We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-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 Galalpha1-3Galbeta1-4GlcNAc-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 alpha1,3-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.

Animals↗

PubMed2001

Down-regulation of the alpha-Gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc.

The down-regulation of the alpha-Gal epitope (Galalpha1,3Galbeta-R) in swine tissues would be highly desirable, in terms of preventing hyperacute rejection in pig-to-human xenotransplantation. In an earlier study, we reported that the introduction of the beta1,4-N-acetylglucosaminyltransferase (GnT) III gene into swine endothelial cells resulted in a substantial reduction in the expression of the alpha-Gal epitope. In this study, we report on the mechanism for this down-regulation of the alpha-Gal epitope by means of structural and kinetic analyses. The structural analyses revealed that the amount of N-linked oligosaccharides bearing the alpha-Gal epitopes in the GnT-III-transfected cells was less than 10% that in parental cells, due to the alteration of the terminal structures as well as a decrease in branch formation. In addition, it appeared that the addition of a bisecting GlcNAc, which is catalyzed by GnT-III, leads to a more efficient sialylation rather than alpha-galactosylation. In vitro kinetic analyses showed that the bisecting GlcNAc has an inhibitory effect on alpha-galactosylation, but does not significantly affect the sialylation. These results suggest that the bisecting GlcNAc in the core is capable of modifying the biosynthesis of the terminal structures via its differential effects on the capping glycosyltransferase reactions. The findings may contribute to the development of a novel strategy to eliminate carbohydrate xenoantigens.

Acetylglucosamine↗

PubMed2001

The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis.

The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human beta2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human beta2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human beta2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.

Animals↗

PubMed2001

A synthetic dDAF (CD55) gene based on optimal codon usage for transgenic animals.

The human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenic mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Delta1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Delta1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenic pigs with high levels of expression.

Animals↗

PubMed2001

[Usefulness of self ligation mediated polymerase chain reaction: a rapid method for fingerprinting in molecular epidemiology of tuberculosis].

Restriction fragment length polymorphism (RFLP) analysis based on the insertion sequence IS 6110 has been used as one of the powerful tools for epidemiological study of tuberculosis. However this technique requires more than 1 micro-gram of DNA and two days for completion. To overcome these inconvenience, we have modified a PCR-based method, self ligation mediated PCR (SL-PCR) on the molecular epidemiological study. This method uses a pair of primers whose orientations are from inside to outside of IS 6110. The DNA fragments flanking IS 6110 are amplified by the PCR by using the Sau 3A I digested and ligated chromosomal DNA of Mycobacterium tuberculosis strains. By using this method, M. tuberculosis strains can be differentiated within 8 hours.

DNA Fingerprinting↗

PubMed2000

Initial T-cell activation required for transplant vasculopathy in retransplanted rat cardiac allografts.

BACKGROUND: A precise understanding of immunological mechanisms is needed to prevent transplant vasculopathy. METHODS: The developing process of transplant vasculopathy was investigated by retransplanting rat cardiac allografts and measuring the expressions of 21 different genes inside the retransplanted allografts under nonimmunosuppressive conditions. RESULTS: Significant transplant vasculopathy developed if WKY hearts were grafted to LEW and retransplanted to WKY 5 days after the initial grafting, but it did not in allografts retransplanted 3 days after the initial grafting. The disease did not progress in retransplanted isografts or if nude rats were used as the initial recipients. However, the development of transplant vasculopathy was not affected by changing the second recipients to the F1 progeny of donor x recipient or to nude animals. Among the expressions of 21 different genes observed in allografts at 1, 14, 30, or 60 days after retransplantation, those of T-cell activation-related genes, such as interferon-y and Fas ligand, showed the earliest and the most dramatic difference between 3- and 5-day-retransplanted allografts whereas macrophage/monocyte activation-related genes showed little difference. Furthermore, reverse transcription-polymerase chain reaction analyses of allografts retransplanted to nude animal indicated that T cells of the initial recipient origin survive and remain activated even 60 days after retransplantation. CONCLUSIONS: The T-cell response occurring between 3 and 5 days after grafting was identified as the critical parameter to the disease progression. Once alloreactive T cells enter a graft, they may be able to survive a long period and promote chronic rejection.

Animals↗

PubMed2000

Importance of donor-derived lymphocytes in the protection of pancreaticoduodenal or islet grafts from recurrent autoimmunity: a role for RT6+NKR-P1+ T cells.

BACKGROUND: A rat pancreas transplantation model with insulin-dependent diabetes mellitus (IDDM) recurrence was established using a Wistar-Furth (WF; RT1u, RT6.2) rat as a donor and diabetes-prone (DP; RT1u, rt6.1 gene carrier) BioBreeding rat as a recipient. Interestingly, NKR-P1+TCRalphabeta+ (NKT) cells have recently been reported to have immunoregulatory functions in preventing autoimmune diabetes. The purpose of this study was to specifically examine the contribution of NKT cells in the prevention of IDDM recurrence. METHODS: Graft survivals with or without anti-intercellular adhesion molecule-1/leukocyte function-associated antigen-1 monoclonal antibodies were examined comparing pancreaticoduodenal (PD) transplantation with islet transplantation or pancreas-alone transplantation excluding duodenum and peripancreatic lymph nodes, in an IDDM recurrent model. The cells of the spleen were analyzed by flow cytometry (including intracellular interleukin (IL)-4 analysis), and serum cytokine levels (IL-4 and interferon-gamma) were determined by enzyme-linked immunosorbent assays (ELISA). RESULTS: Only those DP recipients transplanted with PD grafts with monoclonal antibody treatment were free from IDDM. Flow cytometric analyses of spleen cells showed that NKT cells in them, compared with those in the recurrent DP recipients (mean <7%), increased significantly (13.7+/-3.1% in the total splenic T cells), most of which (85.9+/-4.3%) were derived from the donor (RT6.2+). The absolute number of RT6+NKT cells significantly increased in the nonrecurrent DP recipients, whereas that of RT6-NKT cells was similar with those of the other recurrent DP recipients. These RT6+NKT cells were predominantly CD4+ and showed significantly more expressions of intracellular IL-4 than the other T cells. By ELISA, serum interferon-gamma was not detectable (< 13 pg/ml) in any of the rats. However, IL-4 was detected at 111.6+/-47.8 pg/ml only in the nonrecurrent DP recipients. CONCLUSIONS: Unlike islet or pancreas-alone transplants, NKT cells, especially RT6+NKT cells derived from PD grafts, may have an important immunoregulatory function of preventing IDDM recurrence, involving a Th2 deviation.

ADP Ribose Transferases↗

PubMed2000

A surface-bound form of human C1 esterase inhibitor improves xenograft rejection.

BACKGROUND: The purpose of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH) molecule against human complement attack on a swine endothelial cell (SEC) membrane. Human C1-INH functions as an inhibitor for complement reaction in the first step of the classical pathway in the fluid phase. METHODS: A surface-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a glycosylphosphatidylinositol (GPI) anchor of the decay-accelerating factor (CD55) was constructed, and stable Chinese hamster ovarian tumor (CHO) cell lines and SEC lines expressing C1-INH-PI were then prepared by transfection of the constructed cDNA. The basic function of the transfected molecules on the xenosurface was investigated using CHO transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine-to-human discordant xenograft. RESULTS: Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-PI showed a medium level of expression of these molecules. The C1-INH levels were significantly reduced as a result of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, suggesting that the molecules were present as the PI-anchor form. Approximately 51.3 x 10(4) and 13.3 x 10(4) molecules of C1-INH-PI blocked human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively. In addition, the complement-inhibiting activity of human C1-INH molecules is not homologously restricted. CONCLUSIONS: The results suggest that the surface-bound form of C1-INH represents a good candidate as a safeguard against hyperacute rejection of xenografts.

Animals↗

PubMed2000

Differential expression of proteoglycans biglycan and decorin during neointima formation after stent implantation in normal and atherosclerotic rabbit aortas.

Proteoglycans decorin and biglycan, which bind to TGF-beta, are thought to participate in regulation of extracellular matrix accumulation in arterial intimal hyperplasia. To investigate the correlation of these proteoglycans with the cellular localization and phenotypic modulation of smooth muscle cells (SMCs), we analyzed the spatial and chronological distribution of these proteoglycans and two cytokines, TGF-beta and IL-1beta, in the process of neointima formation after stent implantation in the aortas of rabbits fed a high-cholesterol diet (atherosclerotic group) or a regular diet (control group). We implanted metallic stents in the rabbit aortas and harvested the aortas 4-56 days later for immunohistochemical and mRNA in situ hybridization analyses. In the control group, TGF-beta and biglycan expression was in correspondence with the chronology and localization of embryonic SMCs. In the atherosclerotic group, TGF-beta and biglycan expression was sustained throughout the experimental period, which was in accord with the prolonged expression of embryonic SMCs. Decorin, which did not occur in neointima in the control group, appeared in the atherosclerotic aortas in the confined area of vascular SMCs surrounding the macrophages around the stent wire. These results indicate that biglycan and decorin kinetics during neointima formation after arterial injury are distinct, despite their similar construction; biglycan synthesis correlates with embryonic SMCs.

Animals↗

PubMed2000

Effect of various glycosyltransferases on the swine xenoantigen.

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Animals↗