HLA抗体筛选技术的最新进展及其在器官移植中的应用

人类白细胞抗原(HLA)是介导器官移植排斥反应的主要因素,良好的组织配型是肾移植患者长期存活和术后移植肾功能恢复的重要条件之一。在多次输血、生育史、再次移植的受者受到同种HLA免疫致敏可产生群体反应性抗体(panelreactive antibody,PRA)。PRA是一组特定的人类白细胞抗原(HLA)抗体,有多种类型,包括HLA-A、B、C、DR、DQ等抗体[1],是造成超急性和加速性排斥反应和移植物丢失的危险因素之一。PRA水平的高低更直接地影响移植肾的近期存活率。因此,将抗体筛选技术应用于器官移植实践,对减少移植后排斥反应,提高移植成功率和移植…     

HLA配型和DSA对移植肾功能的影响研究

器官移植中良好的供受者间HLA配型对预防移植肾早期排斥和移植肾的长期存活具有重要意义.肾移植术后发生排斥的重要因素之一是群体反应性抗体(PRA)的参与,特别是肾移植术后产生抗供者特异性抗体(DSA).因此本文对肾移植供受者HLA分型和DSA对移植肾急性排斥反应和近期肾功能进行研究.现报道如下. 材料和方法 一般资料:选择了2007年10月至2009年12月间在我院接受肾移植的128例患者,男性88例,女性40例;最大年龄63岁,最小年龄20岁.     

MICA抗体对肾移植患者长期存活影响的初步研究

目前在肾移植术后研究群体反应抗体与移植肾排斥外,主要组织相容性Ⅰ类相关链A抗原(MICA)抗体也处于研究的热点.MICA基因是MIC基因家族的一员,包括MICA、B、C、D、E、F、G这7个成员,MICA距HLA-B位点最近.在某些肾移植术后抗HLA抗体阴性的患者排除细胞性排斥的情况下仍发生急性排斥,甚至亲属肾移植供受者HLA抗原全相配的患者仍发生急性排斥,因此推断可能有其他抗体参与了移植肾排斥.     

大鼠胸腺内移植肾小球后不用免疫抑制剂使肾移植物长期存活

异体移植物的排斥反应限制了肾移植的临床应用。为有效防止排斥反应,异体移植物受体通常要接受终生免疫抑制治疗,但这又往往导致严重合并症甚至影响病人生命。为建立一种对特异性抗原的不反应状态,促使产生相应的免疫耐受性,作者通过肾小球胸腺内移植诱导出免疫耐受然后再行异体肾移植,使肾移植物能长期存活。     

高PRA介导肾移植后延迟性超急排斥反应

目的：了解HLA-A、B、DR、DQ表现型为纯合子的终末期肾脏疾病患者的PRA水平,并研究其与肾移植预后的关系。方法：对2006年8月至2007年8月间在我院登记等待首次肾移植的438例终末期肾脏疾病患者HLA、PRA情况以及其中1例PRA65的HLA纯合子患者肾移植效果进行分析。HLA基因分型采用PCR-SSP技术,PRA、抗体强度及抗体特异性分析采用ELISA技术,数据统计采用SPSS11.5统计软件。结果：438例患者中有HLA纯合子12例,杂合子426例;12例纯合子中PRA阳性者6例,阳性率50,平均HLA抗体强度为55,广泛致敏率达100;426例杂合子中PRA阳性者65例,阳性率15.26,平均HLA抗体强度为28,广泛致敏率为40;纯合子与杂合子之间的PRA阳性率、平均HLA抗体强度和广泛致敏率相比差异均有统计学意义（P〈0.05）,纯合子高PRA的风险大（OR=5.29,95CI：1.67～16.70）;1例PRA65的纯合子患者术前经血浆置换、蛋白免疫吸附、静滴丙种球蛋白治疗使PRA降低至阴性,在舒莱免疫诱导下行尸体肾移植术,供受者HLA抗原2个错配,术后移植肾功能逐渐恢复,第3天开始PRA呈阳性并逐渐升高,伴随临床逐渐出现尿少、血压升高、移植肾区疼痛等排斥反应征象,血清肌酐逐渐升高,移植肾B超见动脉阻力指数逐渐升高,提示急性排斥反应,采用术前有效清除HLA抗体的所有方法再次治疗无效,于术后第36天因移植物失功行移植肾切除。结论：HLA纯合子产生高PRA的风险大;文中1例PRA阳性的HLA纯合子早期移植肾功能恢复随后出现不可逆排斥反应致移植肾失功的情况,提示近年来随着各种有效清除HLA抗体的治疗方法不断用于临床导致了抗体介导的延迟性超急排斥反应（AMDR）的出现从而使移植肾失功;高PRA患者行肾移植术需高度慎重,应尽可能避开受者PRA峰值时的全部抗体谱来选择供体。     

活体亲属肾移植患者HLA配型配合率的分析

HLA配型在肾移植的应用对提高肾移植的长期存活率及对抗HLA抗体高敏感患者的肾移植具有重要意义.良好的HLA配型是维持移植器官长期存活的必备因素之一,对预测移植肾的长期存活具有一定的临床意义.本研究对亲属肾移植供受者HLA配型的配合率进行了调查.     

肾移植受者HLA体液免疫致敏状态的监测及其临床意义

为探讨肾移植受者同种异体HLA抗原致敏后体液免疫状态与术后排斥反应及移植物存活率的相关性及其临床意义,应用One Lambda混合抗原板(LATM)通过ELISA筛查受者术前血清中的HLA-IgG抗体,对阳性血清进一步用抗原板(LAT1240和LAT1HDS)检测抗体阳性率及其特异性;并采用序列特异性引物聚合酶链反应(PCR-SSP)技术进行HLA基因分型。在1 297例肾移植受者中,HLA-IgG抗体阳性者165例,其中I类抗体阳性126例,II类抗体阳性90例,51例同时存在I类和II类HLA-IgG抗体,抗体阳性率>50%的高致敏受者94例。所有受者术后未发生超急性排斥反应,抗体阳性组受者的急性排斥发生率与阴性组受者比较,没有统计学差异。然而抗体阳性组受者移植物功能延迟恢复(DGF)的发生率显著高于阴性组受者(P<0.001)。抗体阳性组受者的1年、3年和5年移植肾存活率分别为95%、88%和80%;与抗体阴性组受者组比较无统计学差异。女性受者中抗体阳性率明显高于男性受者(P<0.001);再次移植受者中抗体阳性率显著高于初次移植受者(P<0.001)。抗体阳性组受者的供受者配型明显优于抗体阴性组受者。HLA-IgG抗体是反映受者体液免疫致敏状态的敏感指标,供受者间良好的HLA配型能显著降低排斥反应发生率和改善移植物存活。     

再次肾移植疗效评价及预后多因素分析

背景：随着新型免疫抑制的使用和围手术期处理水平的提高,再次肾移植疗效已得到很大提高,然而与首次移植相比,再次肾移植面临更多的危险因素。 目的：评价再次肾移植的疗效,分析影响预后的主要因素。 方法：回顾分析中南大学湘雅三医院湘雅移植医学研究院2001-04/2009-06再次肾移植患者68例的临床资料,用Kaplan-Meier法计算人/肾存活率并与同期首次移植患者的人/肾存活率对比分析,选用可能影响再次肾移植疗效的12个临床指标,Log-rank检验和Cox回归模型分别进行预后单因素和多因素分析。 结果与结论：全组随访6~86个月,随访期间死亡10例(14.7%),移植肾失功18例(26.5%)。再次移植组1,3,5年人/肾存活率分别为94.1%/89.7%,87.3%/80.5%,80.3%/68.7%,对照组为96.3%/94.5%,90.5%/85.4%,83.8%/75.6%,再次肾移植人/肾存活率均低于首次移植,但差异无显著性意义(P > 0.05)。单因素分析显示,首次移植肾存活时间、群体反应性抗体峰值、HLA抗原错配、急性排斥、移植肾功能延迟恢复和免疫诱导与再次移植肾存活有关;COX回归多因素分析显示,首次移植肾存活时间、群体反应性抗体峰值和HLA抗原错配是影响移植肾存活的独立因素。随着再次肾移植前后处理水平的提高,再次移植可获得与首次移植近似的疗效;首次移植肾存活时间、群体反应性抗体峰值和HLA抗原错配是影响再次肾移植预后的最主要因素。     

肾同种移植排斥的移植物洗脱液中的针对供者内皮细胞和单核细胞抗原的抗体

HLA相同的同胞间进行肾移植存活良好,但无亲属关系的供者尸肾移植中HLA配型则效果不显著。通常是用淋巴细胞进行配型,而肾特异抗原的可能作用被忽视。为此,作者测定了二例急性排斥反应的肾移植洗脱液,证明在洗脱液中存在针对相应供者的单核细胞与移植物周围备管和静脉内皮细胞的非淋巴细胞抗体。病人1:HLA分型为A1,A28,B8,B12,Bw4,Bw6,供者:A1,A10,B8,B12,Bw4,Bw6。一月内急性排斥了肾移植物。病人2:Aw19,B12,B13,Bw4,Cw5,Cw6。供者:Aw19,B12,B14,BW4。出现超急排斥。     

群体反应性抗体与器官移植

群体反应性抗体(PRA)是移植中最重要的免疫因素。研究证明移植排斥是由体液免疫介导的,PRA分析能反映受者人类白细胞抗原(HLA)的致敏状态,在移植前预测排斥反应的发生和移植后预警移植物丧失,使器官移植后各种排斥反应的发生率显著降低。目前PRA尤其是特异性抗供者抗体(DSA)在肾、心脏、肺等实体器官移植中的作用及其前瞻性应用研究成为热点。本文就PRA与器官移植的研究进展进行综述。     

Clinical relevance of lung-restricted antibodies in lung transplantation

Lung transplant is a definitive treatment for several end-stage lung diseases. However, the high incidence of allograft rejection limits the overall survival following lung transplantation. Traditionally, alloimmunity directed against human leukocyte antigens (HLA) has been implicated in transplant rejection. Recently, the clinical impact of non-HLA lung-restricted antibodies (LRA) has been recognized and extensive research has demonstrated that they may play a dominant role in the development of lung allograft rejection. The immunogenic lung-restricted antigens that have been identified include amongst others, collagen type I, collagen type V, and k-alpha 1 tubulin. Pre-existing antibodies against these lung-restricted antigens are prevalent in patients undergoing lung transplantation and have emerged as one of the predominant risk factors for primary graft dysfunction which limits short-term survival following lung transplantation. Additionally, LRA have been shown to predispose to chronic lung allograft rejection, the predominant cause of poor long-term survival. This review will discuss ongoing research into the mechanisms of development of LRA as well as the pathogenesis of associated lung allograft injury.     

The impact of non-HLA antibodies on outcomes after lung transplantation and implications for therapeutic approaches

The role of donor-specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) in lung allograft rejection has been recognized over the past 20 years. During this time, there has been growing experience and recognition of an important role for non-HLA antibodies in lung allograft rejection. Multiple self-antigens have been identified that elicit autoimmune responses including collagen V, K-α 1 tubulin, angiotensin type 1 receptor, and endothelin type A receptor, but it is likely that other antigens elicit similar responses. The paradigm for the pathogenesis of these autoimmune responses consists of exposure of sequestered self-antigens followed by loss of peripheral tolerance, which then promotes allograft rejection. Studies have focused mainly on the impact of autoimmune responses on the development of Bronchiolitis Obliterans Syndrome or its mouse model surrogate. However, there are emerging data that illustrate that non-HLA antibodies can induce acute antibody-mediated rejection (AMR) after lung transplantation. Treatment has focused on antibody-depletion protocols, but experience is limited to cohort studies and appropriate controlled trials have not been conducted. It is noteworthy that depletion of non-HLA antibodies has been associated with favorable clinical outcomes. Clearly, additional studies are needed to identify the optimal therapeutic approaches to non-HLA antibodies in clinical practice.     

Relevance of MICA and other non-HLA antibodies in clinical transplantation

The clinical importance of HLA-specific antibodies for organ allograft outcome is well established. In the past few years, there has been an increasing interest in non-HLA antigens as targets of injury in organ transplant recipients. This increased interest has been spurred by the fact that HLA-identical kidney transplants also undergo immunological rejections. Polymorphisms within non-HLA genes associated with evoking an immune response to alloantigens are currently being studied for their association with transplant outcome. Non-HLA antigens, such as the polymorphic MHC class I-related chain A (MICA), expressed on endothelial cells have been implicated in the pathogenesis of hyperacute, acute and chronic organ allograft rejections. Use of endothelial cells as targets may clarify the specificities of other clinically relevant non-HLA antibodies in graft rejections. This review summarizes past and current knowledge of the clinical importance and specificities of non-HLA antibodies, and mechanisms by which these antibodies may contribute to graft destruction in clinical transplantation. The aims of current research into the role of non-HLA antigens and their genetics in predicting outcome are to develop an improved insight into the basic science of transplantation and to develop a risk or prognostic index for use in the clinical setting. Non-HLA antibody responses are receiving increasing interest in acute and chronic rejection and specificity, affinity, and pathogenicity need to be investigated to estimate their contribution. Undoubtedly, this will continue to be an area of interest in terms of fully understanding the role of non-HLA antigens as targets of immune-mediated injury and the potential for clinical intervention.     

非HLA因素致大鼠肾移植CAN加快模型的建立

目的建立大鼠异体肾移植慢性移植肾肾病(CAN)加快模型,为研究非HLA免疫因素及非免疫因素致CAN的病因、病理及病理生理机制提供平台。方法采用雄性Fisher大鼠和Lewis大鼠分别作为供-受体,并以假手术组作为对照,进行异体原位肾移植。受体移植术前对供肾进行强化冷缺血处理1 h,于术后4 w,8 w,12 w分别观察各受体血肌酐和移植肾组织病理变化情况。结果移植术后4周Fisher-Lewis组开始出现血肌酐的升高及移植肾CAN的病理改变,8~12周病变渐趋明显,与对照组比较有统计学差异(P<0.05)。结论以Fisher和Lewis近交系大鼠作为供-受体建立的大鼠异体肾移植CAN加快模型是一种可靠、实用和研究价值高的肾移植实验动物模型。     

Immunology of renal allograft rejection

Allograft rejection remains the critical problem of renal transplantation. The immunologic mechanisms that underlie renal allograft rejection are heterogeneous and involve the humoral and cellular limbs of the immune response. Antibody-mediated hyperacute rejection is now rare owing to improved prospective cross-matching. Chronic rejection, characterized by intrarenal arterial fibrosis, is still poorly understood. Knowledge of the afferent and efferent processes involved in rejection has led to effective therapeutic and experimental strategies that employ monoclonal antibodies and other pharmacologic agents to reverse, or prevent, acute allograft rejection. In addition, allospecific tolerance has been achieved experimentally and clinically in a variety of manners. Preliminary studies on the mechanism of allograft tolerance induced by donor-specific blood transfusions before transplantation suggest a role for an immunoregulatory cell population that specifically down regulates cytotoxic lymphocyte responses to donor antigens in some recipients. A role for noninherited maternal antigens and anti-idiotypic antibodies in down regulating immune responses to allografts have also been reported by several studies. An improved understanding of allograft rejection and tolerance may identify approaches to prolong allograft survival without the morbidity and mortality associated with present-day immunosuppression.     

ARHGDIB and AT1R autoantibodies are differentially related to the development and presence of chronic antibody-mediated rejection and fibrosis in kidney allografts

The role of non-HLA autoantibodies in chronic-active antibody-mediated rejection (c-aABMR) of kidney transplants is largely unknown. In this study, the presence and clinical relevance of non-HLA autoantibodies using a recently developed multiplex Luminex-based assay were investigated. Patients with a kidney allograft biopsy at least 6 months after transplantation with a diagnosis of c-aABMR (n = 36) or no rejection (n = 21) were included. Pre-transplantation sera and sera at time of biopsy were tested for the presence of 14 relevant autoantibodies.A significantly higher signal for autoantibodies against Rho GDP‐dissociation inhibitor 2 (ARHGDIB) was detected in recipients with c-aABMR as compared to recipients with no rejection. However, ARHGDIB autoantibodies did not associate with graft survival. Levels of autoantibodies against angiotensin II type 1-receptor (AT1R) and peroxisomal trans‐2‐enoyl‐CoA reductase (PECR) were increased in recipients with interstitial fibrosis in their kidney biopsy. Only the signal for AT1R autoantibody showed a linear relationship with the degree of interstitial fibrosis and was associated with graft survival.In conclusion, anti-ARHGDIB autoantibodies are increased when c-aABMR is diagnosed but are not associated with graft survival, while higher levels of AT1R autoantibody are specifically associated with the presence of interstitial fibrosis and graft survival.     

移植肾穿刺活组织检查：72例的病理及临床分析

背景：肾移植后慢性排斥反应及各种移植肾病变是移植肾失功能的常见原因,但对移植肾予以准确评估往往非常困难,活检仍是目前的主要手段。 目的：分析肾移植后出现合并症时移植肾穿刺活检的病理结果。 方法：对72例移植肾进行肾穿刺活组织检查,并进行病理诊断及分类,结合移植后情况进行分析。 结果与结论：72例中发生急性细胞介导性排斥反应35例,急性抗体介导性排斥反应12例,移植肾急性药物毒性损伤10例,慢性T细胞介导性排斥反应6例,慢性抗体介导性排斥反应2例,急性肾小管坏死4例,慢性移植肾肾病3例。移植肾组织活检的病理报告与穿刺前临床诊断的符合率在75%以上。移植肾穿刺活检未发生明显的不良反应。提示移植肾活检安全可靠,对肾移植后难以根据临床化验资料作出准确判断肾脏损害的并发症及治疗方案的选择有极为重要的指导意义。     

Role of anti-interleukin-2 receptor antibodies in kidney transplantation

From the early 1960s, the mainstay of immunosuppression for kidney transplantation has been corticosteroids. Since then, many new drugs have been developed to maintain the renal allograft. Current maintenance immunosuppression commonly consists of corticosteroids, antiproliferative agents and calcineurin inhibitors (e.g. cyclosporin). More recently, antihuman antibodies, either monoclonal or polyclonal, have been developed to use for induction at the time of transplantation or to treat rejection. With the advances in molecular technology, a new class of antihuman antibodies [the anti—interleukin-2 receptor (IL-2R) antibodies] has emerged that incorporate a murine antigen-binding site on to a human immunoglobulin backbone. Such methodology creates antihuman antibodies with high affinity for the epitope and with prolonged serum antibody half-lives. Interleukin-2 and its receptor are central to lymphocyte activation and are the main targets of calcineurin inhibitors. In addition, the anti—IL-2R antibodies inhibit a key target in immune activation. Daclizumab and basiliximab have been shown to significantly reduce the incidence of acute rejection in kidney transplantation. Since these anti—IL-2R antibodies are well tolerated and since calcineurin inhibitors are intrinsically nephrotoxic, anti—IL-2R antibodies have been used in an attempt to avoid cyclosporin after transplantation. Data from clinical trials seem to indicate that the addition of an anti-IL-2R antibody is not sufficient to warrant complete withdrawal of calcineurin inhibitors for more than a very short period after transplantation. A more promising role for anti—IL-2R antibodies may be in renal transplant recipients with delayed graft function (DGF). Recent data on the use of either low-dose calcineurin inhibitors or sirolimus (rapamycin) in conjunction with the anti-IL-2R antibodies for patients with DGF showed no increased risk of acute rejection. Long-term graft survival with use of these low-dose calcineurin inhibitor protocols has yet to be established.     

Association of vimentin antibody and other non-HLA antibodies with treated antibody mediated rejection in heart transplant recipients

Non-human leukocyte antigen (HLA) antibodies have been implicated in heart transplantation rejection. However, targets of non-HLA antibodies remain elusive. Here, we utilized a panel of multiplex beads-based assay to determine the specificity of non-HLA antibodies following heart transplantation. We utilized a selected cohort of recipients who did not have HLA donor specific antibodies, but were diagnosed with antibody mediated rejection and treated for antibody mediated rejection. We found the presence of vimentin antibody was associated with treated antibody mediated rejection. Our results suggest that, in heart transplant recipients who are suspected of AMR but in the absence of HLA donor specific antibodies, non-HLA antibodies should be examined.     

Pre-transplant Screening for Non-HLA Antibodies: Who should be Tested?

Retrospective studies of angiotensin II type 1 receptor antibodies (AT1R-Ab) and anti-endothelial cell antibodies (AECA) have linked these antibodies to allograft injury. Because rising healthcare costs dictate judicious use of laboratory testing, we sought to define characteristics of kidney transplant recipients who may benefit from screening for non-HLA antibodies. Kidney recipients transplanted between 2011 and 2016 at Johns Hopkins, were evaluated for AT1R-Ab and AECA. Pre-transplant antibody levels were compared to clinical and biopsy indications of graft dysfunction. Biopsies were graded using the Banff' 2009–2013 criteria. AT1R-Ab and AECA were detected using ELISA and endothelial cell crossmatches, respectively. AT1R-Ab levels were higher in patients who were positive for AECAs. Re-transplanted patients (p?<?0.0001), males (p?=?0.008) and those with FSGS (p?=?0.04) and younger (p?=?0.04) at time of transplantation were more likely to be positive for AT1R-Ab prior to transplantation. Recipients who were positive for AT1R-Ab prior to transplantation had increases in serum creatinine within 3?months post-transplantation (p?<?0.0001) and developed abnormal biopsies earlier than did AT1R-Ab negative patients (126?days versus 368?days respectively; p?=?0.02). Defining a clinical protocol to identify and preemptively treat patients at risk for acute rejection with detectable non-HLA antibodies is an important objective for the transplant community.