膀胱尿路上皮癌患者外周血T淋巴细胞凋亡蛋白Fas的表达

目的 探讨膀胱尿路上皮癌与Fas介导的血T淋巴细胞凋亡的关系.方法 应用流式细胞仪检测52例膀胱癌患者和37例健康对照者外周血CD4Fas、CD8Fas阳性T细胞.结果 膀胱癌患者外周血CIM、CD8Fas阳性T细胞分别为(20.74±9.02)%、(7.51±5.93)%,对照组分别为(15.68±5.58)%、(4.27±3.08)%,肿瘤组较对照组明显增加(P<0.01).浸润组CD8Fas阳性T细胞数为(5.83±3.95)%,表浅组为(5.83±3.95)%,浸润组较表浅组明显降低(P<0.05).复发组CD4、CD8Fas阳性T细胞与初发组比较筹异无统计学意义(P>0.05).结论 血T淋巴细胞Fas表达的异常可能在膀胱癌的发生发展中起重要的作用.     

Fas/FasL在异源融合瘤诱导结肠癌细胞早期凋亡中的作用

目的 探讨Fas/FasL通路在人异源树突状细胞（dendritic cell,DC）/结肠癌细胞融合瘤诱导结肠癌细胞凋亡中的作用.方法 使用50%聚乙二醇（PEG）将健康人外周血来源DC与结肠癌细胞SW480融合,用其致敏T淋巴细胞,采用流式细胞术检测致敏细胞毒性T淋巴细胞（CTL）对结肠癌细胞凋亡的影响;使用免疫组化法分别检测SW480细胞Fas分子的表达以及T淋巴细胞活化前后FasL的表达情况.结果 结肠癌SW480细胞的早期凋亡率为46.88%;结肠癌SW480细胞高表达Fas分子用融合瘤致敏T细胞后,其FasL分子的表达明显增加.结论 异源树突状细胞融合瘤可能通过Fas/FasL通路来诱导结肠癌细胞的早期凋亡.     

腹主动脉瘤患者外周血CD4+CD28-调节性T细胞的变化及意义

目的：探讨CD4+ CD28-T细胞在不同阶段腹主动脉瘤患者血清中的数量变化及临床意义。
方法:腹主动脉瘤患者20例,其中小腹主动脉瘤10例,大腹主动脉瘤10例,正常健康对照组10例,采用流式细胞术测定3组受检者外周血CD4+,CD8+,CD4+ CD28-T亚群数量及占淋巴细胞的比例,运用酶联吸附实验（ELISA）方法检测上述各组人群血浆中Th1细胞因子（IFN-γ,TNF-α）表达水平。
结果:腹主动脉瘤组CD4+ CD28-T细胞表达水平显著升高,其中CD4+ CD28-T细胞在小腹主动脉瘤组升高最为显著（P＜0.05）。Th1细胞因子（IFN-γ,TNF-α）表达水平亦呈显著升高。
结论:不同发展阶段腹主动脉瘤患者血清中CD4+ CD28-T细胞表达升高,AAA患者外周血T淋巴细胞处于活化状态,在AAA发生发展的过程中,机体存在全身免疫反应的激活,外周血中T细胞亚组发生改变,导致炎症及自身免疫反应性扩展。

     

转化生长因子-β1差异性诱导CD4+和CD8+T细胞表达转录因子FOXP3的研究

目的 研究CD4+T细胞和CD8+T细胞被转化生长因子-β1(TGF-β1)诱导表达转录因子FOXP3的差异性.方法 给予CD4+T细胞和CD8+T细胞抗T细胞受体(TCR)刺激的同时,加入TGF-β1培养4d后,通过胞内细胞因子染色,分析二者被诱导表达FOXP3的情况.同时通过CFSE标记实验检测被TGF-β1诱导表达FOXP3的淋巴细胞的增殖情况;通过annexin V流式染色检测TGF-β1对于T淋巴细胞凋亡的影响.结果 相对于CD8+T淋巴细胞,TGF-β1主要诱导CD4+T淋巴细胞阳性表达FOXP3[外周血单个核细胞(PBMC):29.66±3.624比7.430±0.643;癌旁组织浸润淋巴细胞(NIL):31.74±2.612比8.637±1.146].被TGF-β1诱导表达FOXP3的淋巴细胞增殖活跃(94.39±1.179),受到活化刺激后依旧能够有效分泌干扰素-γ(IFN-γ)效应细胞因子(39.58±1.611).TGF-β1能够有效降低CD8+T淋巴细胞活化后的细胞凋亡率(25.39±2.158比9.320±0.3219).结论 与肝癌患者FOXP3表达阳性的淋巴细胞＞95％(97.15±0.3807,n=10)集中在CD4+T淋巴细胞的结果一致,TGF-β1优先选择性诱导CD4+T细胞表达FOXP3.与天然Treg(调节性T细胞)低增殖活性和低细胞因子分泌活性不同,被TGF β1阳性诱导表达FOXP3的T细胞依旧具有活跃的细胞增殖和分泌IFN γ效应细胞因子的功能.CD8+T淋巴细胞相对于CD4+T淋巴细胞,更易发生活化后细胞凋亡,而TGF-β1能够有效降低这种细胞凋亡率.     

不同免疫抑制剂诱导外周血活化T淋巴细胞凋亡的研究

目的探讨各种免疫抑制剂在体外对人外周血淋巴细胞凋亡的诱导作用及其机制。方法从健康人外周血中分离T淋巴细胞，经抗原刺激诱导淋巴细胞活化，将霉酚酸酯（MMF）、环孢素A（CsA）、他克莫司（FK506）、西罗莫司、泼尼松（Pred）、达利珠单抗及硫唑嘌呤（Aza）单独或联合加入静止T淋巴细胞和活化T淋巴细胞中共同培养，3d后，采用共聚焦显微镜、流式细胞仪、DNA片段化电泳分析及逆转录聚合酶链反应基因扩增方法检测淋巴细胞凋亡发生率，酶联免疫吸附试验检测试剂盒测定细胞培养基中自细胞介素2（IL-2）和Fas的表达水平。结果各种免疫抑制剂中，仅Pred能明显促进静止T淋巴细胞凋亡。MMF、Aza及Pred均能促使活化T淋巴细胞发生凋亡（P〈0．05，P〈0．01），而CsA、FK506、西罗莫司及达利珠单抗均抑制活化T淋巴细胞的凋亡（P〈0．01）。两种或三种不同的免疫抑制剂同时加入活化T淋巴细胞的培养基中，其凋亡细胞明显少于对照组（P〈0．01）。FK506和CsA能显著抑制活化T淋巴细胞的Fas和IL-2表达（P〈0．05）。结论MMF、Aza及Pred可能通过Fas／FasL信号通路诱导活化T淋巴细胞的凋亡，而CsA和FK506通过抑制IL-2的产生而阻碍活化T淋巴细胞的凋亡，西罗莫司和达利珠单抗则可能通过阻断IL-2的活化效应而抑制活化T淋巴细胞的凋亡。     

大鼠腹主动脉瘤形成过程中T细胞及CD4+CD28-T细胞的动态变化及意义

目的 探讨T淋巴细胞及其亚群在腹主动脉瘤发病中的作用.方法 50只Wistar大鼠随机分成A、B、C、D、E5组,每组10只,A组为正常对照组,B、C、D、E组分别为术后3、7、14、28 d组,运用猪胰弹力酶灌注法建立腹主动脉瘤模型.免疫组化方法检测上述各组大鼠腹主动脉局部T、B淋巴细胞及巨噬细胞的表达水平,流式细胞术检测各组大鼠外周血中CD4+ CD28-T淋巴细胞亚群的分布变化.结果 大鼠腹主动脉局部T、B淋巴细胞及巨噬细胞表达的变化:术后7dT细胞显著升高(40±15,P＜0.05),B细胞及巨噬细胞有升高,但差异无统计学意义(P＞0.05);术后14 d,3种细胞表达升高最为显著(P＜0.05);术后28 d,3种细胞的表达较术后14 d明显下降.术后各组大鼠外周血CD4+、CD28-T细胞均有升高(P＜0.05),以术后7d比例最高(8.1％±2.1％,P＜0.05).结论 T淋巴细胞是腹主动脉瘤形成过程中最重要的炎症细胞之一,T淋巴细胞的浸润及其亚群分布变化所致的免疫及炎症反应在腹主动脉瘤形成早期可能起着重要的触发及调节作用.     

Fas配体在膀胱肿瘤细胞系免疫逃逸中的作用

目的　探讨膀胱肿瘤细胞系的免疫逃逸机制。　方法　采用免疫细胞化学方法检测膀胱癌细胞系BIU 87中Fas配体 (FasL)的表达 ,应用BIU 87与JurkatT淋巴细胞共培养的方法体外研究FasL诱导T淋巴细胞凋亡的作用。　结果　膀胱癌细胞系BIU 87FasL表达阳性。BIU 87与JurkatT淋巴细胞共培养T细胞凋亡率为 (33.2± 6 .5 ) % ,应用抗体NOK 2中和BIU 87细胞FasL后 ,T细胞凋亡率减少为 (10 .1± 2 .7) % ,两组相比 ,差别有非常显著性意义 (P <0 .0 1)。　结论　膀胱肿瘤细胞系可能通过表达FasL主动诱导T淋巴细胞凋亡从而在膀胱肿瘤的免疫逃逸中发挥作用     

可溶性Fas阻断肝癌细胞通过Fas/FasL 途径触发T淋巴细胞的凋亡

目的　通过阻断Fas介导T细胞凋亡 ,建立快速大量激活制备肿瘤特异性细胞毒性T淋巴细胞 (CTL)的方法。方法　分离肝癌细胞和肿瘤浸润淋巴细胞 (TIL)。选择FasL表达阳性的肝癌标本 ,在体外将两者混合 ,并在CD2 8单抗共刺激下 ,激活制备特异性CTL。应用可溶性Fas受体阻断肝癌细胞通过Fas FasL途径触发激活T细胞凋亡 ,与对照组比较观察阻断凋亡作用 ,通过3H掺入法和51 Cr释放法了解T细胞增殖杀伤活性。结果　流式细胞术仪检测未阻断组较阻断组和未阻断对照组凋亡率明显升高 ,未阻断组凋亡率达 4 7 82 %± 0 13% ,静息性T淋巴细胞组为 3 76 %± 0 2 5 % ,阻断组为 8 2 2 %± 0 2 6 % (P <0 0 1) ,DNAladder显示未阻断组T淋巴细胞出现明显梯状条带 ,阻断组为阴性。51 Cr释放法表明阻断后T淋巴细胞杀伤活性增加 ,较未阻断组及未阻断对照组差异有显著性 (P<0 0 1)。3H掺入法检测证实可溶性Fas受体阻断激活T细胞凋亡后 ,细胞增殖明显升高 ,较未阻断组差异有显著性 (P <0 0 1)。结论　体外实验可获得大量激活T细胞 ,并可杀伤肿瘤细胞 ,     

养胃抗瘤冲剂对胃癌患者外周血T淋巴细胞rDNA的转录活性和凋亡相关蛋白影响的研究

目的 :观察养胃抗瘤冲剂对胃癌患者外周血T淋巴细胞rDNA转录活性和凋亡相关蛋白变化的影响及在肿瘤转移中的意义。 方法 :采用KL型肿瘤免疫图像分析系统和流式细胞仪测定正常人和胃癌患者养胃抗瘤冲剂治疗前后外周血T淋巴细胞rDNA的转录活性和调亡相关蛋白 (Fas/FasL)的表达变化。 结果 :胃癌患者外周血T淋巴细胞rDNA转录活性明显降低 ,凋亡相关蛋白Fas表达升高 ,与健康人相比 ,差异显著 (P <0 0 1) ,随着病情的进展 ,这种趋势更为明显。养胃抗瘤冲剂治疗后外周血T淋巴细胞的Ag -NORs升高、Fas表达降低 ,与治疗前比较差异显著 (P <0 0 1)。 结论 :养胃抗瘤冲剂可以降低外周血T淋巴细胞的凋亡能力 ,对增殖能力则有增强作用 ,从而提高机体的细胞免疫功能。外周血T淋巴细胞rDNA转录活性和凋亡相关蛋白变化可以从淋巴细胞增殖和凋亡活性两方面评价肿瘤患者的免疫状态 ,在胃癌转移的临床监测中具有重要意义。     

双套腹主动脉分支支架对腹主动脉瘤合并双侧髂动脉瘤的近期疗效

<正>腹主动脉瘤(abdominal aortic aneurysm,AAA)是由于腹主动脉退行性变而产生的动脉瘤样扩张,主动脉腔内修复术(endovascular aneurysm repair,EVAR)已成为腹主动脉瘤(abdominal aortic aneurysm,AAA)的主要治疗方式,11%～12%的AAA合并双侧髂总动脉瘤(common iliac aneurysm,CIA)^[1]。     

Impaired Fas-induced apoptosis of T lymphocytes in patients with abdominal aortic aneurysms

OBJECTIVE: Homeostasis of the immune system is maintained by apoptotic elimination of potentially pathogenic autoreactive lymphocytes. Emerging evidence shows that Fas-mediated apoptosis is impaired in activated lymphocytes from patients with autoimmune disease. The aim of this work was to assess apoptosis mediated by the cell death receptor Fas in peripheral T lymphocytes from patients with abdominal aortic aneurysms (AAA). METHODS: The apoptotic pathway was triggered by anti-Fas monoclonal antibodies in cultured and activated peripheral T-cell lines from 20 AAA patients with control groups of 15 patients with aortic atherosclerotic occlusive disease (AOD) and 25 healthy individuals. Cell survival and death (apoptosis) rate were assessed. RESULTS: Cross-linkage of Fas receptor exerted a strong apoptotic response on T cells from AOD patients and healthy controls, but a much less pronounced effect on T cells from AAA patients. The evaluation of cell survival rate showed a significantly higher percentage in AAA group (98.9% +/- 10.3%) than in the AOD subjects (58.9% +/- 15.2%) or the healthy group (59.4% +/- 12.9%; P < .001). Apoptosis assessment by annexin V and propidium iodide staining and flow cytometry showed similar results. The defect in AAA group was not due to decreased Fas expression, since Fas was expressed at normal levels. Moreover, it specifically involved the Fas system because cell death was induced in the normal way by methylprednisolone. Complementary DNA sequencing identified no causal Fas gene mutation, but two silent single nucleotide polymorphisms with higher frequency were found in the AAA group. CONCLUSIONS: Fas-induced apoptosis in activated T cells from AAA patients is impaired. This may disturb the normal down-regulation of the immune response and thus provide a new insight into possible mechanisms and routes in the pathogenesis of AAA.     

Apoptosis resistant bronchoalveolar lavage (BAL) fluid lymphocytes in sarcoidosis

BACKGROUND: Sarcoidosis is a chronic granulomatous lung disease of unknown origin. The accumulation of activated T cells at sites of inflammation represents an early stage in granuloma formation. Since mechanisms governing the normal resolution of inflammatory processes are poorly understood, this study aimed to investigate the apoptotic phenotype of peripheral blood and lung T lymphocytes from patients with sarcoidosis. METHODS: Bronchoalveolar lavage (BAL) was performed in 10 patients with active sarcoidosis and five healthy controls. RESULTS: Virtually no lymphocyte apoptosis, as determined by annexin V or Hoechst staining, was seen in either patients or controls. Sustained caspase-3 activity in non-apoptotic BAL fluid lymphocytes of the patients was detected, however, in agreement with in vitro studies demonstrating caspase activation after T cell receptor (TCR) triggering as a physiological response required for efficient T cell activation. Only 11.0% (range 7.7-17.6) of the BAL lymphocytes from sarcoidosis patients were annexin V positive after exposure to the apoptotic stimulus tributyltin compared with 55.0% (range 42.0-62.0) of BAL lymphocytes from healthy controls (p<0.001). After anti-Fas treatment only 8.5% (range 6-10) of BAL fluid lymphocytes from patients but 45.5% (range 38-62) from healthy controls were apoptotic. CONCLUSION: BAL fluid lymphocytes from patients with sarcoidosis display a non-apoptotic morphology associated with endogenous caspase-3 activity. They seem to be resistant to apoptosis, which might contribute to the accumulation of inflammatory cells in the lungs, persistence of inflammation, and the development and maintenance of granuloma.     

Abdominal aortic aneurysm and aortic occlusive disease: a comparison of risk factors and inflammatory response.

OBJECTIVE: to compare patients with abdominal aortic aneurysm (AAA) and aortic occlusive disease (AOD) with regard to risk factors for atherosclerosis, co-morbid conditions and inflammatory activity. PATIENTS AND METHODS: a total of 155 patients undergoing abdominal aortic surgery between January 1993 and October 1997: 82 (53%) had aneurysmal disease and 73 (47%) had occlusive disease. Principal risk factors were compared: age; gender; smoking; hypertension; hyperlipidaemia; diabetes mellitus; severe peripheral vascular disease (PVD) and ischaemic heart disease. Aortic wall tissue samples were obtained during surgery. A prospective blind analysis was performed for the presence of inflammatory cytokines TNF-alpha, IL-1 beta, IL-6 and TGF-beta. RESULTS: the average age of AAA patients was 74 years (50-88), while that of AOD patients was 61 years (43-82) (p<0.0001). Diabetes mellitus was found to be much more prevalent in the AOD group (p<0.001), while hypertension and severe PVD were more prevalent in the AAA group (p<0.001). No differences were found concerning any of the risk factors. Inflammatory cytokine activity: AAA tissue samples contained significantly higher mean TNF-alpha and IL-6 levels compared to the AOD samples (5.6+/-2.7 x 10 E-4 vs. 4.4+/-2.7 x 10 E-5 atmoles/microl (p=0. 01), and 0.6+/-0.4 vs. 0.01+/-0.006 atmoles/microl (p=0.02) respectively). No differences were found related to IL-1 beta and TGF-beta. CONCLUSIONS: (1) Patients with AAA have fewer atherosclerotic risk factors than do patients with AOD. (2) Patients with AAA and AOD have significantly different inflammatory activity. (3) The data supports the hypothesis that AAA and AOD are probably two different pathological entities.     

Lymphopenia in dialysis patients: a preliminary study indicating a possible role of apoptosis

Lymphopenia is a common finding in dialysis patients. Since infection rate and mortality associated with infection are high in dialysis patients, lymphopenia may be one of the contributing factors. In the present study, we evaluated the mechanism responsible for lymphopenia in these patients. Lymphocytes isolated from dialysis patients showed increased apoptosis (p < 0.001) when compared to lymphocytes isolated from healthy subjects (healthy subjects, 0.5 +/- 0.2% vs. dialysis patients, 8.8 +/- 0.7% apoptotic cells/field). Sera from dialysis patients promoted lymphocyte apoptosis in a time- and dose-dependent manner. These sera also enhanced lymphocyte DNA fragmentation into multiple integers of 180 base pairs in the form of a ladder pattern. Cellulose acetate membranes promoted T cell apoptosis when compared to polysulfone membranes and to control. Cellulose acetate dialysis membranes also appear to promote lymphocyte FasL expression. Similarly, dialysis sera enhanced T cell Fas as well as FasL expression. Neither the cellulose acetate nor polysulfone membranes could induce FasL expression on B cells. Similarly, dialysis sera failed to induce FasL expression on B cells. On the other hand, anti-FasL antibodies attenuated dialysis sera-induced apoptosis in T as well as B cells. Interestingly, dialysis serum showed a 5-fold increase in FasL content when compared with control serum. These results suggest that dialysis-associated factors can induce autocrine death in T cells but the help of activated T cells is required to induce death in B cells.     

Monocyte apoptosis in dialysis patients is Fas ligand-mediated

BACKGROUND: The mononuclear phagocyte system plays an important role in host defense. Since dialysis patients have been reported to show enhanced leukocytes apoptosis, we evaluated the mechanism of increased apoptosis of monocytes in dialysis patients. METHODS: Apoptotic studies were carried out on monocytes isolated from dialysis patients as well as healthy subjects. The effect of dialysis sera and membranes was evaluated on monocyte apoptosis as well as monocyte expression of proapoptotic proteins such as Fas and FasL. To confirm the role of FasL, we evaluated the effect of activated secretory products on T cell apoptosis. In addition, we studied FasL content of dialysis sera and supernatants of activated monocytes. RESULTS: Monocytes isolated from dialysis patients (MDP) showed a greater magnitude of apoptosis when compared to monocytes isolated from healthy subjects (MHS) (MHS, 3.6 +/- 1.1% vs. MDP, 24.3 +/-1.4%). Sera of hemodialysis patients (SHD) promoted (p < 0.001) apoptosis of MHS when compared to pooled control sera (HPS) (HPS, 0.8 +/- 0.5% vs. SHD, 11.5 +/- 0.5% apoptotic cells/field). Dialysis membranes, cellulose acetate membranes in particular, promoted monocyte apoptosis. Interestingly, anti-FasL antibodies partly inhibited dialysis sera-induced monocyte apoptosis. Dialysis membranes also modulated monocyte expression of both Fas and FasL. Secretory products of activated monocytes also promoted T cell apoptosis. Dialysis sera and activated monocyte secretory products showed increased FasL content. CONCLUSIONS: These results suggest that dialysis patients have an increased rate of monocyte apoptosis, which is mediated through a uremic milieu (serum factors). One of these serum factors seems to be FasL. In addition, dialysis membranes seem to promote apoptosis independent of the uremic milieu. The present study provides a mechanistical insight into the enhanced apoptosis of monocytes in dialysis patients.     

Monitoring of skeletal muscle oxygenation using near-infrared spectroscopy during abdominal aortic surgery

Purpose. To examine the utility of near-infrared spectroscopy (NIRS) in assessing lower-leg perfusion, NIRS was performed on the calf muscles of patients who underwent abdominal aortic surgery. Methods. Thirty patients undergoing elective infrarenal abdominal aortic surgery for abdominal aortic aneurysm (AAA group; n = 16) and aorto-occlusive disease (AOD group; n = 14) were studied. Before induction of anesthesia, NIRS probes were placed over both calf muscles, and muscle oxygen saturation (S_tO2) was continuously monitored throughout the surgery. Results. The preoperative S_tO2 value was significantly lower in the AOD group (57.0 ± 11.2%) than in the AAA group (68.7 ± 7.0%). In both groups, S_tO2 significantly decreased after aortic cross-clamping; the maximal ischemic value of S_tO2 in the AAA group (17.8 ± 7.2%) was significantly lower than that in the AOD group (46.7 ± 17.1%). The time taken to reach maximal ischemia was significantly longer in the AAA group (30 ± 12 min) than in the AOD group (19 ± 12 min). After release of the aortic clamp, the decreased S_tO2 returned to the preoperative level in the AAA group, whereas it increased above the preoperative value in the AOD group. Conclusion. NIRS performed on the calf muscles is a useful method for assessing the changes in lower-leg perfusion during and after abdominal aortic surgery. Received: June 6, 2001 / Accepted: December 5, 2001     

Simultaneous coronary bypass and abdominal aortic surgery in patients with severe coronary disease — indication and results

In patients with severe coronary artery disease (CAD) abdominal aortic surgery is still associated with high morbidity and mortality rates. Some patients will present with both symptomatic CAD and large, symptomatic abdominal aortic aneurysms (AAA) or end-stage aortic occlusive disease (AOD) that does not allow for a two-stage procedure. We report a series of 29 patients who underwent simultaneous coronary artery bypass graft surgery (CABG) and abdominal aortic surgery (25 AAA, 4 AOD). In the AAA group there were 23 males and 2 females with a mean age of 68 years (50–80). Sixteen patients presented with severe three-vessel disease. Ten patients had unstable angina. Aortic stenosis or insufficiency was present in two and one patient, respectively. Four patients with three-vessel disease and an ejection fraction below 30% presented with end-stage AOD and critical limb ischemia. Coronary bypass graft surgery was performed first. With the patient still on partial cardiopulmonary bypass, abdominal aortic surgery was carried out. Patients received an average of 3.1 coronary bypass grafts. Additionally, three aortic valves were implanted. Fourteen tube grafts and 15 bi-iliacal or bifemoral bifurcation grafts were placed in the abdominal aortic position. Additional vascular surgery was performed in five patients. Intraoperative management was without complication in all but one patient, who had intraoperative myocardial infarction (AOD group). Hospital mortality was 8% (2/25) in the AAA group. There was however substantial hospital morbidity (52.2%). The mean follow-up is 20.5±2.5 months. The actuarial survival rate at 3 years is 84.9%. It is concluded that combined CABG and abdominal aortic surgery is a reasonable option for patients who present with both severe CAD and symptomatic abdominal aortic disease. The continuation of CPB during aortic surgery may effectively prevent the adverse effects of infrarenal aortic clamping on a failing ventricle.