肾移植早期尿淋巴细胞表型分析的应用价值

目的：探讨尿淋巴细胞表型分析在肾移植早期免疫监测中的临床应用价值。方法：以流式细胞技术对37例肾脏移植患者的105份尿样本进行尿淋巴细胞表型分析,测定CD3、CD4、CD8、CD19、CD25、HLA－DR阳性细胞百分率,其结果在急性排斥反应组和肾功能稳定组之间进行比较。结果：与肾功能稳定组比较,急性排斥反应组尿淋巴细胞表达HLA－DR数增多非常显著（P＜0．01）,CD8和CD25细胞增多显著（P＜0．05）,而CD3、CD4、CD19细胞数变化不显著（P＞0．05）。在诊断移植肾急性排斥反应上,HLA－DR阳性样本的诊断敏感性和特异性分别达86．96％和90．24％,CD8和CD25阳性标本的阴性预测值分别达86．79％和85．71％。结论：尿淋巴细胞表型分析可间接反映出移植肾内的免疫状态,是诊断和鉴别诊断移植肾急性排斥反应的有效方法,适用于肾移植早期的免疫监测。     

肾移植患者外周血CD4^＋CD25^＋调节性T细胞的变化及临床意义

目的观察肾移植患者外周血中CD4^＋CD25^＋调节性T细胞水平的变化，探讨其在诊断移植肾急性排斥反应中的作用。方法采用流式细胞仪检测26例肾移植患者及30例正常对照组外周血中CD4^＋CD25^＋调节性T细胞水平。结果①慢性肾衰竭患者外周血CD4^＋CD25^＋调节性T细胞水平与对照组比较，差异有统计学意义（P〈0．05）。②非排斥组移植后1、2、4、8周CD4^＋CD25^＋调节性T细胞水平明显高于移植前（P〈0．01）。③急性排斥组排斥反应主要发生在术后第7～21d，其CD4^＋CD25^＋调节性T细胞水平明显低于同期的非排斥组（P〈0．01）。结论CD4^＋CD25^＋调节性T细胞水平的测定可以作为肾移植患者移植后急性排斥反应诊断和预测预后的重要指标。     

外周血及移植肾内嗜酸性粒细胞变化的临床意义

为了解移植肾在急性排斥时外周血和移植肾内嗜酸性粒细胞（ＥＯ）变化的意义，动态观察３１例同种异体肾脏移植病人的外周血和移植肾内ＥＯ的变化。结果发现在急性排斥反应时，移植肾内ＥＯ数＞２％者占８０．９％，明显高于肾功能稳定时，Ｐ＜０．０１；重度排斥中血ＥＯ数＞４％者占８２．６％，明显高于肾功能稳定时和中度以下排斥者，Ｐ＜０．０１。结果认为，测定移植肾内的ＥＯ变化可以做为监测急性排斥反应的可靠指标，外周血中的ＥＯ明显增多常提示排斥反应较为严重。     

尿流式细胞术检测对移植肾急性排斥反应的诊断

目的:探讨尿流式细胞术检测对移植肾急性排斥反应的诊断价值。方法:对63例肾移植术后患者在住院期间出现血肌酐值升高,均行尿流式细胞术检测,结果与临床诊断作对比分析。结果:急性排斥反应患者的尿液细胞数超过10000以上的占97.6%,CD54阳性细胞的占81.8%,CD103阳性细胞占57.6%,HLA-DR阳性细胞占90.9%;在其他患者中,缺少特异性抗原的表达。结论:尿液中HLA-DR、CD54、CD103阳性细胞可作为急性排斥反应的特异标志。尿流式细胞术能区分急性排斥反应与其他因素引起的移植肾功能损伤。尿流式细胞术可作为评价移植肾功能的检测指标。     

人白细胞抗原交叉反应组配型在肾移植中的应用

目的：探讨人白细胞抗原（HLA）交叉反应组（CREGs)配型原则在临床肾移植中的应用及意义。方法：312例肾移植患者分为两组，一组按传统配型原则错配2个以上位点并采用CREGs配型原则选择供受者，149例；另一组为同期按传统配型原则错配0－2个位点者，163例。比较两者移植肾1年存活率及术后1个月内急性排斥发生率。结果：按CREGs配型原则，供受者HLA－I类抗原0、1、2错配百分比分别为16．7％、41．6％和34．2％，传统配型者分别为6．7％、21．5％和71．8％。CREGs 0、1错配组相配率显著高于传统配型对应组（P＜0．01）。术后1个月急性排斥反应发生率各对应组间相比差异无显著性意义（P＞0．05），但CREGs 0错配组明显低于A、B2错配组（P＜0．05）。移植肾1年存活率各对应组间差异无显著性意义（P＞0．05），但CREGs 0错配组明显高于A、B2错配组（P＜0．05）。结论：采用CREGs配型原则选择供受者，可以获得满意的移植肾1年存活率，并不增加术后排斥反应发生率，为等待供肾的患者提供了更多机遇。     

肾移植术后供者特异性抗体对移植肾近期效果的影响

目的 评价肾移植术后供者特异性抗体(Ds-Ab)对移植肾近期效果的影响。方法 对2001年1月至2002年7月间进行尸肾移植的92例受者，使用酶联免疫吸附(ELISA)法，检测受者血清中HLA抗体水平，随访1年。结果 16例(17．4％)受者术后出现供者特异性抗体。抗体阳性组急性排斥发生率(56．3％)高于抗体阴性组(11．9％)，P=0．000；移植肾功能延迟恢复的发生率(12．5％)与抗体阴性组(9．2％)比较，差异无显著性，P=0．102；供者特异性抗体阳性组受者发生急性排斥后，移植肾肌酐水平高于抗体阴性组或无急性排斥组。结论 供者特异性抗体与肾移植术后急性排斥有关，可能影响近期移植肾功能。     

慢性排斥移植肾中细胞间粘附分子-1与HLA-DR表达的关系

目的：探讨慢性排斥移植肾中细胞间粘附分子－1（ICAM－1）和HLA－DR表达与间质淋巴细胞浸润的关系。方法：对20例慢性排斥肾移植受者进行肾活检，采用免疫组化技术（ABC法）检测移植肾内ICAM－1和HLA－DR的表达。结果：ICAM－1在慢性排斥移植肾肾小管上皮细胞和间质微小动脉内皮细胞表达增强，而HLA－DR表达则普遍上调，尤其在远曲小管。此外，在ICAM－1和HLA－DR表达增强的局部血管周围和小管间质伴有大量淋巴细胞浸润。结论：慢性排斥移植肾中ICAM－1和HLA－DR表达增强可能在排斥反应中起诱导作用，尤其是间质炎细胞的浸润及抗原递呈，同时它们又可能使表达上调的细胞成为免疫反应效应支的靶细胞，从而参与慢性排斥的细胞免疫损伤及移植肾间质损害过程。     

rhIL-10抑制小肠移植急性排斥反应的研究

目的观察rhIL-10对小肠移植急性排斥反应和T细胞增殖的抑制作用。方法将移植后的大鼠随机分为同基因组、对照组和3种不同剂量的rhIL-10治疗组，各组n=6。术后3、5、7d取移植肠管，行病理检查，测外周血T细胞亚群及术后7d肝、肾功能的重要参数：天冬酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、血肌酐（Crea）和血尿素（BUN）。结果对照组术后3d发生轻度排斥，5d发生中度排斥，7d发生重度排斥；中、高剂量组除部分标本在术后5、7d发生轻度排斥外，无排斥征象；低剂量组与对照组改变相似，但排斥的病理改变发生较晚、较轻。术后3、5、7d，低、中、高剂量组外周血CD3^＋T细胞数及CD4^＋／CD8^＋比值与对照组比较差异有统计学意义（P〈0．05）。各组AST、ALT、Crea和BUN差异均无统计学意义（P〉0．05）。结论（1）对小肠移植急性排斥的抑制，低剂量作用是明确的，但疗效有限。中、高剂量作用较明显；与同基因组、对照组比较，不增加肝、肾毒副作用；（2）动态检测外周血CD3^＋T细胞数及CD4^＋／CD8^＋比值可作为器官移植术后监测排斥反应的重要指标之一。     

血小板/内皮细胞粘附分子在急性排斥反应时表达的研究

应用免疫级化SP法和计算机图像分析系统观察分析18例急性排斥反应时移植肾活检标本中血小板／内皮细胞粘附分子（PECAM－1，CD31）的表达。结果显示急性排斥反应时肾小球毛细血管内皮细胞中PECAM－1阳性反应强度较正常明显减低或由阳性转为阴性，而在肾小管上皮中表达增强；PECAM－1与HLA－DR抗原在肾小球和肾小管中的表达呈平行关系。提示细胞粘附分子PECAM－1对移植肾急性排斥反应有一定诊断意义。     

HLA配型对群体反应性抗体阳性的肾移植受者人/肾存活率的影响

目的探讨HLA交叉反应组（CREGs）配型对群体反应性抗体（PRA）阳性肾移植受者人／肾存活率的影响。方法应用美国莱姆德公司LAT1240、LM720R、SSP2LB试剂，准确检测112例PRA阳性肾移植受者体内PRA的水平及其抗体的特异性，评估其致敏状态，应用CREGs配型标准选择最匹配的供者。结果112例受者中，HLA-Ⅰ类抗体阳性43例，Ⅱ类抗体阳性39例，Ⅰ、Ⅱ类抗体均为阳性30例；HLA配型0～5个位点错配数分别为6、39、38、21、7、1例，术后移植肾发生加速性排斥反应2例、急性排斥反应18例、慢性排斥反应5例、移植肾功能延迟恢复（DGF）4例，因排斥反应导致移植肾切除1例，死亡13例（其中移植肾带功能死亡5例）。目前人存活99例，肾存活96例，5年、3年和1年肾存活率分别为86．21％、86．96％和91．96％。结论运用CREGs配型原则，能使供、受者间的HLA相配率显著提高，可减少PRA对肾移植的不良影响，提高PRA阳性受者的人／肾存活率。     

定期检测肾移植患者尿中供者细胞DNA的临床意义

目的 探讨肾移植患者尿中供者细胞的出现与急性排斥反应的相关关系及其临床意义。方法 以供者为男性、受者为女性或HLA—DB抗原有错配的80例肾移植患者为研究对象，定期收集尿液标本，从中提取DNA，利用聚合酶链反应及序列特异性引物—聚合酶链反应分别检测Y染色体上特异的基因片段DYZ—l和HLA-DR抗原的基因DRB1。结果 手术当天受者的尿中即有供者细胞出现，随着时间的推移，尿中供者细胞DNA的基因表达强度逐渐减弱，直至术后30d，仍有90．0％患者的尿中有供者细胞DNA的基因表达，其中8例(29．6％)发生了急性排斥反应；出院后发生急性排斥反应者，90．0％的尿液标本中能检测到供者细胞DNA，抗排斥治疗结束后2周，83．3％仍为阳性，治疗过程中尿中供者细胞DNA的基因表达强度逐渐减弱，直至3个月后，88．9％转为阴性；肾功能良好的稳定期患者，仅6．7％的患者尿中DYZ—l或HLA-DRB1基因阳性。结论 肾移植患者尿中供者细胞DNA的检测可以作为诊断急性排斥反应，并与药物性肾功能损伤进行鉴别的一种方法，其基因表达强度变化为定量评价排斥反应提供了可能性。     

Relationship of HLA-DR expression to rejection and mononuclear cell infiltration in renal allograft biopsies

Research on renal biopsies has shown that HLA class I antigens are distributed throughout the renal parenchyma, but that the distribution of HLA-DR varies greatly. We investigated HLA-DR expression in biopsies of 90 renal transplants, and also semiquantitatively assessed the proportions of CD68-, CD3-, and HLA-DR-positive infiltrating cells by immunohistochemistry. The relationships between tubular DR expression and interstitial lymphocyte and macrophage infiltration were examined. Forty of the biopsies showed acute rejection (AR), 33 showed chronic rejection (CR), 10 showed suspected rejection (SR), and 7 showed no evidence of rejection (NR). HLA-DR expression was noted in 35/40 (87.5%) of the AR cases, 22/33 (66.6%) of the CR cases, and 6/10 (60%) of the SR cases. Only 1 (14.3%) of the NR cases exhibited HLA-DR antigen expression in the renal tubules. The proportions of lymphocyte and macrophage infiltration observed in the interstitium were significantly correlated with tubular DR expression in all cases (p<0.01). At 6 months after biopsy was done, 24/35 (68.6%) of the AR patients with tubular DR expression had showed second episode of rejection or showed deteriorated renal function. The remaining 11 AR cases with tubular DR expression had stable renal function at this stage. The cases that had no significant tubular DR expression had no problems with rejection or functional deterioration. These findings are consistent with the theory that expression of HLA-DR antigens on renal tubular cells may be a marker of rejection and poor graft outcome.     

HLA致敏原性错配对肾移植受者急性排斥反应发生率的影响

目的：探讨供受者HLA致敏原性错配(IM)对肾移植受者急性排斥反应发生率的影响。方法：回顾性分析196例首次肾移植受者IM对肾移植术后肾功能的恢复时间及1年内排斥反应发生率情况。结果：IM对肾移植术后肾功能恢复时间无明显影响；IM患者1年内急性排斥率明显增加；各类位点IM对肾移植术后急性排斥反应的影响进行比较，A位点影响不大，B位点与急性排斥反应有关，DR位点IM可致急性排斥反应明显增加。结论：在临床采用氨基酸残基配型标准判断组织配型的同时，IM不容忽视，HLA-B位点IM与肾移植术后急性排斥反应相关，HLA-DR位点IM明显影响肾移植术后排斥反应发生率。     

HLA‐G on peripheral blood CD4+ T lymphocytes: a potential predictor for acute renal rejection

HLA‐G Expression in grafts and serum has been shown to improve graft acceptance. However, its expression on peripheral blood lymphocytes (PBLs) during acute rejection (AR) remains unknown. In this study, we serially monitored HLA‐G expression on CD4⁺ and CD8⁺ PBLs of 66 recipients undergoing renal transplantation using flow cytometry at different time points before and after transplantation, as well as during AR episode. In stable recipients, HLA‐G expression on CD4⁺ PBLs declined during the first week after transplantation and increased continuously with immunosuppressive therapy. Then, expression declined gradually after 1 month and remained at a higher level compared with pretransplantation. When AR occurred, HLA‐G expression decreased significantly compared with the stable level. In three recipients suffering from recurrent rejection, it remained at a low level despite impact immunosuppressive treatment. With mix lymphocyte assay, HLA‐G⁺ CD4⁺ T cells showed inhibitory role on proliferation of peripheral blood mononuclear cell. HLA‐G expression on CD8⁺ PBLs was almost undetectable at different time points in the recipients and healthy controls. Our results suggest that HLA‐G on CD4⁺ PBLs might provide a potential marker for the early diagnosis of renal AR and for the immunosuppressive status of recipients.     

肾移植术后测定外周血CD3/HLA-DR及CD3/CD(16+56)的临床意义

目的探讨肾移植患者术后外周血ＣＤ３／ＨＬＡ－ＤＲ及ＣＤ３／ＣＤ（１６＋５６）的变化及其意义。方法提取患者的外周血淋巴细胞,加入双荧光标记的鼠抗人单克隆抗体ＣＤ３／ＣＤ（１６＋５６）、ＣＤ３／ＨＬＡＤＲ,流式细胞分析仪进行测定。结果术前患者的ＣＤ＋３／ＣＤ＋（１６＋５６）、ＣＤ－３／ＣＤ＋（１６＋５６）及ＣＤ－３／ＨＬＡＤＲ＋高于健康人,而ＣＤ＋３／ＣＤ－（１６＋５６）和ＣＤ＋３／ＨＬＡＤＲ－低于健康人;术后３天肾功能稳定者的全部淋巴细胞亚群下降,尤以ＣＤ＋３／ＣＤ＋（１６＋５６）为著;术后排斥者的ＣＤ－３／ＣＤ＋（１６＋５６）、ＣＤ＋３／ＣＤ＋（１６＋５６）、ＣＤ－３／ＨＬＡＤＲ＋及ＣＤ＋３／ＨＬＡＤＲ＋较稳定者显著升高,而急性肾小管坏死者的上述４个指标异常增高,是排斥组的２倍。结论术后动态测定ＣＤ３／ＨＬＡＤＲ和ＣＤ３／ＣＤ（１６＋５６）有助于急性排斥和急性肾小管坏死的早期诊断及鉴别诊断,对及时治疗和抗排斥疗效的评价具有一定意义     

Analysis of urinary donor-derived DNA in renal transplant recipients with acute rejection

Abstract: In renal transplantation we usually diagnose an acute rejection by based on the results of a needle biopsy; however, this takes time and findings in some cases are not definite. We analysed the urine of renal recipients for the presence of donor DNA in an attempt to establish a diagnostic means of acute rejection. Sixty-four renal transplant recipients were examined. Thirty-seven patients had no trouble after transplantation and 22 patients developed acute rejection, diagnosed based on serum creatinine levels and/or needle biopsy findings of the graft. Five patients had drug-induced renal dysfunction. In female recipients with a male graft we examined urine for the presence of Y-chromosome (SRY and DYZ-1) and in recipients receiving a HLA mismatched graft we investigated the HLA-DR gene (DRB1) by the polymerase chain reaction (PCR) method. Among female recipients with a male graft there were 14 patients with stable renal function and SRY and DYZ-1 on Y-chromosome were negative in 13 (93%) and positive in one, whereas SRY and DYZ-1 of urine were positive in the four female patients with acute rejection and these DNA fragments disappeared in three after rejection therapy. One patient was subjected to haemodialysis. Among 23 recipients of a graft from HLA mismatched donors with stable renal function, DRB1 was negative in 21(91%). Among 18 patients with acute rejection DRB1 was positive in 16 (93%) and negative in two. These DNA fragments disappeared in 13 patients after rejection therapy. In all patients with drug-induced renal dysfunction donor-derived DNA was negative. Presence of donor-specific DNA in the urine of the recipient is associated strongly with acute rejection and analysis of DNA derived from donor cells in urine might be an effective and accurate method for the diagnosis of acute rejection of a renal transplant.     

Relationship between the expression levels of CD61, CD63, and PAC-1 on platelet surface in peripheral blood and the transplanted kidney function

OBJECTIVE: To analyze the relationships between the expression levels of CD61, CD63, and PAC-1 on the platelet surface and the incidences of acute rejection and tubular necrosis as well as the recovery of graft function after renal transplantation. METHODS: The expression levels of CD61, CD63, and PAC-1 on platelet surfaces were assayed by flow cytometry in 86 patients with different stages of uremia before and after transplantation. Patients were divided into three groups: 29 patients with normal graft function, 30 with acute rejection, and 27 with acute tubular necrosis. Patients with acute rejection were randomly assigned into groups treated with or without anticoagulants. RESULTS: The expression levels of CD61, CD63, and PAC-1 on platelet surfaces significantly increased (P <.05) among patients with acute rejection, as compared with those with normal graft function or acute tubular necrosis. Compared with controls, the expression levels of CD61, CD63, and PAC-1 were lower among acute rejection patients who, received anticoagulant therapy. The recovery time for graft function shorter and, the 1-year patients and graft survival rates higher. CONCLUSIONS: The pretransplant expression levels of CD61, CD63, and PAC-1 on platelet surface were significantly higher among patients with acute rejection, suggesting that this complication rather than acute tubular necrosis may be related to platelet activation. Patients with acute rejection displayed benefit from anticoagulant therapy.     

CD69 expression on peripheral CD8 T cells correlates with acute rejection in renal transplant recipients

BACKGROUND: The development of a noninvasive method to diagnose renal allograft rejection could prevent the complications associated with graft biopsy and allow more accurate surveillance of allograft function. The present study determines whether expression of CD69 on peripheral T lymphocytes of renal allograft recipients correlates with the presence of acute graft rejection. METHODS: Peripheral blood T lymphocytes from healthy volunteers, renal allograft recipients with elevated creatinine but no evidence of rejection on biopsy, and renal allograft recipients with biopsy-proven rejection were analyzed by flow cytometry for the expression of CD69 and various intracellular cytokines (interleukin-2, interferon-gamma). Results were then compared with the degree of rejection on biopsy. RESULTS: CD69 expression on CD3+, CD4+, and CD8+ T-cell subsets was low in controls and transplant recipients without allograft rejection. In contrast, patients with renal allograft rejection showed significantly elevated percentages of CD69+ cells in the CD3+ (P<0.01) and CD8+ subsets (P<0.01). The fraction of CD69+ and CD8+ T cells was found to be a more clinically useful test based on receiver-operator characteristics. CD69 expression on CD4+ T cells did not correlate with rejection. Significant intracellular cytokine levels were not detected in unstimulated T cells from any of the groups; stimulation with mitogens increased expression equally among the three groups. CONCLUSIONS: We demonstrate that expression of CD69 on CD3+ and CD8+ peripheral blood T cells correlates closely with the presence of acute graft rejection in renal allograft recipients. Measurement of this surface marker may provide a rapid, noninvasive, and accurate means by which graft rejection can be identified.     

Flow cytometry evaluation of urinary sediment in renal transplantation

The value of exfoliative urinary cytology for the diagnosis of different pathological conditions in renal transplantation is widely recognized. The method, however, has not yet gained full acceptance, mainly because identification of the different cells is not always possible by means of standard staining techniques. In view of its characteristics, flow cytometry (FC) seems to represent a consistently reliable, rapid and innovative approach for differentiating the various cells present in the urinary sediment and assessing their number. This study gives the examination result of 223 urinary specimens from 127 transplanted patients selected according to pathology. Sediment cells, collected from fresh urine samples, were washed, treated with a lysing solution, resuspended in saline solution and directly analysed in a FACSCAN cytometer. Morphological evaluation showed: a small number of cells in patients with stable renal function; a larger number of cells, with predominance of lymphocytes, during acute rejection episodes; an absolute predominance of neutrophils during bacterial infection; large-sized cellular debris in cases of post-transplant tubular necrosis; and small cell debris in cases of cyclosporine cytotoxicity. Lymphocyte surface-marker evaluation made it possible to differentiate lymphocyte populations observed during acute rejection episodes (cytotoxic T-cell, CD8 and HLA class II and NK cells) from those detected during bacterial infection (T-cell CD4 positive). These results suggest that urinary FC may be a reliable diagnostic tool in clinical renal transplantation.