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Mi Y, Li R, Xu K, Jiang H, Sun X. Gene transfer of antisense B7.1 attenuates acute rejection against liver allografts in rats. J INVEST SURG 2010; 23:87-93. [PMID: 20497010 DOI: 10.3109/08941930903469359] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
OBJECTIVE Blockade of CD80-CD28 costimulatory pathway induces unresponsiveness of T cells to alloantigens and protects allografts against immune rejection. The aim of this study was to investigate whether downregulating the expression of B7.1 (CD80) in the donor livers by antisense B7.1 gene transfer could attenuate the acute immune rejection against liver allografts in rats. METHODS The liver grafts from 60 Dark Agouti rats were intraportally perfused with antisense B7.1 expression vector, before they were transplanted into Lewis rats. Empty vector pcDNA3 served as control to be perfused into livers of another group of 60 Dark Agouti rats. The orthotopic liver transplantation was performed. The rats were randomly sacrificed at scheduled time points to collect liver allografts, or monitored to record the survival rate. The livers were histologically examined to calculate Banff rejection activity index, or subjected to Western blot analysis or immunohistochemistry for examining the expression of B7.1, or counting CD4+ and CD8+ cells. RESULTS Antisense gene transfer resulted in markedly downregulation of B7.1 in the donor livers, attenuated acute immune rejection against liver allografts, prolonged the survival time of rats, and decreased the number of infiltrating CD4+ and CD8+ cells in livers. CONCLUSIONS Blocking expression of B7.1 in liver by antisense gene therapy may represent a potential strategy to attenuate acute rejection against liver allografts.
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Affiliation(s)
- Yuetang Mi
- Department of Hepatobillary Surgery, Qilu Hospital of Shandong University, Jinan, China
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Laurence JM, Allen RDM, McCaughan GW, Logan GJ, Alexander IE, Bishop GA, Sharland AF. Gene therapy in transplantation. Transplant Rev (Orlando) 2009; 23:159-70. [PMID: 19428235 DOI: 10.1016/j.trre.2009.04.001] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Gene therapy is an exciting and novel technology that offers the prospect of improving transplant outcomes beyond those achievable with current clinical protocols. This review explores both the candidate genes and ways in which they have been deployed to overcome both immune and non-immune barriers to transplantation success in experimental models. Finally, the major obstacles to implementing gene therapy in the clinic are considered.
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Affiliation(s)
- Jerome M Laurence
- Collaborative Transplantation Research Group, Bosch Insitute, Royal Prince Alfred Hospital and University of Sydney, NSW 2006, Australia
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Liu C, Pan S, Jiang H, Sun X. Gene transfer of antisense B7.1 attenuates acute rejection against splenic allografts in rats. Transplant Proc 2007; 39:3391-3395. [PMID: 18089391 DOI: 10.1016/j.transproceed.2007.08.094] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2006] [Revised: 06/18/2007] [Accepted: 08/08/2007] [Indexed: 01/01/2023]
Abstract
Blockade of CD80-CD28 costimulatory pathway induces unresponsiveness of T cells to alloantigens and protects allografts against immune rejection in numerous animal models. The aim of this study was to investigate whether blocking expression of B7.1 (CD80) on donor splenocytes by an antisense technique protected splenic allografts against immune rejection. Splenic grafts from Wistar-Furth rats were intra-arterially transfused with an antisense B7.1 expression vector, before they were transplanted into Sprague-Dawley rats. The rats were sacrificed at scheduled times, and the splenic allografts histologically examined. Antisense gene transfer resulted in marked down-regulation of B7.1 in donor spleens, hyporesponsiveness of recipient T cells, and attenuated acute immune rejection against splenic allografts. No obvious damage to skin, liver, or gut due to graft-versus-host disease was detected in the recipients. In conclusion, blocking expression of B7.1 in donor spleens by antisense gene therapy represented a potential alloantigen-specific immunosuppressive strategy to inhibit acute rejection against splenic allografts.
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Affiliation(s)
- C Liu
- Department of General Surgery, the Fourth Affiliated Hospital, and the First Clinical Medical School, Harbin Medical University, China
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Tsoulfas G, Takahashi Y, Liu D, Yagnik G, Wu T, Murase N, Geller DA. Hydrodynamic plasmid DNA gene therapy model in liver transplantation. J Surg Res 2006; 135:242-249. [PMID: 16926028 DOI: 10.1016/j.jss.2006.04.020] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2006] [Revised: 04/20/2006] [Accepted: 04/21/2006] [Indexed: 02/08/2023]
Abstract
BACKGROUND There is great interest in the field of transplantation to genetically modify grafts to decrease preservation injury or allograft rejection. Although adenoviral gene transfer has been effective in experimental liver transplantation, viral toxicity and safety concerns limit potential use in clinical trials. Therefore, the purpose of this study was to develop a model of nonviral gene transfer in the liver transplant setting, allowing for efficient transgene expression. MATERIALS AND METHODS Orthotopic syngeneic rat liver transplantation was performed with 3 h cold ischemia using University of Wisconsin (UW) preservation. A hydrodynamic gene transfer technique was developed where plasmid DNA was delivered to the liver graft by ex vivo rapid infusion of DNA in UW via the IVC with other vessels clamped. Expression plasmids for the marker genes luciferase and secreted human alpha1-antitrypsin (alpha1-AT) were used. Hepatic injury was assessed by graft histology and liver transaminases. Transgene expression was determined by hepatic luciferase relative light units activity (RLU) and serum alpha1-AT protein levels. Variables examined included the effect of (a) volume injected on the intravenous pressure in the liver graft; (b) injury to the liver, as measured by hepatic enzymes and histopathology; (c) variable expression between lobes; (d) volume of UW that the plasmid is administered in; (e) amount of DNA plasmid; (f) type of the promoter used; (g) clamp time; as well as (h) the time course of the marker gene expression. RESULTS Control rats underwent standard orthotopic syngeneic rat liver transplantation and had no detectable hepatic luciferase activity or serum human alpha1-AT. The optimal DNA plasmid dose was found to be 400 mug/liver graft, as there was no increase in the luciferase expression by increasing the dose. Furthermore, cytomegalovirus promoter yielded greater expression than Rous sarcoma virus. A high injection pressure gradient allowed for more efficient transgene expression, but produced greater liver injury shown by elevated transaminases and centrilobular necrosis. Lowering injection volume from 75 to 50% of graft weight decreased liver injury by 4.5-fold. Although higher UW injection volumes were associated with increased expression, volumes of only 50% led to luciferase expression up to 10,000,000 RLU/mg; this expression was homogeneous between the different liver lobes. Human alpha1-AT was detected in recipient blood as early as 6 h, peaked at 24 h, and remained high for 5 days. CONCLUSIONS We have developed a nonviral gene transfer technique where hydrostatic pressure across the cold-preserved liver vascular bed allows for efficient plasmid DNA delivery. This simple strategy should prove useful to genetically modify liver grafts in the transplantation setting.
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Affiliation(s)
- George Tsoulfas
- Department of Surgery, T.E. Starzl Transplantation Institute, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
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Martins PNA, Chandraker A, Tullius SG. Modifying graft immunogenicity and immune response prior to transplantation: potential clinical applications of donor and graft treatment. Transpl Int 2006; 19:351-9. [PMID: 16623870 DOI: 10.1111/j.1432-2277.2006.00301.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Many studies have shown a strong association between initial graft injury and poor long-term graft outcome. Events initiated by unspecific immune-activating processes including brain death and ischemia/reperfusion injury occurring prior to transplantation reduce graft functionality and amplify the host immune response. These events may be particularly relevant for less than optimal grafts with reduced resistance to unspecific injuries. Several approaches to ameliorate immune activation of the graft by treating the donor or the graft have been studied. While various substances have been shown to have protective effects in experimental transplantation, only a few drugs have been tested clinically and have demonstrated beneficial effects. We review the results of experimental and clinical studies on donor or graft immunomodulation prior to transplantation and analyze the evidence to support clinical application of these strategies.
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Affiliation(s)
- Paulo N A Martins
- Department of Surgery, Division of Transplant Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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Hua N, Yamashita K, Hashimoto T, Masunaga T, Fujita M, Furukawa H, Uede T, Todo S. Gene Therapy-Mediated CD40L and CD28 Co-stimulatory Signaling Blockade plus Transient Anti-xenograft Antibody Suppression Induces Long-Term Acceptance of Cardiac Xenografts. Transplantation 2004; 78:1463-70. [PMID: 15599310 DOI: 10.1097/01.tp.0000144324.83846.a9] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
BACKGROUND The authors have previously demonstrated that inhibition of CD28 and CD40 ligand (CD40L) co-stimulatory signals by adenovirus-mediated cytotoxic T-lymphocyte-associated (CTL) antigen 4 (A4) immunoglobulin (Ig) and CD40Ig gene therapies induces tolerance or long-term acceptance in rat liver and heart allograft transplantation. In this study, the authors examined whether co-stimulation blockade with a brief course treatment of FK779, a novel leflunomide derivative, would be an ideal strategy for controlling xenograft rejection. METHODS Hamster hearts were transplanted into Lewis rats. Adenovirus vector coding (Ad) CD40Ig, CTLA4Ig, or LacZ gene (1 x 10(9) plaque-forming units) was administered intravenously to recipient rats 2 days before or immediately after xenografting. FK779 (10 mg/kg/day) was administered orally to recipients for 7 days beginning on day -1. Graft survival, graft histology, and xenoreactive antibodies were examined. RESULTS : Both untreated and AdLacZ-treated control rats rejected cardiac xenografts, with a median survival time (MST) of 3 days. Co-stimulatory blockade alone by AdCTLA4Ig, AdCD40Ig, or both could not overcome such delayed xenograft rejection (DXR) (MST, 3-4 days). Under a short-course FK779 treatment that suppressed T-cell-independent xenoreactive antibodies, administration of AdCD40Ig (MST, 30.5 days) but not AdCTLA4Ig (MST, 9 days) significantly prolonged xenograft survival as compared with the FK779 monotherapy (MST, 7 days). In contrast, DXR and cellular rejection were controlled successfully and all xenografts were accepted for over 100 days when AdCTLA4Ig and AdCD40Ig were administered under FK779 induction therapy. However, chronic rejection was present in all long-term surviving xenografts. CONCLUSIONS : Gene therapy-based co-stimulation blockade with FK779 induction treatment seems to be an attractive strategy with which to control xenograft rejection.
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Affiliation(s)
- Nan Hua
- First Department of Surgery, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
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Wang YF, Xu AG, Hua YB, Wu WX. Effect of local CTLA4Ig gene transfection on acute rejection of small bowel allografts in rats. World J Gastroenterol 2004; 10:885-8. [PMID: 15040038 PMCID: PMC4727007 DOI: 10.3748/wjg.v10.i6.885] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To evaluate the local expression of CTLA4Ig gene in small bowels and its effect on preventing acute rejection of the small bowel allografts.
METHODS: Groups of Wistar rats underwent heterotopic small bowel transplantation from SD rats. The recipients were randomly divided into experimental group (allografts were transfected with CTLA4Ig gene) and control group (non CTLA4Ig gene transfected). In the experimental group, the donor small bowels were perfused ex vivo with CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery before transplantation, and the CTLA4Ig expression in the small bowel grafts post-transplantation was assessed by immunohistology. On d 3, 7 and 10 post-transplantation, the allografts in each group were harvested for the examination of histology and detection of apoptosis.
RESULTS: Small bowel allografts treated with CTLA4Ig cDNA showed abundant CTLA4Ig expression after transplantation. Acute rejection of grade I on d 7 and grade II on d 10 after transplantation was noticed in the control allografts, and a dramatically increased number of apoptotic enterocytes in parallel to the progressive rejection could be recognized. In contrast, the allografts treated with CTLA4Ig cDNA showed nonspecific histological changes and only a few apoptotic enterocytes were found after transplantation.
CONCLUSION: Local CTLA4Ig gene transfection of small bowel allograft is feasible, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.
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Affiliation(s)
- Yi-Fang Wang
- Department of Gastrointestinal Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
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Wang YF, Xu AG, Hua YB, Wu WX. Effect of local CTLA4Ig gene transfection on acute rejection of small bowel allografts in rats. Shijie Huaren Xiaohua Zazhi 2004; 12:685-688. [DOI: 10.11569/wcjd.v12.i3.685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the local expression of CTLA4Ig gene in small bowels and its action on preventing acute rejection of the small bowel allografts.
METHODS: Wistar rats underwent heterotopic small bowel transplantation from SD rats. The recipients were divided into experimental group (allografts were transfected with CTLA4Ig gene) and control group (CTLA4Ig gene not transfected) randomly. In the experimental group, the donor small bowels were perfused in vitro with CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery before transplantation, and the CTLA4Ig expression in the small bowel grafts post-transplantation was assessed by immunohistology. On d 3, 7 and 10 post-transplantation, the allografts in each group were harvested for the examination of histology and assay of apoptosis.
RESULTS: Small bowel allografts treated with CTLA4Ig cDNA showed abundant CTLA4Ig expression after transplantation. Acute rejection grade Ⅰ on day 7 and grade Ⅱ on day 10 after transplantation was noticed in the control allografts, and a dramatically increased number of apoptotic enterocytes in parallel to the progressive rejection could be recognized. In contrast, the allografts treated with CTLA4Ig cDNA showed nonspecific histological changes and only a few of apoptotic enterocytes were found after transplantation.
CONCLUSION: Local CTLA4Ig gene transfection of small bowel allograft is feasible, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.
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Bartlett ASR, McCall JL, Ameratunga R, Howden B, Ramadas R, Yeong ML, Benjamin CD, Hess D, Munn SR. Intragraft gene and protein expression in rat liver allografts treated with costimulatory blockade alone or in combination with CyA. J Surg Res 2003; 115:1-8. [PMID: 14572766 DOI: 10.1016/s0022-4804(03)00131-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND Costimulatory blockade has been shown to prevent acute rejection (AR) and promote long-term graft survival in a number of animal models including nonhuman primates. The effect of concomitant administration of conventional immunosuppressives on long-term liver allograft survival and intragraft expression of immune mediators has not previously been examined. MATERIALS AND METHODS A high-responding Dark Agouti to Lewis orthotopic liver transplant (LEW OLT) model was used to compare anti-CD154 alone, or in combination with cyclosporin (CyA) on allograft survival. Donor-specific reactivity was assessed by mixed lymphocyte reaction (MLR) and allogeneic skin grafts. Surviving rats were euthanized on day 150 and intragraft gene (CD80, 86, 152, 154, IFN-gamma, IL-2, IL-6, IL-10, IL-13, TNF-alpha, TGF-beta, IL-7, Fas-ligand, Granzyme B, bax, and bcl(2)) and protein (CD4, CD8, ED1, CD154, CD80, CD86) expression was measured. RESULTS Untreated control recipients had a median survival time of 5 days. Recipients treated with anti-CD154 survived to beyond 150 days with no evidence of AR. Concomitant administration of CyA did not alter the long-term survival. There was no difference in the serum aspartate aminotransferase between treatment groups or a change over time. All treated recipients showed a reduction in donor-specific MLR at day 40 and 60 but had persistence of donor reactivity to skin grafts at day 100. Histologically, liver architecture was well preserved despite the presence of a nondestructive mononuclear cell infiltrate. Analysis of intragraft gene expression revealed an inverse relationship between the duration of anti-CD154 therapy and the gene expression of costimulatory molecules and Th1 cytokine transcripts. The pro-apoptotic gene, bax, was increased in recipients treated with anti-CD154, but not CyA, compared with normal liver. CONCLUSIONS These data demonstrate that anti-CD154 therapy either alone or in combination with CyA allows for the long-term survival of liver allografts in the rat despite there being a difference in the intragraft gene and protein profile.
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Affiliation(s)
- Adam S R Bartlett
- Department of Surgery, University of Auckland, Auckland, New Zealand
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Yamashita K, Masunaga T, Yanagida N, Takehara M, Hashimoto T, Kobayashi T, Echizenya H, Hua N, Fujita M, Murakami M, Furukawa H, Uede T, Todo S. Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies. Transplantation 2003; 76:1089-96. [PMID: 14557758 DOI: 10.1097/01.tp.0000085651.20586.30] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.
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Affiliation(s)
- Kenichiro Yamashita
- First Department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan
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Bartlett AS, McCall JL, Ameratunga R, Howden B, Yeong ML, Benjamin CD, Hess D, Peach R, Munn SR. Costimulatory blockade prevents early rejection, promotes lymphocyte apoptosis, and inhibits the upregulation of intragraft interleukin-6 in an orthotopic liver transplant model in the rat. Liver Transpl 2002; 8:458-68. [PMID: 12004346 DOI: 10.1053/jlts.2002.32979] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Costimulatory pathways have a pivotal role in the T-cell response to alloantigen. The role of costimulatory blockade with anti-CD154 in orthotopic liver transplantation (OLT) has not been examined previously. This study aims to investigate effects of anti-CD154 and CTLA4-immunoglobulin (Ig) in the early post-OLT period using a major histocompatibility complex-disparate fully arterialized OLT model in the rat. Lewis rats underwent OLT with Dark Agouti liver allografts. Recipients were randomized to receive (1) isotype control, (2) anti-CD154, (3) CTLA4-Ig, or (4) cyclosporine A (CyA). Rats were killed day 8, and specimens were obtained for histological examination, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, immunohistochemistry, and quantitative reverse-transcriptase polymerase chain reaction. An additional five transplant recipients were treated with anti-CD154 for 14 days postoperatively to assess long-term allograft survival. All isotype control animals died on or before day 6 of acute rejection. Apart from four deaths caused by nonimmunologic causes, all treated recipients survived to day 8. The median survival of rats treated for 14 days with anti-CD154 was greater than 150 days. Serum aspartate aminotransferase and bilirubin levels normalized by day 3 in the CyA group and day 5 in transplant recipients treated with costimulatory blockade. Histologically, there was no difference between isotype controls and CTLA4-Ig-treated animals, whereas anti-CD154-treated transplant recipients had a lower Banff score. CD4+ and CD8+ T-cell infiltrates were prominent in transplant recipients treated with costimulatory blockade. Intragraft analysis showed an increase in lymphocyte apoptosis, Fas ligand messenger RNA expression, and reduction in interleukin-6 gene expression in transplant recipients treated with costimulatory blockade. Costimulatory blockade did not alter intragraft gene expression of other mediators of T-cell priming, differentiation, and effector function compared with isotype control animals. In conclusion, costimulatory blockade prevented acute rejection, enabled long-term survival, and increased intragraft lymphocyte apoptosis in a high-responding rat OLT model.
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