治疗后慢性乙型肝炎病毒感染者中低病毒血症人群外周血淋巴细胞状态分析

目的评估治疗后慢性乙型肝炎病毒(hepatitis B virus, HBV)感染者中低病毒血症人群外周血T、B、NK淋巴细胞情况, 为解决低病毒血症提供思路。方法回顾性收集344例治疗后慢性HBV感染者的基本情况、生化、凝血、HBV DNA、外周血淋巴细胞计数、T淋巴细胞PD1及CD28、NK淋巴细胞穿孔素及颗粒酶B等数据, 分为低病毒血症(low-level viremia, LLV)组与完全病毒学应答(complete virological response, CVR)组, 比较两组外周血淋巴细胞计数、T淋巴细胞PD1及CD28表达、NK淋巴细胞穿孔素及颗粒酶B表达, 倾向性评分匹配分析, 验证结果的准确性。结果对162例LLV者和182例CVR者进行疾病诊断、谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(aspartate aminotransferase, AST)、白蛋白(albumin, ALB)水平比较, 差异无统计学意义(P>0.05), 性别和年龄差异有统计学意义(P<0.05);两组外周血CD3+、CD4+、C...     

血液透析对终末期肾病合并HBV病毒感染患者免疫功能的影响

目的:探讨了血液透析对终末期肾病合并HBV病毒感染患者免疫功能的影响。方法:随机选取2011年6月至2013年6月期间我院接收救治的50例终末期肾病合并HBV病毒感染患者作为观察组,给予持续性血液透析治疗,同时选取50例单纯终末期肾病患者作为对照组,与观察组给予相同的持续性血液透析治疗方案。分别对两组患者治疗前后24 h尿蛋白量、血清白蛋白、尿素氮、肌酐进行分析;采用流式细胞术分析两组患者治疗前后外周血调节性T淋巴细胞比例和CD8+T淋巴细胞比例变化,同时对穿孔素/颗粒酶B进行分析。结果:经过透析治疗后,两组患者临床症状及实验室指标均较治疗前有显著性改善,差异具有统计学意义(P<0.05)。两组治疗后临床症状和实验室指标比较无统计学意义(P>0.05)。对照组患者治疗后免疫功能与治疗前相比,无明显改变(P>0.05);观察组患者治疗前调节性T淋巴细胞(Treg细胞)比例和CD8+T淋巴细胞比例与对照组相比呈显著性升高趋势(P<0.05);治疗前观察组患者外周血中T细胞活化分子穿孔素/颗粒酶B表达率较对照组高(P<0.05),经过治疗后,Treg细胞和CD8+T淋巴细胞比例均显著下降(P<0.05),穿孔素/颗粒酶B表达率呈显著下降趋势(P<0.05)。结论:血液透析能够显著改善合并HBV病毒感染患者的免疫功能。     

mHLA-G 联合PCT、hs-CRP 在肾移植排斥反应及感染中的诊断价值分析

目的:探讨膜型人类白细胞抗原(mHLA-G)联合血清降钙素原(PCT)、超敏C反应蛋白(hs-CRP)在肾移植排斥反应及感染中的诊断价值。方法:以我院2015年1月～2018年10月126例肾移植患者为研究对象,其中肾功能稳定组42例,术后发生急性排斥反应(AR)组40例,术后发生感染组44例,另选同期健康体检者50例作为正常对照组;对比4组外周血mHLA-G、血清PCT、 hs-CRP水平,分析这些指标在肾移植排斥反应及感染中的诊断价值。结果:肾移植后4周,AR组mHLA-G表达水平最低,与稳定组、感染组、对照组相比差异均有明显统计学意义(P0.05);感染组mHLA-G表达水平最高,明显高于对照组、稳定组、AR组(P0.05);稳定组mHLA-G表达水平略低于对照组,但差异无统计学意义(P0.05)。肾移植后4周,AR组血清PCT水平明显高于稳定组与对照组(P0.05),但稳定组与对照组间无明显差异(P0.05);感染组血清PCT水平明显高于AR组、稳定组、对照组(P0.05)。肾移植后4周,AR组与感染组血清hs-CRP均明显高于稳定组与对照组(P0.05),且AR组与感染组无明显差异(P0.05)。mHLA-G联合PCT、 hs-CRP与在肾移植排斥反应及感染中的诊断价值明显高于单一检测指标。结论:mHLA-G联合PCT、 hs-CRP在肾移植AR及感染中具有重要诊断价值。     

肾移植急性排斥反应时B7-2/CD28信号通路的表达*☆

背景：以往动物研究表明,在器官移植急性排斥反应时共刺激分子的表达与急性排斥反应密切相关。 目的：观察急性排斥反应时患者移植肾脏组织和外周血中B7-2/CD28信号通路的表达。 方法：对53例同种异体肾移植患者于移植前1 d、移植后1,3,7,14,21,28 d分别取外周血以及在临床诊断急性排斥反应当天和抗排斥治疗1周后额外采血,用流式细胞仪检测共刺激分子B7-2/CD28在外周血淋巴细胞中的表达;同时,行经皮肾穿刺活检供肾修整结束时、移植后7 d、1个月、6个月、1年或以上获取活检肾脏组织,用免疫组织化学方法检测活检组织中B7-2/CD28的表达情况。 结果与结论：移植后1,3 d内所有患者外周血中CD28+,CD4+/CD28+,CD8+/CD28+细胞比率均有显著下降(P < 0.05),一二周后恢复到术前水平;移植后7 d未发生急性排斥反应的患者肾脏组织B7-2阳性表达率显著上升(P < 0.05),1个月后下降至移植前水平(P > 0.05)。移植后发生急性排斥反应的患者外周血CD28+,CD4+/CD28+,CD8+/CD28+细胞比率及肾脏组织B7-2阳性表达率明显上升(P < 0.05),经抗排斥治疗1周后均好转。结果证实,在肾移植后出现急性排斥反应时,肾脏组织以及外周血中共刺激分子B7-2/CD28的表达上调与急性排斥反应的发生密切相关。       

颗粒酶B和穿孔素免疫组织化学检测诊断肝移植急性排斥反应的敏感性与特异性

目的　探讨颗粒酶B和穿孔素两种免疫活化分子在肝移植急性排斥诊断中的作用,及其与Banff急性排斥反应组织学诊断标准之间的对应关系。方法　在常规组织学诊断基础上,将41份肝穿刺标本用颗粒酶B与穿孔素单克隆抗体进行免疫组织化学EnVision二步法标记,IPP图像分析软件计算阳性细胞数/mm2 作为免疫活化细胞指数(AI),以组织学诊断作为评判有无急性排斥反应的标准。结果　在41份肝穿刺标本中,组织学诊断为急性排斥反应21份,缺乏急性排斥反应组织学改变20份。急性排斥反应组颗粒酶B与穿孔素AI值显著高于无排斥反应组(P<0. 001),中重度排斥反应组AI值显著高于轻度及其非确定性排斥反应组(P<0. 001)。与组织学诊断比较,颗粒酶B的敏感性、特异性、阳性预测值、阴性预测值及诊断一致率分别达到90. 0%、95. 2%、94. 7%、90. 9%以及92. 7%;穿孔素的各指标也分别达到80. 0%以上。结论　颗粒酶B与穿孔素是急性排斥反应免疫效应细胞活化标志,在临床肝移植急性排斥反应时表达明显升高,作为组织学诊断急性排斥反应的辅助指标具有相当高的敏感性及特异性,可用于肝移植后肝穿刺标本的鉴别诊断。     

应激对鼠脾NK细胞穿孔素及颗粒酶的mRNA表达的影响

目的：阐明躯体性应激和心理性应激降低NK细胞杀伤活性的机制及异同。方法：应用Communication box系统分别使小鼠连续负荷躯体性应激和心理性应激后，以放免法检测鼠血浆类固醇激素水平：以^51Cr释放法检测鼠脾NK细胞杀伤活性；以流式细胞术检测鼠脾NK细胞数量；以RT-PCR技术检测鼠脾细胞中杀伤效应介质——穿孔素、颗粒酶A及B的mRNA表达水平。结果：负荷躯体性应激和心理性应激后，小鼠血浆类固醇激素水平升高，分别于第3天和第5天达高峰后，逐渐下降；两者均可导致鼠脾NK细胞数量减少，杀伤活性降低（P〈0．05，P〈0．01）；两者均可导致穿孔素的mRNA表达下降；但躯体性应激降低颗粒酶A的mRNA表达，而颗粒酶B的mRNA表达反而升高；心理性应激可降低颗粒酶B的mRNA表达，虽然也降低颗粒酶A的mRNA表达，但无显著性差异。结论：躯体性应激和心理性应激均可降低脾NK细胞数量及其杀伤活性，但对杀伤效应介质——穿孔素、颗粒酶A及B的mRNA表达的影响不同，提示躯体性应激和心理性应激对NK细胞功能影响的机制不同。     

实时荧光定量PCR检测淋巴细胞中NKG2D、穿孔素和颗粒酶B基因的表达

目的:建立可用于测定淋巴细胞免疫功能的SYBRGreen I实时荧光定量PCR技术。方法:根据NCBI基因库中4种基因(NKG2D、穿孔素、颗粒酶B和内参照GAPDH)的序列,设计合成相应的引物,扩增上述基因。建立SYBRGreen I实时荧光定量PCR方法,检测肿瘤患者外周血淋巴细胞和诱导培养的患者CIK细胞(cytokine induced killer,CIK)中NKG2D、穿孔素和颗粒酶B mRNA的含量。结果:应用设计的引物扩增NKG2D、穿孔素和颗粒酶B基因后,经琼脂糖凝胶电泳和溶解曲线分析表明具有特异性。SYBR Green I实时荧光定量PCR检测结果表明,肿瘤患者的淋巴细胞中颗粒酶B基因的表达降低,而经细胞因子和单克隆抗体诱导培养的肿瘤患者CIK细胞与其淋巴细胞相比,细胞中穿孔素和颗粒酶B基因的表达明显增加(P0.01)。结论:该SYBRGreen I实时荧光定量PCR方法可用于检测淋巴细胞中NKG2D、穿孔素和颗粒酶B的mRNA的表达、作为研究淋巴细胞免疫功能的有力手段。     

白藜芦醇抑制肾移植大鼠肾损伤

目的探究白藜芦醇对肾移植大鼠肾损伤的改善作用及作用机制。方法将40只大鼠分为假手术组、模型组(同种异体肾移植手术)、白藜芦醇高和低剂量组[40和20 mg/(kg·d),腹腔注射,连续7 d]。术后24 h,检测血清Cr、BUN、SOD和MDA水平,RT-qPCR检测肾组织Bax和Bcl-2 mRNA表达,Western blot检测IKKα、p-IκBα和NF-κB p65蛋白表达。结果与假手术组比较,模型组血清Cr、BUN和MDA含量增高,SOD活性降低,肾组织Bax mRNA表达增加,Bcl-2 mRNA表达降低,IKKα、p-IκBα和NF-κB p65蛋白表达增加(P0.05);与模型组比较,白藜芦醇显著降低血清Cr、BUN和MDA含量,提高SOD活性,降低Bax mRNA、IKKα、p-IκBα和NF-κB p65蛋白表达,提高Bcl-2 mRNA表达(P0.05)。结论白藜芦醇对肾移植大鼠肾损伤具有保护作用,其作用机制与抑制NF-κB信号通路的活化有关。     

结核杆菌H37Ra免疫小鼠后脾淋巴细胞的杀伤活性及免疫机制的研究

目的探讨结核杆菌H37Ra免疫小鼠后,产生特异性的脾淋巴细胞杀伤活性及其免疫机制。方法(1)分别以结核杆菌H37Ra、BCG和PBS免疫小鼠后第30天及第60天,分离各组小鼠脾淋巴细胞作为效应细胞,以表达Ag85B分泌蛋白的Sp2/0细胞为靶细胞,用MTT比色法检测免疫小鼠脾淋巴细胞的杀伤率;(2)在免疫后第60天,小鼠脾淋巴细胞经PPD刺激后,以RT-PCR法检测穿孔素mRNA及颗粒酶BmRNA的表达。结果(1)结核杆菌H37Ra组脾淋巴细胞的杀伤率明显高于PBS对照组(P<0.05),略高于BCG组;(2)H37Ra组和BCG组穿孔素、颗粒酶mRNA的表达量均显著高于PBS组(P<0.05),H37Ra组穿孔素mRNA的表达量显著高于BCG组(P<0.05),但颗粒酶BmRNA的表达量两组无差异。结论结核杆菌H37Ra免疫小鼠后,能增强脾淋巴细胞的杀伤活性,其机制可能与增加穿孔素、颗粒酶B的表达有关。     

儿童系统性红斑狼疮患者外周血淋巴细胞表面CD95的表达及临床意义

目的:探讨儿童系统性红斑狼疮(systemic lupus erythematosus,SLE)外周血淋巴细胞表达CD95的特征及与疾病活动性和其他免疫学指标间的关系.方法:使用流式细胞术检测60例SLE患儿和20例对照外周血T淋巴细胞亚群和B淋巴细胞表面CD95的表达,并分析其与SLE疾病活动性以及实验室检查之间的关系.结果:初发SLE患儿外周血中CD4+T细胞表面CD95的表达显著高于对照组,差异有统计学意义(P<0.05);初发SLE患儿外周血中CD19+B细胞表面CD95的表达显著高于健康儿童(P<0.05);CD19+CD95+B细胞的比例和SLE疾病活动性呈正相关(r=0.4,P<0.05);CD4+CD95+T细胞的比例和SLE疾病活动性呈正相关(r=0.3,P<0.05),CD4+CD95+T细胞的比例和外周血抗双链DNA抗体(anti-dsDNA Abs)的水平呈正相关(r=0.2,P<0.05);治疗后SLE患儿外周血中CD19+CD95+B细胞和CD4+CD95+T细胞的比例均有显著下降,差异有统计学意义(P<0.05).结论:儿童SLE患者外周血中淋巴细胞表达CD95的水平显著升高,且与SLE的疾病活动性及血清中抗双链DNA抗体相关,可以作为SLE的评价指标.     

Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine

BACKGROUND: Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation. METHODS: We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status. RESULTS: The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed. CONCLUSIONS: Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.     

Optimization of RNA yield,purity and mRNA copy number by treatment of urine cell pellets with RNAlater

BACKGROUND: We have shown that measurement of mRNA for cytotoxic attack proteins perforin and granzyme B in urinary cells is a noninvasive means of diagnosing acute rejection of human renal allografts. Urinary cell mRNA studies have yielded useful information in other patient populations such as patients with cancer. The isolation of sufficient and high quality ribonucleic acid (RNA) from urinary cells however is problematic. RNAlater, an RNA stabilization solution, has been reported to optimize RNA isolation from tumor tissues stored at room temperature and from pigment-rich ocular tissues. METHODS: We explored whether the addition of RNAlater to urine cell pellets improves RNA yield, enhances purity and facilitates measurement of low abundance mRNAs. We measured, with the use of real-time quantitative polymerase chain reaction (PCR) assay, levels of expression of a constitutively expressed gene 18S rRNA and mRNA for granzyme B and transforming growth factor-beta(1) (TGF-beta(1)) in urine specimens and renal biopsies obtained from renal allograft recipients. RESULTS: RNA yield (P<0.01, Wilcoxon signed rank test) and the A260/A280 ratio (P<0.01) were both higher with urine cell pellets treated with RNAlater prior to snap freezing compared to cell pellets that were not treated with RNAlater prior to snap freezing. Levels (copy number per 1 microg of total RNA) of 18S rRNA (P<0.02), granzyme B mRNA (P=0.002) and TGF-beta(1) (P=0.02) were all higher with treated urine cell pellets compared to untreated cell pellets. Kruskall-Wallis one way analysis of variance and pair-wise comparisons with Student-Newman-Keuls test showed that the levels of mRNA for granzyme B (P<0.05) and TGF-beta(1) (P<0.05) are significantly different between renal allograft biopsies and untreated urine cell pellets but not between the biopsy specimens and RNAlater-treated urine cell pellets. CONCLUSIONS: The addition of RNAlater to urine cell pellets improves RNA isolation from urinary cells and facilitates measurement of low abundance mRNAs.     

Noninvasive diagnosis of cellular and antibody-mediated rejection by perforin and granzyme B in renal allografts

A major milestone in transplantation would be the use of biomarkers to monitor rejection. We examined the association between perforin and granzyme-B gene expression detected in the peripheral blood of renal allograft recipients with cellular and antibody-mediated rejection. Furthermore, we judged the appropriateness of assigning negative rejection statuses to persons without a biopsy whose grafts were functioning well clinically. Of the 46 patients who completed the study, recipients with cellular rejection had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.006). Interestingly, recipients with antibody-mediated rejection also had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.04). Patients with high levels of granzyme B had a probability of rejecting that was 26.7 times greater than those patients with low levels of granzyme B. Perforin and granzyme B had sensitivities of 50% and specificities of 95% in predicting rejection (cutoff value = 140). Assigning negative rejection statuses to recipients without a biopsy whose grafts were functioning well did not have a major effect on the direction or significance of covariate values. This study suggests that perforin and granzyme-B gene expressions in peripheral blood are accurate in detecting both cellular and antibody-mediated rejection.     

Nephritogenic T cells use granzyme C as a cytotoxic mediator

Cytoplasmic granules of cytotoxic T lymphocytes contain several proteins that may be involved in cell-mediated cytotoxicity. We have previously described nephritogenic T cell clones that are cytotoxic to cultured renal proximal tubular epithelial cells (MCT). One of these clones, M52.34.1, expresses perforin, a cytotoxic mediator. We investigated the expression of other granule-associated proteases of M52.34.1. Granzymes A and B have been extensively studied in T cell-mediated cytotoxicity, and associated with tissue destruction in models of transplantation. However, the activity of other granzymes has not been as extensively investigated. We focused our studies on granzyme C. Northern blots showed very high levels of granzymes B and C mRNA expression in M52.34.1 cells 3 days following T cell activation. There was no expression of granzyme A mRNA. An antisense oligonucleotide made from the 5′-upstream region of the murine granzyme C exon 1 inhibited granzyme C mRNA expression in M52.34.1 when added at a concentration of 50 μM to the culture medium for 2 days. There was no inhibition of granzyme C mRNA expression with the sense oligonucleotide. The granzyme C antisense oligonucleotide inhibited M52.34.1 cytotoxicity to MCT at effector: target ratios of 20:1 and 40:1. M52.34.1 cells mediate inflammatory interstitial nephritis following adoptive transfer. If T cells were resuspended in 200 μM of the antisense oligonucleotide prior to subcapsular transfer, the recipient kidneys showed markedly diminished tubular cell destruction, suggesting that granzyme C can also be an important mediator of cytotoxicity in vivo.     

Effects of pulsing procedure of interleukin-12 in combination with interleukin-2 on the activation of peripheral blood lymphocytes derived from patients with hepatocellular carcinoma

In patients with hepatocellular carcinoma (HCC), natural killer (NK) cell activity decreases significantly, and the reduced activity may be associated with the progression of HCC. In this study we evaluated the effects of pulsing with interleukin (IL)-2 and/or IL-12 on the activation of freshly isolated peripheral blood lymphocytes (PBL) derived from patients with HCC. PBL obtained from 9 HCC patients, 4 liver cirrhosis patients, and 9 normal subjects were cultured in the presence of IL-2 and/or IL-12. After 24 h of incubation, the levels of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha presented in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA). The IFN-gamma and TNF-alpha production of PBL pulsed by a combination of IL-2 and IL-12 was significantly higher than those of PBL stimulated by either IL-2 or IL-12 alone. The mRNA encoding perforin, granzyme B, as well as IFN-gamma and TNF-alpha, were markedly enhanced in PBL stimulated with a combination of IL-12 and IL-2. The pulsing procedure of IL-12 in combination with IL-2 resulted in the increase of IFN-gamma and TNF-alpha, and the expression of perforin and granzyme B mRNA in PBL obtained from HCC patients, as well as in those obtained from normal subjects. These results indicate that adoptive immunotherapy based on PBL pulsed with a combination of IL-2 and IL-12 may be a promising adjunctive strategy for HCC treatment.     

HLA-mismatched renal transplantation without maintenance immunosuppression

Five patients with end-stage renal disease received combined bone marrow and kidney transplants from HLA single-haplotype mismatched living related donors, with the use of a nonmyeloablative preparative regimen. Transient chimerism and reversible capillary leak syndrome developed in all recipients. Irreversible humoral rejection occurred in one patient. In the other four recipients, it was possible to discontinue all immunosuppressive therapy 9 to 14 months after the transplantation, and renal function has remained stable for 2.0 to 5.3 years since transplantation. The T cells from these four recipients, tested in vitro, showed donor-specific unresponsiveness and in specimens from allograft biopsies, obtained after withdrawal of immunosuppressive therapy, there were high levels of P3 (FOXP3) messenger RNA (mRNA) but not granzyme B mRNA.     

移植肾急性排斥与移植前后血清细胞因子表达的变化

背景：目前异体器官移植后细胞因子变化己经有较多的报道,但有关细胞因子在肾移植患者中的动态变化规律及其与移植急性排斥反应的关系鲜有报道。 目的：观察肾移植受者移植前后血清细胞因子表达变化,并探讨其与移植肾急性排斥的关系。 方法：选择2008年9月至2011年9月武警后勤学院附属医院收治的接受肾移植患者48例,均为首次肾移植,分为肾功能稳定组和急性排斥反应组。另选择健康体检者30人为对照组。 结果与结论：肾功能稳定组、急性排斥反应组移植前1 d血清肿瘤坏死因子α、白细胞介素6、白细胞介素8水平与对照组相比差异无显著性意义(P > 0.05)。肾功能稳定组血清3种细胞因子水平均于移植后第1天即开始逐渐升高,在3 d时显著升高(P < 0.05),5 d时开始下降,7 d下降显著(P < 0.05),14 d左右趋于降至移植前水平,21-28 d表达稳定在移植前水平。急性排斥反应组血清3种细胞因子水平于移植后第1天即显著升高(P < 0.05),7-14 d维持在高水平 (P < 0.05),21-28 d稳定下降,但仍明显高于移植前(P < 0.05)。相同时间段,急性排斥反应组血清3种细胞因子水平均明显高于肾功能稳定组(P < 0.05)。提示移植后血清肿瘤坏死因子α、白细胞介素6、白细胞介素8动态水平变化在一定程度上反映肾移植受者的免疫反应状态,可作为辅助早期诊断急性排斥反应的免疫生物学指标。