Honjo T. Takigawa M. Sasaki M. Minato N. Tsudo M. Uchiyama T. Maeda M. J. Immunol. 1985; 134: 1623-1630PubMed Google Scholar), at various effector:target ratios. Each assay was performed in triplicate. After a 4-h incubation at 37 °C, the released LDH was measured using an MTX LDH kit (Kyokuto). The spontaneous release of LDH activity from effector cells and target cells were less than 10 and 5%, respectively. The results are expressed as the percent of specific lyses (36Miyagawa S. Nakai R. Yamada M. Tanemura M. Ikeda Y. Taniguchi N. Shirakura R. J. Biochem. 1999; 126: 1067-1073Crossref PubMed Scopus (39) Google Scholar). Examinations were carried out for according to the guidelines for the handling of animals from the Research Institute of the HAMRI Co., Ltd. (Ibaraki, Japan). The heterozygous transgenic pigs with GnT-III and wild-type controls, either sex (18–22 days old, 2.5–6.0 kg), were used as donors for all experiments. Cynomolgus monkeys, Macaca fascicularis, obtained from HAMRI Co., Ltd., weighing 2.5–7.0 kg, were used as recipients. Preoperative serum from all monkeys was assayed for the anti-pig endothelial cell antibody titer and complement hemolytic activity (CH50 unit) (37Mayer M.M. Experimental Immunochemistry. 2nd Ed. Charles C Thomas Publisher, Springfield, IL1961: 133Google Scholar). The anti-PEC antibodies, IgG and IgM, were checked, using PEC as a target. The PECs from control pigs were incubated with 10% serum from each recipient monkey at 4 °C for 1 h, washed, and then incubated with 1.25 µg of FITC-conjugated anti-human IgG or IgM (Cappel) as a second antibody for 1 h at 4 °C. Stained cells were analyzed with a FACS Calibure flow cytometer. CH50 was determined by a microtiter method, according to the methodology described by Mayer (37Mayer M.M. Experimental Immunochemistry. 2nd Ed. Charles C Thomas Publisher, Springfield, IL1961: 133Google Scholar, 38Miyagawa S. Hirose H. Shirakura S. Naka Y. Nakata S. Kawashima Y. Seya T. Matsumoto M. Uenaka A. Kitamura H. Transplantation. 1988; 46: 825-830Crossref PubMed Scopus (229) Google Scholar). In this procedure, CH50 was assayed in gelatin Veronal buffer by using sensitized sheep erythrocytes. After incubation at 37 °C for 60 min, 50 µl of the same buffer was added, and the mixtures were centrifuged. The hemoglobin content of each supernatant was estimated spectrophotometerically. The CH50 unit was defined as the serum volume sufficient to lyse 50% of the erythrocytes added to each well, and the complement activity in a test serum was then calculated as the number of CH50 units. After the donor pigs had been anesthetized with thiopental sodium (20 mg/kg) and Stresnil (2 mg/kg), a median sternotomy was performed. The inferior vena cava was divided above the diaphragm, and 200 ml of glucose-potassium cardioplegic solution with heparin (1,000 IU) (39Sawa Y. Matsuda H. Shimazaki Y. Kadoba K. Ohtake S. Takami H. Onishi S. Kawashima Y. Circulation. 1988; 78: 191-197Google Scholar) was then infused from the ascending aorta. The heart was excised under topical cooling with PBS (4 °C) after division of the superior vena cava, the pulmonary aorta and veins, and the ascending aorta. Recipient cynomolgus monkeys were anesthetized using ketamine hydrochloride (10 mg/kg) and xylazin (1 mg/kg). A heterotopic heart transplantation was performed in the abdomen, according to the Ono-Lindsey method (40Ono K. Lindsey E.S. J. Thorac. Cardiovasc. Surg. 1969; 57: 225-229Abstract Full Text PDF PubMed Google Scholar). A midline abdominal incision was used to expose the aorta and inferior vena cava below the renal vessels. The donor heart was placed in the abdomen of the recipient monkey, the end-to-side anastomosis being donor aorta to recipient abdominal aorta and donor pulmonary aorta to recipient inferior vena cava. No immunosuppression nor anticoagulants was administered before or after the procedure. The rejection of the cardiac transplants was defined based on the cessation of beating. The following time was 4 h after the operation. Samples of graft tissue were removed at the time of rejection or 4 h after transplantation for immunohistochemical analysis. Data are presented as the mean ± S.E. Student's t test was used to ascertain the significance of differences within groups. Differences were considered statistically significant when p < 0.05. The pCX promoter was used for the ubiquitous expression of GnT-III in transgenic mouse. Eight founder pCX-GnT-III transgenic mice were obtained from 73 live pups from 576 microinjected oocytes. A founder that was determined to express high levels of human GnT-III was mated with B6 mice to propagate transgenic offspring for the analysis of transgene expression in various tissues. Transgenic pigs were also obtained by means of the pCX-GnT-III construct. Five founders of pCX-GnT-III transgenic pigs were obtained from 59 live pups from 583 microinjected oocytes. Of the founder pigs, one was stillborn, and another died shortly after birth. Two founders, Gx-1 and Gx-2, showed GnT-III enzyme activity in their tails, and the other, Gx-3, had only a very low level of GnT-III activity. The transgenic pig, Gx-1, was successfully bred, and three offspring, 3 weeks (pig 1), 3 months (pig 2), and 6 months (pig 3), with the transgene were examined for in vitrostudy. Two, 3 weeks, were used in the transplantation experiments. The copy number of transgenes in hemizygous offsprings (F1) of transgenic mouse and pig lines was examined by Southern hybridization. The copy number of transgenic mouse, line 2, which was used in this study, had seven copies of pCX-GnT-III constructs, and the pig lines GX-1 and GX-2 had three and two copies, respectively. The level of activity of GnT-III in each animal was not correlated with the copy numbers of the transgene. The profiles of the GnT-III activities of each organ in wild-type and the GnT-III transgenic animals were investigated. Although the wild-type mouse showed GnT-III activity only in kidney tissue, GnT-III was expressed ubiquitously in the GnT-III transgenic mice (Fig.1A). As shown in Fig. 1B, the wild-type pigs showed very low levels of GnT-III activity in the kidney, but a slightly higher level in the brain. The CAG promoter led to the nearly ubiquitous expression of GnT-III in the organs of the transgenic pig. To analyze the alteration of antigenicity in the transgenic mouse, immunostaining of each organ was performed using NHS and GS-IB4 lectin and M86 monoclonal antibody (Table I). Characteristic of the transgenic mouse, changes of antigenicity were found in most organs except for the kidney, which has endogenous GnT-III activity (Fig. 2, c and d). In particular, the antigenicity of the liver from the GnT-III transgenic mouse is clearly down-regulated despite its relatively lower expression of GnT-III activity (Fig. 2, e andf).Table IImmunohistochemical analysisAnimalNHSGS-IB4M86Wild-typeTransgenicWild-typeTransgenicWild-typeTransgenicTransgenic miceHeart1+++∼±++±+++2++++++∼±+++Kidney1++++++2+++±++Liver1+±∼−+±∼−+±∼−2++±++−+±∼−Lung1+−+±∼−+−2+−±−±±∼−Pancreas1±∼−−±−±∼−−2+∼±−+−+−Muscle1+∼±−+±+±∼−2+++∼±++±+++∼±Transgenic pigsHeart1++±+±+−2+±+±+±3++±+±+−Kidney1++∼+±+±++∼+±2+±+++∼±+∼±−3++±∼−++±∼−+−Liver1++++±++±2++±+±∼−+++∼±3+++++++±Lung1+±±±+±∼−2+−+±+±3+±+∼±±+±∼−Pancreas1+−+±+−2ND1-aND, not determined.NDNDNDNDND3±−±−−−Spleen1+±+±±−2+++−+±3+±+±+±Muscle1+±±±++∼+±2±−+−+∼±±3++∼+++±+±∼−Skin1±−−−±±2−−±−−−3+±−−±−Brain1−−−−−−2−−±−−−3±−−−−−Aorta1++++++±2NDND+++++∼+±3+++++±++++∼+Grading scale: −, not stained; ±, stained equivocally or weakly; +, stained moderately; ++, stained intensely.1-a ND, not determined. Open table in a new tab Grading scale: −, not stained; ±, stained equivocally or weakly; +, stained moderately; ++, stained intensely. In the case of pigs, compared with wild-type pig, GnT-III transgenic pigs indicated a lower susceptibility to NHS, GS-IB4 lectin, and M86 mAb in many organs except brain, which also had GnT-III endogenous enzyme activity (Fig. 2, m and n). To determine whether the pancreatic islets from the transgenic pig have elevated GnT-III activity, double staining with anti-GnT-III Ab and anti-insulin Ab was carried out. Double staining of the pancreas revealed that the islets from the transgenic pig have a high level of expression of the GnT-III enzyme (Fig. 3). The α1,3GT and GnT-IV, and GnT-V enzyme activity in the control animals and the influences of an excess of GnT-III over the enzymes in each organ were measured. The average GnT-IV activities in many organs of the transgenic mouse and pig were lower than those in wild, but the differences were not significant (Fig. 4, A andD). It was not possible to predict changes in GnT-V and α1,3GT activities in both mice and pigs. GnT-III and α1,3GT activities in the PEC from transgenic piglets were examined by HPLC, and the amelioration of antigenicity of the PEC was also analyzed by flow cytometry, using NHS, GS-IB4 lectin, and M86 (Fig.5, A–E).Figure 5Features of the PEC from transgenic pigs. Enzyme activities, GnT-III (panel A) and α1,3GT (panel B) of PECs from transgenic pigs were measured by HPLC. Each value is expressed as the mean ± S.E. of three to four independent experiments. Xenoantigenicity of transgenics to NHS (panel C), GS-IB4 (panel D), and M86 (panel E) were investigated by flow cytometry. PECs from control and transgenic pigs were treated with 10 or 20% NHS as the first antibody and anti-human immmunoglobulin second antibodies. The reduction of α-Gal on the cell surface was also analyzed using GS-IB4 lectin and M86 mAb. PECs from control and transgenic pigs were treated with FITC-conjugated GS-IB4 lectin, M86 mAb, and FITC-conjugated anti-mouse IgM secondary antibody. The FACS mean shift value of the PEC treated with polyclonal chicken anti-H-D antigen antibody is indicated (panel F). The H-D antigen of the PEC from transgenic pigs was down-regulated significantly. Each value is expressed as the mean ± S.E. of six to eight independent experiments. The amelioration of complement-mediated lysis by from wild-type and transgenic pigs was estimated by 20 or 40% NHS, which served as a source of natural antibodies and complem" @default.
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• W2139676396 date "2001-10-01" @default.
• W2139676396 modified "2023-10-12" @default.
• W2139676396 title "Remodeling of the Major Pig Xenoantigen by N-Acetylglucosaminyltransferase III in Transgenic Pig" @default.
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• W2139676396 doi "https://doi.org/10.1074/jbc.m104359200" @default.
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