中性粒细胞与淋巴细胞比值对急性脑梗死患者预后的预测价值

目的探讨中性粒细胞与淋巴细胞比值(NLR)对急性脑梗死患者预后的预测价值。方法回顾性连续纳入2014年1月至2015年12月郑州大学第五附属医院神经内科住院的急性脑梗死患者307例,其中女80例,男227例。根据改良Rankin量表评分标准,分为预后良好组(195例)和预后不良组(112例)。记录入院时年龄、性别、既往病史、美国国立卫生研究院卒中量表(NIHSS)评分等资料,根据入院中性粒细胞计数与淋巴细胞计数计算出NLR值。采用Logistics回归分析急性脑梗死预后不良的影响因素,采用受试者工作特征(ROC)曲线评价入院时NLR水平对急性脑梗死患者预后不良的预测作用。结果 (1)与预后良好组比较,预后不良组患者年龄、复发性脑梗死发生率、入院时NIHSS评分、入院NLR水平均较高,组间差异均有统计学意义[(69±12)岁比(62±14)岁,25.0%(28/112)比14.4%(28/195),5.00(3.00,9.00)分比3.00(1.75,5.00)分,3.66(2.62,7.91)比2.47(1.94,3.40),均P0.05],其余基线资料和临床特征的组间差异均无统计学意义(均P0.05)。(2)多因素Logistics回归分析结果显示,年龄、入院NIHSS评分及入院时NLR水平的升高,是预后不良的独立危险因素(OR值分别为1.030,1.148,1.427,95%CI分别为1.007~1.053,1.059~1.246,1.247~1.634,均P0.05)。(3)入院时NLR水平对急性脑梗死患者预后不良的诊断界值为2.84,其敏感度为69.6%,特异度为64.6%。结论入院时NLR水平增高对评估急性脑梗死患者预后不良具有一定的参考价值。     

聚合酶链反应方法检测外周血淋巴细胞HLA-A mRNA方法学的建立及在肾移植中的初步应用

目的通过建立检测宿主外周血淋巴细胞(PBL)表面HLA-AmRNA表达程度的实时荧光定量逆转录聚合酶链反应(RT-FQ-PCR)方法,检测肾移植患者移植后外周血淋巴细胞表面HLA-AmRNA的表达程度,探讨其在诊断移植后早期排斥反应的发生的价值及机体免疫状态的关系。方法以6-磷酸葡萄糖脱氢酶(G6PDH)作内参照,用Taqman探针荧光定量技术,建立检测PBLHLA-AmRNA的RT-FQ-PCR方法;并检测35例随访移植患者的不同时间的60个标本和60名正常人的阈循环数(Ct),然后用REST软件分析PBLHLA-AmRNA的表达水平。结果正常人和51个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量分别为0·036±0·012和0·026±0·015(双侧t=1·981,P<0·05),提示服用免疫抑制药(目前临床用药剂量均偏大)后患者PBLHLA-AmRNA表达减弱,机体免疫状态低,无排斥反应发生但易导致感染、肿瘤等并发症的产生;其中9个移植标本的PBLHLA-AmRNA对G6PDH的相对表达量为0·042±0·017(双侧t=2·002,P<0·05),提示较其他51例移植标本患者PBLHLA-AmRNA表达增强的患者,经病例回访查询发现该5例移植患者在这期间有排斥反应(根据临床表现、肾功能检查、影像学及核医学检查的临床排斥反应期)的发生,其中1例在移植后6个不同时间的标本显示2次有增高,每次经治疗后均降低。结论建立的宿主PBLHLA-AmRNART-FQ-PCR检测方法简便、特异、重复性好、可信度高,可用于肾移植后PBLHLA-AmRNA表达水平的检测,评估机体的免疫状态,预测排斥反应的发生。     

CFSE和AnnexinV双标记技术检测细胞毒性T淋巴细胞的杀伤效应

目的 利用CFSE和AnnexinV对靶细胞进行染色,建立一种通过流式细胞仪技术检测细胞毒性T淋巴细胞(CTL)杀伤效应的新方法.方法 应用磁珠分 OT-Ⅰ T细胞受体(TCR)转基因小鼠CD8+CTL细胞和脾脏树突状细胞(sDC),将2种细胞和鸡卵蛋白(OVA)抗原肽共培养65 h后分析其细胞分裂增殖、IFN-γ和IL-4细胞内因子及穿孔素、颗粒酶等效应分子的表达特性,收获具备杀伤效应的CD8+CTL细胞.利用预先激活的小鼠CD8+CTL细胞作为效应细胞,CFSE标记的同系小鼠脾细胞作为靶细胞,体内体外2种条件下利用AnnexinV染色靶细胞检测特异性CTL杀伤效应,并与CFSEhigh和CFSElow标记靶细胞检测体内CTL杀伤效应的经典方法进行比较.结果 OT-Ⅰ初始CD8+CTL细胞体外活化培养后有4个分裂峰,54.1%的CTL细胞表达IFN-γ,0.78%的细胞表达IL-4,穿孔素、颗粒酶2种CTL效应分子均为阳性表达,CD8+CTL细胞已经具备CTL杀伤活性.体外实验结果显示OVA+靶细胞在共培养条件下,AnnexinV阳性细胞比例明显高于分离培养条件下[(62.4±3.5)%比(28.3±2.2)%,P＜0.01],OVA-的AnnexinV阳性靶细胞比例在不同培养条件下差异无统计学意义[(20.3±4.8)%比(17.3±2.9)%,P＞0.05].共培养条件下OVA+靶细胞其AnnexinV阳性细胞比例也明显高于OVA-靶细胞(P＜0.01),在分离培养条件下差异则无统计学意义(P＞0.05).活化的CD8+CTL细胞所介导的杀伤效应具有抗原依赖性和部分的细胞接触依赖性.体内CTL实验中,利用CFSE和AnnexinV双标记的方法检测出靶细胞的杀伤率高于未孵育OVA抗原肽的对照组靶细胞[(52.63±8.12)%比(13.84±4.37)%,P＜0.01].而同等条件下利用CFSEhigh和CFSElow双标记的方法检出靶细胞杀伤率为41%.结论 CFSE和AnnexinV双标记靶细胞的方法检测CTL杀伤效应与单纯的使用CFSEhigh和CFSElow标记靶细胞方法有很好的可比性,该策略为利用流式细胞仪技术检测细胞杀伤功能的方法学提供了新的选择.     

Isolation and phenotypic characterization of mucosal nasal lymphocytes by direct ex vivo analysis

Cellular inflammation of the nasal mucosa demonstrates a local immune response which plays an important role in allergic rhinitis. The aim of the present study was to characterize nasal mucosal lymphocytes regarding their activation and differentiation state by direct ex vivo flowcytometric analysis. Lymphocytes from the inferior turbinates were isolated by a mechanical method of preparation and, for comparison, from peripheral blood by Ficoll gradient centrifugation. Patients suffering from rhinitis or difficulty in nasal breathing were divided into an allergic (pollen-allergy, n = 13) and non-allergic group (n = 24). Expression of different T- and B-cell markers was determined by flowcytometric analysis. CD4+ T-cells from the nasal mucosa exhibited a memory phenotype (CD45RO+, 97%), were highly activated (CD69+, 43–73%), and showed low expression of the cutaneous lymphocyte antigen (CLA+, 5%). Nasal CD20+ B-lymphocytes expressed significantly higher levels of mIgE and lower levels of CD23 and CD80 than peripheral B-cells. Subsets of CD80+ (4%) and CD86+ (6%) CD20+ B-lymphocytes were identified in the nasal mucosa. No significant differences between allergic and non-allergic individuals were determined. As expected, the data show profound phenotypical differences between circulating peripheral blood and nasal mucosal lymphocytes. Activated memory lymphocytes are present in the nasal mucosa from allergic, but also non-allergic patients and may indicate to a significant role of a local inflammatory state without systemic criteria for allergy. In our study, we show that direct ex vivo isolation of lymphocytes is practicable method and offers a new technique to examine the local nasal allergic immune response using a multiparametric phenotypical analysis. Christin Wolfram and Claudia Rasche contributed equally to this work.     

Lymphocyte measures in treatment-naïve 13-15-year old adolescents with alcohol use disorders

Many adolescents have chronic exposure to hazardous levels of alcohol. This is likely to be a significant predictor of health outcomes, including those related to immunity. We assessed substance use and biochemical immunological parameters in heavy drinking adolescents (meeting DSM-IV criteria for alcohol dependence) and light/nondrinking control adolescents in Cape Town. Lifetime alcohol dose, measured in standard units of alcohol, was orders of magnitude higher in alcohol-dependent (AD) participants than controls. All adolescent AD had a “weekends-only” style of alcohol consumption. The AD group was chosen to represent relatively “pure” AD, with minimal other drug use and no psychiatric diagnoses. With these narrow parameters in place, we found that AD adolescents were lymphopenic compared with controls, with significantly lower mean numbers of absolute circulating CD3+, CD4+, and CD8+ T-lymphocytes. On conclusion, we found that adolescent AD individuals with excessive alcohol intake, in a weekend binge-drinking style but without comorbid drug or psychiatric disorders, may be at increased risk of lymphopenia. This alcohol misuse may increase infectious disease susceptibility (including TB and HIV) by reducing immune system capabilities. Complex interactions of alcohol with other documented high-risk activities may further compound health risks.     

周期联合甲氨蝶呤 环磷酰胺对类风湿关节炎患者外周血淋巴细胞周期及凋亡的影响

目的探讨甲氨蝶呤(MTX)和环磷酰胺(CTX)及两者联合治疗对类风湿关节炎(RA)患者外周血淋巴细胞(PBL)周期及凋亡的影响。方法入选活动期RA患者30例,随机分为3组(MTX组、CTX组及MTX+CTX联合组),应用流式细胞术检测MTX、CTX及联合治疗前后PBL周期及凋亡的变化,同时对10名健康志愿者PBL的周期及凋亡情况进行检测。结果活动期RA患者基线PBL各期细胞比例与正常人相比,G0/G1期细胞比例低于正常对照组,S期细胞比例高于正常对照组,差异均有统计学意义;基线早期凋亡率与正常人相比差异无统计学意义。MTX组患者用药后较用药前G0/G1期细胞比例增加,S期细胞比例减少,PBL早期凋亡率明显增加,差异均有统计学意义。CTX组患者用药前后PBL各期细胞比例差异无统计学意义;用药后较用药前PBL早期凋亡率明显增加,差异有统计学意义。MTX+CTX联合组用药后较用药前PBLG0/G1期细胞比例增加,S期细胞比例减少,早期凋亡率较MTX组、CTX组明显增加,差异均有统计学意义。MTX组及MTX+CTX联合组用药前后外周血淋巴细胞G0/G1期细胞比例变化与早期凋亡率变化之间呈显著正相关。结论MTX+CTX联合治疗能更加有效地诱导阻滞于G0/G1期的PBL发生凋亡。     

Defective B-cell and regulatory T-cell function in Wiskott-Aldrich syndrome

We report two Chinese boys with Wiskott-Aldrich syndrome presenting with gastro-intestinal bleeding, eczema and recurrent infection. They had thrombocytopenia and the mean platelet volume was small. Serum IgG and IgA were elevated and lymphocyte proliferation in response to phytohaemagglutinin, concanavalin A and pokeweed mitogen was defective. Despite documented herpes simplex virus type 1 and cytomegalovirus infection in one patient, he did not mount any humoral response. The generation of antibody-secreting cells in response to pokeweed mitogen was markedly defective in a plaque-forming cell assay. Both patients' regulatory T-cell and B-cell functions were defective in this assay. The genetic defect in Wiskott-Aldrich syndrome therefore affects T-cells, B-cells and platelets.     

Clinical aspects and distribution of immunologically active cells in the nasal mucosa of patients with nasal polyps after endoscopic sinus surgery and treatment with topical corticosteroids

Summary Clinical parameters of 72 patients who were operated upon for nasal polyps were evaluated as well as biopsy specimens of the mucosa of the middle and inferior turbinates of 41 of these patients. Biopsies were taken at the time of endoscopic sinus surgery (ESS), after 6 months and after 1 year in 23 patients. During the follow-up period the patients were treated with topical corticosteroids (budesonide). At the time of ESS significantly more CD8+ (suppressor/cytotoxic) cells than CD4+ (helper/inducer) cells were found in the middle and inferior turbinates. At 6 months significantly more CD4+ cells were found than at the time of ESS, whereas at 1 year the number of CD4+ cells had decreased and was lower than at 6 months. These data support the theory that the occurrence of nasal polyps is associated with T-cell-dependent disturbances. Clinical evaluation revealed that most of the patients with chronic airway obstruction had better pulmonary functions postoperatively or required less medication for lung disease. These findings show that ESS combined with topical corticosteroids has a positive effect on upper and lower respiratory tract pathology. Correspondence to: A. E. Stoop     

鼻咽癌外周血淋巴细胞基因表达谱的研究

背景与目的：近年来兴起的DNA芯片技术能在一次杂交实验中同时监测数以千计的基因表达,使分析免疫细胞的基因表达模式成为可能。本研究旨在应用这一先进技术检测鼻咽癌患者与健康人外周血淋巴细胞基因表达的差异,以探讨鼻咽癌患者免疫细胞的基因表达特点。方法：应用含2000点cDNA的BioDoor10s/D 睛研究基因表达的差异。提取鼻咽癌患者淋巴细胞和健康人淋巴细胞的mRNA,分别用标有不同荧光素的dUTP反转录制备cDNA探针,将探针与芯片杂交后扫描荧光强度,从而筛选出差异性表达的基因。结果：发现鼻咽癌患者淋巴细胞有62个基因呈现差异表达。差异性表达主要涉及编码信号传递蛋白的基因,表达上调的5个基因中有3个基因与编码信号传导的蛋白相关。其余57个基因呈现表达下调,在cy5/cy3值为0．3左右的9个下调基因中,编码信号传导蛋白的相关基因占了5个。结论：与健康人相比,鼻咽癌患者的外周血淋巴细胞基因表达呈下调趋势。淋巴细胞信号传导功能异常和基因表达下调可能是鼻咽癌免疫功能有别健康人的原因之一。