• Title, Summary, Keyword: Hyperacute Rejection

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Progress in Transgenic Cloned Pig for Xenotransplantation

  • Park, Kwang-Wook
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • pp.9-19
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    • 2003
  • Pig organ is thought to be the most suitable nonhuman organ for xenotransplanstation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of a(1,3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute rejection. ry1any efforts have made characterization of GGTA1 in structure and function. improvement in the technique of DNA transfection of somatic cells and advancement of the pig NT, a specific modification has been made to one copy of the GGTA1 gene by Missouri group in 2002. To date because homozygousity of the genetic modification has been achieved in this gene, the role of gala(1,3) gal specific natural antibody in HAR and the efficacy of xenotransplantation in a nonhuman primate model will be addressed. If other genes are found to be involved in rejection of pig donors by primates, the technology will be available to modify those genes so that rejection can be overcome.

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Progress in Transgenic Cloned Pig for Xenotransplantation

  • Park, Kwang-Wook
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • pp.9-19
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    • 2003
  • Pig organ is thought to be the most suitable nonhuman organ for xenotransplanstation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of a(1,3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute rejection. Many efforts have made characterization of GGTA1 in structure and function, improvement in the technique of DNA transfection of somatic cells and advancement of the pig NT, a specific modification has been made to one copy of the GGTAl gene by Missouri group in 2002 To date because homozygousity of the genetic modification has been achieved in this gene, the role of gala(1,3) gal specific natural antibody in HAR and the efficacy of xenotransplantation in a nonhuman primate model will be addressed. Of other genes are found to be involved in rejection of pig donors by primates, the technology will be available to modify those genes so that rejection can be overcome.

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Complement-dependent Cytotoxic Crossmatch for Prevention of Hyperacute Rejection in Canine Renal Allografts (개의 신장 동종 이식에서 초급성 거부반응의 예방을 위한 보체의존성 세포독성 DLA 플래스 I 교차 반응)

  • Ghil Heh Myung;Woo Heung-Myung
    • Journal of Veterinary Clinics
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    • v.22 no.4
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    • pp.322-327
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    • 2005
  • DLA class I complement-dependent cytotoxicity (CDC) cross-match method was established to control hyperacute rejections in organ transplantation. The aim of the present study is to investigate if DLA class 1 CDC corssmatch method is effective to prevent hyperacute rejections in canine renal allografts. Seven mongrel dogs of similar age and weight were used. Erythrocyte crossmatch was first performed and only the negatives were used. Among the same blood types, CDC cross-match was performed. Anti-dog serum, Hank's balanced salt solution(HBSS), and tile self-serum was used as a positive-, a negative-, and all auto-control respectively. After the reaction with class I complement, it was stained with eosin and scored by international cytotoxicity scoring system under inverted phase microscope. According to these results, kidneys oi CDC negatives among same blood types were cross-transplanted to observe the incidence of hyperacute rejections. One of four 1.2 B blood type dogs had autoantibodies. here were negative CDC results among each blood type, and also there were negative results between different blood types. Two pairs with the same blood types and negative CDC results underwent allo-transplantation each other. There were no hyperacute rejections. DLA cross-match method studied in this experiment for canine renal allograft can be effective to prevent hyperacute rejections. it may be applicable for the future studies of histocompatibility testing in canine renal allografts.

Cooperation between Human DAF and CD59 in Protecting Cells from Human Complement-mediated Lysis

  • Xu, Li;Wu, Wenlan;Zhao, Zhouzhou;Shao, Huanjie;Liu, Wanhong;Liu, Hui;Li, Wenxin
    • BMB Reports
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    • v.39 no.6
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    • pp.743-748
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    • 2006
  • The complement (C) regulatory proteins decay accelerating factor (DAF, CD55) and CD59 could protect host cells using different mechanisms from C-mediated damage at two distinct levels within the C pathway. Co-expression of DAF and CD59 would be an effective strategy to help overcome host C-induced xenograft hyperacute rejection. In this study, we made a construct of recombinant expression vector containing DAF and CD59 cDNA and the stable cell lines were obtained by G418 selection. Extraneous genes integration and co-expression were identified by PCR, RT-PCR and Western blot analysis. Human c-mediated cytolysis assays showed that NIH/3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-DAF, and pcDNA3-CD59DAF-DP were protected from C-mediated damage and that synchronously expressed human CD59 and DAF provided the most excellent protection for host cells as compared with either human CD59 or DAF expressed alone. Therefore, the construct represents an effective and efficacy strategy to overcome C-mediated damage in cells and, ultimately, in animals.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus

  • Kim, Ji-Woo;Kim, Hye-Min;Lee, Sang-Mi;Kang, Man-Jong
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.10
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    • pp.1473-1480
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    • 2012
  • The Galactose-${\alpha}1$,3-galactose (${\alpha}1$,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of ${\alpha}1$,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Knock-in Somatic Cells of Human Decay Accelerating Factor and α1,2-Fucosyltransferase Gene on the α1,3-Galactosyltransferase Gene Locus of Miniature Pig (α1,3-Galactosyltransferase 유전자 위치에 사람 Decay Accelerating Factor와 α1,2-Fucosyltransferase 유전자가 Knock-in된 미니돼지 체세포)

  • Kim, Ji Woo;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.39 no.3
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    • pp.59-67
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    • 2015
  • Galactose-${\alpha}1,3$-galactose (${\alpha}1,3$-Gal) epitope is synthesized at a high concentration on the surface of pig cells by ${\alpha}1,3$-galactosyltransferase gene (GGTA1). The ${\alpha}1,3$-Gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesize $Gal{\alpha}$(1,3)Gal. To prevent hyperacute graft rejection in pig-to-human xenotransplantation, previously, we developed ${\alpha}1,3$-galactosyltransferase gene-knock-out somatic cell by homologous recombination. In this study, we established cell lines of ${\alpha}1,3$-GT knock-out expressing hDAF and hHT gene from minipig fibroblasts to apply somatic cell nuclear transfer. The hDAF and hHT mRNA were expressed in the knock-in somatic cells and ${\alpha}1,3$-GT mRNA was suppressed. However, the knock-in somatic cells were increased resistance to human serum-mediated cytolysis.

Nuclear Transfer using Human CD59 and IL-18BP Double Transgenic Fetal Fibroblasts in Miniature Pigs

  • Ryu, Junghyun;Kim, Minjeong;Ahn, Jin Seop;Ahn, Kwang Sung;Shim, Hosup
    • Journal of Embryo Transfer
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    • v.31 no.1
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    • pp.1-7
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    • 2016
  • Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

Successful Heart Transplantation across an ABO Blood Group Barrier -One case report- (ABO 부적합성 심장이식술 -치험 1례 보고-)

  • 원태희;신윤철
    • The Korean Journal of Thoracic and Cardiovascular Surgery
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    • v.29 no.3
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    • pp.322-325
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    • 1996
  • ABO incompatible allografting is contraindicated in most organ transplantations including heart because of the hyperacute and acute rejections caused by preexisting antibodies. However several reports showed that ABO incompatible organ transplantation could be managed successfully by plasmapheresis, antibody adsorption, immunosuppression, splenectomy, and so on. We experienced one success in ABO incompatible cardiac transplantation by means of plasmapheresis and immunosuppression. However, this does not justify heart transplantation across ABO blood group barriers. Because the effect of ABO incompatibility on continued acute rejection or chronic rejection has not been fully understood, long-term follow-up study is required.

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Characterization of α-Gal Epitope in Cells and Tissues from Homozygous α-1,3-Galactosyltransferase Knockout Pigs

  • Hwang, In-Sul;Kwon, Dae-Jin;Kwak, Tae-Uk;Oh, Keon Bong;Ock, Sun-A;Chung, Hak-Jae;Im, Gi-Sun;Hwang, Seongsoo
    • Reproductive and Developmental Biology
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    • v.39 no.4
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    • pp.127-132
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    • 2015
  • To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred ${\alpha}$-1,3-galactosyltransferase knock-out ($GalT^{-/-}$) pigs. In this study, the somatic cells and tissues from the $GalT^{-/-}$ pigs were characterized by an analysis of the expression of Gal${\alpha}$-1,3-Gal (${\alpha}-Gal$) epitope. Briefly, ear fibroblast cell lines of 19 homozygous $GalT^{-/-}$ pigs were established and cryopreserved. The expression of ${\alpha}-Gal$ epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous ($GalT^{-/-}$) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of ${\alpha}-Gal$ epitope expression. The results showed that the expression of ${\alpha}-Gal$ epitope in $GalT^{-/-}$ cells (0.2 %) were significantly (p<0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous ($GalT^{-/+}$) (9.3 %) and wild type ($GalT^{+/+}$) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of ${\alpha}-Gal$ epitope was detected a partly in $GalT^{-/+}$ cells and mostly in $GalT^{+/+}$ cells, it was almost not detected in the $GalT^{-/-}$ cells. Also, immunostaining results from various tissues of the $GalT^{-/-}$ pig showed that the expression of ${\alpha}-Gal$ epitope was not detectable, whereas various tissues from $GalT^{+/+}$ pig showed a strong expression of ${\alpha}-Gal$ epitope. Our results demonstrated that ${\alpha}-Gal$ epitope expressions from $GalT^{-/-}$ pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.