Molecular mechanisms associated with donor-specific microchimerism in peripheral blood of Brazilian patients after liver transplantation

A large number of studies in liver transplantation have demonstrated allogeneic microchimerism. The clinical and immunologic implications of this finding remain inconclusive, just as the influence of HLA mismatch and donor alloreactivity also are controversial. The present study analyzed the presence of allogeneic microchimerism in liver transplant recipients in relation to donor leukocyte kinetics and rejection episodes. The study was extended to determining the influence of immunogenetic factors in patients after liver transplantation. The presence of allogeneic microchimerism was analyzed on peripheral blood of 50 recipients. DNA extracted from the samples was subjected to typing for HLA-DRB1 and -DQB1 alleles by polymerase chain reactions using sequence-specific primers (PCR/SSP). Microchimerism was identified by nested PCR/SSP. Microchimerism was detected in 72% of patients. There was significant effect of microchimerism on rejection episodes (P=.002), while HLA mismatches did not show significance for one or two mismatches (P=.98). Allogeneic microchimerism detected in the majority of liver transplant patients was observed to be significantly associated with rejection episodes.     

The suppressive effect of resveratrol on protein kinase C theta in peripheral blood T lymphocytes in a rat liver transplantation model

Our aim was to study the effect of resveratrol on the expressions of protein kinase C isotypes (PKC alpha, theta) in peripheral blood lymphocytes and on the expression of IkappaB kinase-beta (IKK beta) in lymphocytes in allografts in a rat liver transplantation model. METHODS: Orthotopic liver transplantations (OLT) were performed from Sprague Dawley rats to Wistar rats. The recipients were divided into two groups after OLT. In the RES group, resveratrol was given intraperitoneally once a day (100 mg kg(-1)) after OLT, whereas in the control group vehicle buffer was given. The expressions of PKC alpha, theta in peripheral blood lymphocytes, expression of IKK beta in lymphocytes in allograft, and survival periods were compared between the groups. RESULTS: The mean survival period after OLT in the RES group was significantly longer than that in control group (P < .05). On posttransplant day 7, the expression of PKC theta in peripheral blood lymphocytes in the RES group was significantly decreased compared with that in the control group (P < .05), whereas there was no obvious difference in the expressions of PKC alpha between the two groups (P > .05), and the positive rate of IKK beta protein in lymphocytes in allografts in RES group was significantly decreased compared with that in the control group (P < .05). CONCLUSION: Resveratrol showed an immunosuppressive effect on lymphocytes for allograft rejection in the rat. Down-regulation of the expression of PKC theta in peripheral blood lymphocytes may be part of the mechanism.     

Pretransplant donor peripheral blood mononuclear cells infusion induces transplantation tolerance by generating regulatory T cells

BACKGROUND: It was suggested that maintenance of tolerance to organ transplantation may depend on the formation of T regulatory cells. METHODS: Lewis (LW) rats were made tolerant to a Brown Norway kidney by pretransplant donor peripheral blood mononuclear cells (PBMC) infusion. At greater than 90 days after transplantation, lymph node cells (LN) and graft-infiltrating leukocytes (GIL) alloreactivity was tested in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype was analyzed by FACS. mRNA expression of cytokines and other markers was analyzed on CD4+ T cells from LN. The tolerogenic potential of tolerant cells in vivo was evaluated by adoptive transfer. RESULTS: Tolerant LN cells showed a reduced proliferation against donor stimulators but a normal anti-third-party alloreactivity. In coculture, these cells inhibited antidonor but not antithird-party reactivity of na?ve LN cells. Interleukin (IL)-10 and FasL mRNA expression was up-regulated in tolerant CD4+ T cells, but an anti-IL-10 monoclonal antibody (mAb) only partially reversed their inhibitory effect. Immunoregulatory activity was concentrated in the CD4+ CD25+ T-cell subset. In a transwell system, tolerant T cells inhibited a na?ve MLR to a lesser extent than in a standard coculture. Regulatory cells transferred tolerance after infusion into na?ve LW recipients. CD4+ T cells isolated from tolerized grafts were hyporesponsive to donor stimulators and suppressed a na?ve MLR against donor antigens. CONCLUSIONS: Donor-specific regulatory T cells play a role in tolerance induction by donor PBMC infusion. Regulatory activity is concentrated in the CD4+ CD25+ subset and requires cell-to-cell contact. Regulatory CD4+ T cells accumulate in tolerized kidney grafts where they could exert a protective function against host immune response.     

肝癌肝移植术后肿瘤复发与外周血调节性T细胞及细胞因子表达水平的关系

目的  分析肝细胞癌(肝癌)肝移植术后肿瘤复发与调节性T细胞(Treg)及细胞因子表达水平的关系。方法  以解放军第309医院2010年至2014年56例肝移植患者为研究对象。根据术后病理资料分为肝癌肝移植组(28例)和肝硬化肝移植组(肝硬化组,28例),肝癌肝移植组再根据术后患者有否肿瘤复发分为未复发组(8例)和复发组(20例)。比较各组患者外周血中Treg及调节性细胞因子[血管内皮生长因子(VEGF)、白细胞介素(IL)-2、IL-10、IL-12、转化生长因子(TGF)-β、干扰素(IFN)-γ]的表达水平。结果  与肝硬化组比较,未复发组的IFN-γ、IL-12水平显著升高(均为P＜0.05);复发组的Treg%、VEGF、IFN-γ、IL-10、TGF-β水平均显著升高,IL-2和IL-12水平明显降低(均为P＜0.05)。与未复发组比较,复发组的Treg%、VEGF、IL-10、TGF-β水平均显著升高,IFN-γ、IL-2、IL-12水平明显降低(均为P＜0.05)。结论  Treg及细胞因子水平可作为肝癌肝移植术后肿瘤复发的预测指标。     

大鼠小肠移植后外周血T淋巴细胞亚群的变化

利用免疫荧光染色技术及流式细胞仪对大鼠小肠移植后外周血Ｔ淋巴细胞亚群变化进行连续监测，以探讨细胞免疫功能变化在排斥反应中的意义。结果表明，排斥反应时首先出现ＣＤ４阳性细胞显著增高，随后出现ＣＤ８阳性细胞明显下降及ＣＤ４阳性细胞／ＣＤ８阳性细胞比值增高，在排斥反应后期其比值下降；使用环孢素Ａ作免疫抑制的大鼠Ｔ淋巴细胞各亚群均无显著性变化。本实验证明ＣＤ４阳性细胞及ＣＤ８阳性细胞共同参与了排斥反应，根     

肝移植术后合并脓毒症患者外周血调节性T细胞比例及功能变化的分析

目的探讨肝移植患者在脓毒症不同阶段外周血调节性T细胞（Treg）比例和功能的变化。方法选取自2009年1月至2010年12月期间在中山大学附属第三医院行外科手术,术后合并脓毒症的47例患者作为研究对象,根据手术方式和美国胸科和危重症医师协会制订的脓毒症诊断和分期标准指南分为4组：肝移植脓毒症组（sepsis after liver transplantation group,TS组;11例）、肝移植严重脓毒症组（severe sepsis after liver transplantation group,TSS组;10例）、非肝移植脓毒症组（sepsis without liver transplantation group,NTS组;15例）、非肝移植严重脓毒症组（severe sepsis without liver transplantation group,NTSS组;11例）,另外选取20名健康正常人作为健康对照组。4组脓毒症患者通过急性生理和慢性健康评估（acute physiology and chronic health evaluation,APACHE）Ⅱ和感染相关的序贯器官衰竭评估（sequential organ failure assessment,SOFA）来评价与比较脓毒症严重程度。分别采集各组研究对象的外周血,采用流式细胞术检测CD4＋CD25＋Foxp3＋调节性T细胞比例（Treg％）,采用荧光定量逆转录聚合酶链反应检测Foxp3信使核糖核酸（messengerRNA,mRNA）。结果TSS和NTSS组的APACHEⅡ评分、SOFA评分均高于TS组和NTS组（均为P〈0．01）,且TS组的APACHEⅡ评分高于NTS组,TSS组的APACHEⅡ评分和SOFA评分均高于NTSS组（均为P〈0．01）。与健康对照组比较,NTS组Treg％明显降低（P〈0．001）,NTSS组明显升高（P=0．003）;而TS组与健康对照组相比差异无统计学意义（P=0．398）,TSS组也高于健康对照组（P=0．006）,但变化幅度不如NTSS组显著。4组脓毒症组的组间比较发现,NTSS组患者Treg％显著高于NTS组（P〈0．01）,而TSS组与Ts组两组比较差异无统计学意义（P=0．099）,NTS组患者Treg％低于Ts组（P=0．05）,而NTSS组则显著高于TSS组（P=0．002）。与健康对照组比较,NTSS组的Foxp3mRNA表达显著升高,差异有统计学意义（P〈0．05）。4组脓毒症组的组间比较发现,TSS组和NTSS组Foxp3mRNA表达值均高于TS组和TSS组,但移植组内（TS组与TSS组）的差异没有非移植组（NTS组与NTSS组）显著（分别为P=0．038、P〈0．001）;另外NTSS组的Foxp3mRNA表达显著高于TSS组（P=0．012）。结论免疫抑制剂的应用使移植患者在发生脓毒症时Treg的比例和功能的变化有别于普通人群,评估机体免疫状态时需要综合多个免疫指标。     

肝移植患者外周血辅助性T 17细胞的变化及意义

目的探讨肝移植术后患者外周血辅助性T(Th)17细胞(CD4+IL-17+T淋巴细胞)与急性排斥反应的关系。方法本文研究对象为2008年6月至2012年12月在首都医科大学附属北京朝阳医院肝胆胰脾外科因良性终末期肝病行原位肝移植术的76例患者。根据患者术后有否发生急性排斥反应,分为排斥组(17例)和非排斥组(59例)。所有患者均按常规定期随访。记录患者排斥反应发生情况及治疗经过。排斥组患者发生急性排斥反应时给予肝穿刺活组织检查确定排斥反应严重程度。所有患者分别于肝移植术前、出院后1年内定期(间隔3~6个月)、或急性排斥反应治疗前和缓解后(3~6个月),检测外周血CD4+IL-17+T淋巴细胞占CD4+T淋巴细胞百分比(CD4+IL-17+T%)。比较两组患者各时间点的CD4+IL-17+T%。分析CD4+IL-17+T%与排斥活动指数(RAI)、免疫抑制剂血药浓度的相关性。结果急性排斥反应发生在术后0.7~12.0个月,中位数2.5个月。肝移植术后,与非排斥组比较,排斥组患者CD4+IL-17+T%明显升高[(1.79±0.44)%比(2.56±0.43)%,P0.001]。排斥组患者发生急性排斥反应时,CD4+IL-17+T%均较未发生急性排斥反应时明显升高[(2.56±0.43)%比(1.50±0.25)%,P0.001)]。非排斥组患者不同时间点的CD4+IL-17+T%变化不明显(P0.05)。排斥组患者发生急性排斥反应时的CD4+IL-17+T%与RAI呈正相关(r=0.72,P=0.001)。排斥组和非排斥组患者他克莫司、环孢素血药浓度与CD4+IL-17+T%无明显相关性(r=0.21,-0.13;均为P0.05)。结论外周血CD4+IL-17+T%可作为诊断和评估肝移植术后急性排斥反应严重程度的监测指标,外周血CD4+IL-17+T%升高提示急性排斥反应严重。     

Effect of oesophagectomy on monocyte-induced apoptosis of peripheral blood T lymphocytes

BACKGROUND: Surgical stress has been reported to induce immunosuppression. The mechanisms giving rise to T-cell dysfunction following surgery are still unclear. The cellular mechanisms behind T-cell dysfunction following surgery were investigated, based on the induction of T-cell apoptosis and downregulation of T-cell signalling molecules. METHODS: Peripheral blood T cells were collected and separated before and after surgery in patients who had oesophagectomy, gastrectomy or cholecystectomy, and studied for their ability to produce cytokines, the induction of T-cell apoptosis with terminal deoxynucleotidyl transferase-mediated dUPT-biotin nick end labelling methods, and the expression of T-cell signalling zeta (TCR zeta) molecules with intracellular staining. RESULTS: The increased degree of T-cell apoptosis, downregulation of TCR zeta molecules and impaired cytokine production of T cells were significant on days 1 and 3 after operation in patients who had oesophagectomy, but not after gastrectomy or cholecystectomy. A higher level of T-cell apoptosis was observed in the co-culture with postoperative monocytes than with preoperative monocytes. CONCLUSION: Peripheral blood T cells obtained after oesophagectomy underwent apoptosis that correlated with the downregulation of TCR zeta molecules. Postoperative monocytes induced by surgical stress were able to mediate the T-cell apoptosis.     

调节性T淋巴细胞在移植免疫耐受领域的临床应用研究

CD4~+ CD25~+ Foxp3~+ 调节性T淋巴细胞(Treg)是一类具有免疫调节功能的T淋巴细胞亚群,其免疫抑制功能成为近年来免疫学领域研究的重要内容~([1]).Treg不仅在维持机体免疫自稳方面发挥关键的作用,而且在移植后诱导和维持免疫耐受方面的作用也越来越受到人们的重视~([2]).