Viral IL-10 gene transfer prolongs rat islet allograft survival

Islet transplantation is a potential cure for diabetes. However, allotransplant rejection severely limits its clinical application. In this study, we sought to transfect rat islets with an adenoviral vector containing the viral IL-10 (vIL-10) gene and examine its efficacy in preventing graft rejection. The immunosuppressive effect of vIL-10 is reported but its efficacy is somehow debatable in transplantation model. vIL-10 transfected islets were transplanted into streptozotocin-induced diabetic rats. Blood glucose, serum vIL-10 concentration, graft histology, and graft cytokine expression were used to monitor graft function up to day 21 after transplantation. Transfected islets released a large amount of vIL-10 protein without affecting their viability and functional integrity. When we transplanted the transfected islets into allogeneic hosts, the survival of grafted islets was not significantly increased. However, the combined use of vIL-10 and subtherapeutic doses of CsA (cyclosporine) significantly prolonged graft survival beyond that achieved with either agent alone (p < 0.001). vIL-10 and CsA-treated rats contain high level of vIL-10 in serum, which is evidenced by the inhibition of allogeneic mixed lymphocyte reaction (MLR). Histological analysis additionally revealed the presence of viable islets up to 21 days. IL-10 mRNA expression in grafted liver was higher and IFN-gamma mRNA was lower in vIL-10 and CsA-treated animals, compared with other groups. The synergistic effect of this combination therapy is potentially correlated with the induction of inhibitory cytokine secretion and downregulation of proinflammatory cytokine secretion from host cells.     

Inactivation of T-cell receptor-mediated integrin activation prolongs allograft survival in ADAP-deficient mice

BACKGROUND: Signaling through the T cell receptor (TCR) leads to profound changes in the function and properties of T cells, including integrin activation. Adhesion and degranulation promoting adapter protein (ADAP) is an adapter protein linking T cell receptor stimulation to integrin activation. We aim to clarify how disruption of TCR-mediated integrin activation affects alloreactive immune responses. METHODS: In vitro T cell proliferation and the cytokine production was determined. In vivo cytotoxic T lymphocyte (CTL) activity was measured as well. Allogenic skin and heart transplantation was used to test the in vivo role of ADAP in alloimmune responses. Histology and flow cytometry was applied to analyze the graft infiltrating lymphocytes. RESULTS: Upon stimulation with allogenic dendritic cells ADAP-deficient T cells displayed impaired proliferative responses compared to wild type (WT) T cells. This was accompanied by significantly decreased production of the cytokine interleukin-2. In contrast, the in vivo CTL activity in ADAP-deficient mice was comparable to that of WT mice. Consistently, we observed a prolongation of fully major histocompatibility complex (MHC)-mismatched heart transplants in ADAP deficient mice. Protection of allogenic heart grafts in ADAP-deficient mice was accompanied by a decrease in the infiltration, proliferation and activation of T cells in the allograft. However, no effect was observed after fully MHC-mismatched skin transplantation. CONCLUSIONS: We have shown that although ADAP is dispensable for the rejection of allografts, ADAP function plays an important role for the efficacy of graft rejection. ADAP's main function appears to affect the induction phase of the immune response.     

输注外源性白细胞介素13延长同种肝移植大鼠的存活时间

目的 探讨外源性重组白细胞介素13(rIL-13)抑制同种肝移植大鼠的排斥反应和延长大鼠存活时间的作用及其机制.方法 以Lewis大鼠为供鼠,BN大鼠为受鼠,采用重建肝动脉的大鼠肝移植方法建立同种大鼠原位肝移植模型.采用随机数字表法将受鼠分为两组,实验组于术后第1、2、3、4和5天经尾静脉注射rIL-13 10μg/d,同期对照组注射等体积生理盐水.术后第7天,检测两组移植肝功能,测定移植肝组织病理改变、细胞因子表达及CD8+T淋巴细胞浸润等情况,并根据Banff标准计算排斥活动指数(RAI),观察术后2周的存活率.结果 与对照组相比,实验组肝功能明显改善,肝组织肿瘤坏死因子α(TNF-α)与E选择素的表达明显降低(P＜0.05),CD8+T淋巴细胞浸润明显减少(P＜0.05),术后2周存活率明显提高(P＜0.05).实验组的RAI为4.8±1.2,明显低于对照组的7.5±1.2(P＜0.05).结论 外源性重组rIL-13可减少促炎症因子TNF-α的释放和抑制E选择素的表达,减少移植物CD8+T淋巴细胞的浸润,从而减轻肝移植后急性排斥反应,延长大鼠的存活时间.     

IL-10/Fc inhibits macrophage function and prolongs pancreatic islet xenograft survival

BACKGROUND: Xenograft rejection is a complex response in which macrophages and other effector cells are activated by CD4+ T cells. Initiation and regulation of this response is in part mediated by cytokines. In this study we test the hypothesis that xenograft destruction is an interleukin- (IL) 10 responsive, macrophage-mediated event. METHODS: To study the effect of the systemic administration of IL-10 on pancreatic islet xenograft rejection, a fusion protein of IL-10/Fc was used. This immunoligand possesses the bioavailability of IL-10 and the long circulating t1/2 in vivo, characteristic of Ig. Wistar rat islets were transplanted into C57BL6 mice. IL-10/Fc was administered either immediately before transplantation or in the posttransplant period. RESULTS: Both therapeutic protocols prolonged xenograft survival. Macrophage effector function was reduced in IL-10/Fc-treated mice, with a reduced macrophage infiltrate, reduced IL-12 and tumor necrosis factor-alpha gene expression and reduced serum NO2- levels. Although the number of T cells infiltrating islet grafts was not reduced, T cell effector function was inhibited in IL-10/Fc-treated animals with reduced interferon-gamma and IL-4 gene expression, reduced anti-donor cytotoxicity by recipient splenocytes and reduced anti-donor IgG1 antibody production. Ultimate rejection of the xenografts appears to be mediated by a CD4+ T cell dependent mechanism probably as a result of inadequate inhibition of IL-12 production by macrophages. CONCLUSION: IL-10/Fc prolonged rat pancreatic islet xenograft survival by inhibiting macrophage mediated immune responses. The effectiveness of this agent when administered pretransplant suggests it may have a role as an induction agent with potential clinical application.     

SOCS3基因转染对小鼠同种异体移植心脏存活时间的影响

目的研究腺病毒载体介导的细胞因子信号抑制因子3(suppressors of cytokine sig-naling3,SOCS3)基因转染对小鼠同种异体移植心脏存活时间的影响。方法以Balb/c小鼠为供体,以C57BL/6小鼠为受体,行同种异体心脏移植。C57BL/6同种异体小鼠心脏移植受体分为3组(21只/组):(1)对照组,单纯同种异体心脏移植;(2)Ad-SOCS3组,供体术前24h转染2×109pfu编码SOCS3基因的腺病毒载体;(3)Ad-GFP组,供体术前24h转染2×109pfu空病毒载体。每组10只受体用于观察生存期;每组5只受体分别于术后第1、3、5、7、9日处死,获取移植物,采用实时定量逆转录酶聚合酶链反应检测SOCS3mRNA表达的变化;每组6只受体于术后第6日处死,获取移植物和脾脏用于组织学检查及Foxp3mRNA、白介素(IL)-17mRNA表达的检测,同时检测脾脏的混合淋巴细胞反应(mixedlymphocytereaction,MLR)强度,采用流式细胞仪分析调节性T细胞(Treg)及Th17细胞的比例。结果供体术前SOCS3基因转染可明显增加术后移植物内SOCS3 mRNA表达;Ad-SOCS3组的移植心脏存活时间明显长于对照组、Ad-GFP组(P0.05)。术后第6日,Ad-SOCS3组移植物淋巴细胞浸润程度较轻、排斥反应强度级别较低、MLR较弱;该组移植物内Foxp3 mRNA水平及脾脏内CD4+CD25+Treg比例与其他两组比较差异无统计学意义(P0.05),但IL-17 mRNA水平及脾脏内CD4+IL-17+Th17比例较其他两组明显降低(均为P0.05)。结论腺病毒载体介导的供体SOCS3基因转染能够延长移植心脏存活时间,其机制可能与抑制淋巴细胞浸润、抑制T淋巴细胞同种抗原反应性以及抑制Th17免疫反应等有关,与Treg的产生及功能无关。     

Peritransplant streptavidin recipient treatment prolongs rat cardiac allograft survival

BACKGROUND: Because streptavidin shows high localization in inflamed tissues, it might also interfere with the proliferation of cells involved in allograft rejection. METHODS AND RESULTS: Treatment of na?ve ACI recipients with 20 mg/kg streptavidin i.p. alone significantly prolonged Lewis cardiac allografts from a mean survival time of 9.8+/-0.7 days in controls to 19.8+/-6.5 days, with one recipient accepting the graft permanently (>250 days). Peritransplant streptavidin treatment combined with 0.5 ml of antilymphocyte serum (ALS) transient immunosuppression led to permanent graft survival (>250 days) in 6 of 10 recipients. Second-set skin grafts performed 60 days after the primary cardiac allograft were prolonged to 45 days, whereas the third party Wistar-Furth (WF) skin grafts were rejected in 15 days without the rejection of the primary Lewis cardiac allografts. Pathology of transplanted cardiac allografts at 100 days showed no mononuclear cell infiltration or chronic allograft vasculopathy. Streptavidin given for 5 days at 20 mg/kg caused a moderate initial weight loss but had no effect on hematologic, biochemical, and histologic parameters in the treated recipients. CONCLUSION: This study demonstrates that peritransplant recipient treatment with streptavidin combined with peritransplant ALS induces prolonged cardiac and second-set skin allograft survival. We conclude that recipient peritransplant streptavidin treatment may provide a new strategy for the induction of transplant tolerance.     

CTLA4Ig combined with anti-LFA-1 prolongs cardiac allograft survival indefinitely

CTLA4Ig and anti-LFA-1 are members of a new generation of immunomodulatory drugs which inhibit important signaling pathways in T cell activation. Both substances target molecules which have pivitol functions in the activation of CD4+ and CD8+ T cells and have been theorized to have an interdependent relationship. These drugs have been used independently in various treatment regimens and have shown great promise in prolonging the survival of allografts. In order to test whether these substances have synergistic or potentiating effects when combined, we performed mixed lymphocyte reactions, skin transplantation and vascularised heterotopic heart transplantation in the Balb/c (H-2(d)) to C3H/HeJ (H-2(k)) strain combination. When anti-LFA-1 and CTLA4Ig were combined at low doses, there was a substantial inhibition of lymphocyte proliferation. When each drug was used as a mono-therapy in skin graft recipients, there was no significant effect on median graft survival (anti-LFA-1, 15 days; CTLA4Ig, 16 days) when compared to untreated controls (13 days), whereas a combination of anti-LFA-1 and CTLA4Ig extended graft survival significantly to 32 days. Untreated vascularised heart grafts rejected at a median of 8 days, CTLA4Ig-treated mice rejected at a median time of 79 days and anti-LFA-1-treated mice rejected at 43 days (n = 9). When CTLA4Ig and anti-LFA-1 were combined, all animals had functioning heart grafts at 100 days after transplantation. Histological analysis of combined-therapy hearts showed no signs or only minor changes associated with chronic rejection. In conclusion, these results indicate a synergistic effect of combining anti-LFA-1 with CTLA4Ig in inhibiting lymphocyte proliferation and prolonging the survival of fully MHC-mismatched allografts.     

NK026680 inhibits T-cell function in an IL-2-dependent manner and prolongs cardiac allograft survival in rats

NK026680 is a triazolopyrimidine derivative that has been shown to inhibit dendritic cell maturation and activation. Here, we examined the immunosuppressive properties of NK026680 on T-cell function and assessed its immunosuppressive efficacy in an ACI (RT1^av1 haplotype) to Lewis (RT1^l) rat heart transplantation model. The effects of NK026680 on T-cell proliferation, activation, and cytokine production were investigated in vitro. Heart transplant recipient rats were administered NK026680 daily for 14 days post-transplantation. In addition to graft survival time, alloimmune responses and graft histology at 4-10 days post-transplantation were assessed. NK026680 was found to inhibit proliferation, CD25 upregulation, IL-2 production, and cell cycle progression in αCD3/αCD28-stimulated murine T cells. These effects were likely due to suppression of the p38 mitogen-activated protein kinase pathway and the subsequent inhibition of p65, c-Fos, and to a lesser extent, c-Jun. Daily NK026680 treatment suppressed alloimmune responses, prevented cellular infiltration into allografts, and prolonged graft survival. The anti-rejection effects of NK026680 were enhanced by tacrolimus. In conclusion, NK026680 inhibits the activation of T cells and prolongs cardiac allograft survival in rats. These features make it a potential candidate immunosuppressant for the treatment of organ transplant patients in the future.     

Blocking of CD44-hyaluronic acid interaction prolongs rat allograft survival

BACKGROUND: Lymphocyte activation and infiltration into a transplanted organ is an integral component of the rejection process. Graft infiltration of lymphocytes requires adhesion of leukocytes to the endothelium, diapedesis, and transmigration. One of several proteins involved in this process is CD44, which is known to interact with endothelial hyaluronan (HA). Blockade of cell-matrix and cell-cell interactions have been used extensively for modulation of immune responses and graft rejection. Based on these observations, we evaluated the effects of blocking CD44-HA interactions in a transplantation model. METHODS: We used a low molecular weight hyaluronic acid formulation (LMWHA) for the treatment of rat renal and cardiac allograft recipients. LMWHA was administered intraperitoneally at 0.5-5 mg/kg for 5-10 days after transplantation with or without a subtherapeutic dose of cyclosporine. RESULTS: LMWHA monotherapy prolonged allograft survival significantly, but only for a few days. In combination with low-dose cyclosporine, long-term survival of allografts was observed in some of recipients. CONCLUSION: Further definition of the underlying mechanism of LMWHA therapy may provide a rationale for the development of novel, nontoxic, nonimmunogenic immunotherapies.     

Preconditioning with ozone abrogates acute rejection and prolongs cardiac allograft survival in rats

Acute cardiac allograft rejection remains a major cause of morbidity and mortality after heart transplantation and predisposes for the development of graft vasculopathy. The aim of this study was to investigate the immunomodulatory effect of preconditioning of the donor and recipient with medical ozone (O(3)/O(2)) on acute allograft rejection. Minimizing the initial ischemia-reperfusion injury may result in a reduction of graft vasculopathy and ameliorate long-term outcomes after cardiac transplantation. Lewis rats were challenged with Wistar-Furth cardiac allograft. In donor and recipient animals a medical ozone (O(3)/O(2))-pneumoperitoneum was induced by single (1x) or repetitive (5x) insufflation (concentration: 50 microg/mL, 80 mL/kg body weight) of medical ozone intraperitoneally. Without immunomodulatory therapy (n = 11) cardiac allograft survival was 5.9 +/- 0.9 days. Preconditioning with medical ozone alone (single bolus as well as repetitive administration, n = 7) of the donor and recipient animals prolonged cardiac allograft survival significantly to 7.6 +/- 1.4 days (P < .05), without any adjunctive immunosuppressive therapy. In this pilot study, the intraperitoneal administration of ozone in donor and recipient animals protected from ischemia-reperfusion injury, reduced the immunogenicity of the graft, and prolonged cardiac allograft survival. Further studies are warranted to elucidate the underlying mechanisms and--more important--to investigate the effect on the development of graft vasculopathy, the major obstacle to long-term graft and patient survivals.