Adriamycin-mediated potentiation of cytotoxicity against freshly isolated bladder cancer cells by autologous non-activated peripheral blood lymphocytes and tumor infiltrating lymphocytes

PURPOSE: Previous studies indicated that cancer patients lack functional anti-tumor cytotoxic lymphocytes. However, anti-tumor cytotoxic lymphocytes may coexist with immunoresistant tumor cells. We reasoned that anti-tumor cytotoxic activity of lymphocytes may be revealed if the tumor cells are sensitized to killing. It has been reported that adriamycin (ADR) exhibits various immunomodulating activities. In the present study, we investigate the effect of ADR on the susceptibility of freshly isolated bladder cancer cells to lysis by autologous non-activated peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. RESULTS: Treatment of ADR-resistant fresh bladder cancer cells with ADR at 0.1 microg./ml. or more for 3 hours or more enhanced their susceptibility to lysis by autologous PBL. This ADR-induced enhancement of susceptibility of fresh bladder cancer cells to lysis by PBL was also observed when lymphokine activated killer cells, purified natural killer cells and T lymphocytes were used as effector cells. Furthermore, while cytotoxicity of freshly derived TIL against autologous bladder cancer cells was minimal, significant cytotoxicity was observed with ADR-treated bladder cancer cells. The ADR analogs, epirubicin and pirarubicin, also enhanced the susceptibility of bladder cancer cells to lysis by autologous PBL. Treatment of bladder cancer cells with ADR had no effect on the expression of MHC class I on the cancer cells or the frequency of bladder cancer target cell conjugates to autologous PBL. Treatment of bladder cancer cells with ADR augmented their sensitivity to anti-Fas monoclonal antibody and tumor necrosis factor-a. Pretreatment of effector cells with ADR had no effect on their cytotoxic function. CONCLUSIONS: These findings demonstrate that PBL and TIL in patients with bladder cancer exhibit anti-tumor cytotoxic function, but their function is not manifested due to development or acquisition of tumor cell resistance to killing. However, the resistance of bladder cancer cells to killing by cytotoxic lymphocytes is overcome if cancer cells are sensitized by subtoxic concentrations of ADR. These findings suggest that treatment of bladder cancer patients with low doses of ADR may sensitize the cancer cells to killing by autologous circulating and tumor infiltrating lymphocytes and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant bladder cancer.     

FTY720对小鼠外周血淋巴细胞的影响

目的　观察FTY72 0对近交系小鼠外周血淋巴细胞的影响。方法　选用近交系雄性C5 7BL/ 6小鼠 ,连续口服FTY72 0 (3mg·kg-1·d-1) 14d。每只动物于服药后 2、4、6、8、12、2 4h以及停药后 1、2、3、4、5、6周自尾静脉采血 ,计数淋巴细胞数。结果　服用FTY72 0后 ,小鼠外周血淋巴细胞迅速减少 ,2h后即由服药前的 (870 0± 6 5 6 ) / μl降至 (3783± 176 ) / μl(P <0 .0 1) ,并于 4h降至最低值 (2 4 6 7± 2 5 2 ) / μl,之后基本保持稳定。停用FTY72 0后小鼠外周血淋巴细胞没有立刻开始恢复 ,在低水平徘徊 2周后 ,第 3周淋巴细胞数开始出现明显回升 ,停药后第 6周恢复到 (7833± 76 4 ) / μl,与服药前相比 ,差异无显著性 (P >0 .0 5 )。结论　FTY72 0起效迅速 ,特异性强 ,且作用可逆。     

Epidural fentanyl effect on cardiac output and hepatic blood flow

Epidural opioids provide high quality analgesia with no clinically apparent effect on the circulation or on specific organ blood flow. Little investigative data is available to support these impressions of circulatory stability. Ten patients presenting for thoracotomy were studied at rest preoperatively to determine if epidural fentanyl had any effect on the systemic circulation or hepatic blood flow. Intravascular pressure measurements, cardiac output estimation using the dye-dilution technique and estimation of altered hepatic blood flow by measuring the clearance of indocyanine green were performed. No significant changes in heart rate, perfusion pressure, cardiac output or hepatic blood flow were detected following the administration of fentanyl 50 micrograms into the epidural space.     

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.     

人羊膜间充质细胞对异体外周血淋巴细胞的免疫抑制作用

目的 探讨人羊膜间充质细胞(human amniotic mesenchymal cells,hAMCs)对异体外周血淋巴细胞的免疫抑制作用,阐明人羊膜间充质细胞调节移植免疫排斥的作用机制.方法 从新鲜足月剖宫产废弃的羊膜组织分离出hAMCs并进行鉴定.采用密度梯度离心从健康志愿者外周血中分离出单个核淋巴细胞(PBMLs),在植物血凝素(PHA)刺激下,分别按1∶0.05,1∶0.10,1∶0.20的比例以hAMCs处理,MTS法检测淋巴细胞的增殖,ELISA法检测细胞因子IFN-γ、TNF-α的分泌.结果 hAMCs能明显抑制异体外周血淋巴细胞的增殖,hAMCs与PBMLs比值为0.05∶1,0.10∶1,0.20∶1时的增殖抑制率分别为16.91％、20.83％、28.19％.hAMCs还可显著抑制异体淋巴细胞IFN-γ和TNF-α的分泌(P＜0.01),并且在一定范围内随着hAMCs数量的增加抑制作用更加明显.结论 hAMCs能抑制异体淋巴细胞增殖并降低淋巴细胞IFN-γ和TNF-α的分泌,这可能是其预防和减轻移植排斥反应发生的机制之一.     

Effects of steroid hormones on phytohemagglutinin-stimulated human peripheral blood lymphocytes.

The interactions of steroid hormones and human peripheral blood lymphocytes (PBLs) have been investigated following glass-wool column separation of PBLs and incubation in a serum-free medium in the presence of phytohemagglutinin (PHA). Addition of hydrocortisone or progesterone above physiological concentrations resulted in inhibition of [14C]2-TdR incorporation. 17beta-Estradiol and 5alpha-dihydrotestosterone inhibited [14C]2-TdR incorporation only at steroid concentrations thousands of times higher than the physiological concentrations. The kinetics of hydrocortisone and progesterone inhibition appeared to be similar and suggested that events occurring early after PHA addition were most sensitive to steroid inhibition and that addition of steroid at 28 hr, at the end of the early prereplicative phase of the cell cycle, inhibited DNA synthesis only at very high concentrations. The PHA-induced increase in RNA synthesis could be prevented by hydrocortisone addition even if delayed for up to 4 hr; morphological transformation was similarly affected. These data, and other investigations showing that progesterone binds to a specific glucocorticoid receptor in PBLs suggest that the progesterone inhibition of macromolecular synthesis in human PBLs is exerted via a glucocorticoid-like mechanism, and that glucocorticoid sensitivity is greatest during the early phases of lymphocyte activation. These results also provide a rationale for the apparent in vivo immunosuppressive capability of progesterone.     

人外周血淋巴细胞Fas的表达及细胞凋亡的诱导

目的 研究Ｆａｓ系统对外周血淋巴细胞的影响。方法 将分离、纯化后的淋巴细胞进行单向淋合淋巴细胞培养,用流式细胞术动态检测新鲜分离和激活后的Ｔ淋巴细胞表面Ｆａｓ表达水平,并用ＤＮＡ电泳和原位末端标记技术分别从定性和定量的角度观察Ｆａｓ系统激活与否对混合培养后淋巴细胞凋亡率的影响。结果 Ｔ淋巴细胞在被激活后Ｆａｓ表达水平升高,加抗Ｆａｓ单克隆抗体组的细胞凋亡率高于末加抗体的对照组（Ｐ〈０．０５）。结论     

Intracellular cytokines of peripheral blood lymphocytes in nephrotic syndrome

Idiopathic nephrotic syndrome (NS) is probably caused by abnormalities in T-lymphocyte function. The presence of several immunological abnormalities in these patients supports this hypothesis, but to date there is no agreement about immunological status and its influence on the course of NS. Thirty-six children with NS [19 with first episode (group I) and 17 in remission (>6 months) of NS (group II), aged 4-17 years, mean 7.1 years] were included in the study. Nineteen age-matched healthy children constituted the control group. Anti-cytokine antibodies were used in conjunction with antibodies against cell surface antigens to study cytokine synthesis in different lymphocyte populations. In the present study the intracellular synthesis of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, and IL-6 was measured. The intracellular synthesis of IL-2 was higher in group I compared with the controls, both in the whole population of T-lymphocytes (12.1+/-6.2% vs. 7.6+/-6.7%, P=0.0281) and in the subpopulation of CD8- lymphocytes (17.3+/-8.5% vs. 7.2+/-4.8%, P=0.0001). No significant differences in IFN-gamma intracellular expression were found. The intracellular synthesis of IL-4 was lower in group I compared with the controls, both in the whole population of T-lymphocytes (1.98+/-1.92% vs. 3.6+/-3.3%, P=0.012) and in the subpopulation of CD8- lymphocytes (2.4+/-2.3% vs. 6.5+/-6.4%, P=0.0002). Similarly, the intracellular expression of IL-6 was lower in group I compared with the control group, in the whole population of T-lymphocytes (0.85+/-0.6% vs. 2.2+/-3.1%, P=0.004), in the CD8- subpopulation (1.1+/-1.1% vs. 2.2+/-2.0%, P=0.006), and in the CD8+ subpopulation (1.1+/-0.9% vs. 2.8+/-3.4%, P=0.0008). The results of this study indicate that the acute episode of NS is associated with increased intracellular synthesis of IL-2 and decreased intracellular synthesis of IL-4 and IL-6.     

Cryopreservation of freshly isolated porcine islet cells

INTRODUCTION: The use of xenogenic islet cells may be a possibility to overcome the shortage of human donor organs to treat diabetes. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor intensive, cryopreservation has emerged as an attractive system of islet banking. The aim of this study was to determine the influence of three different freezing media (FM) on viability of freshly isolated porcine islet cells (FIPIC). METHODS: FIPIC were isolated using a modified Ricordi method and purification performed using a Lymphoprep density gradient. Viability of FIPIC prior to freezing and after thawing was determined using the MTT-based Cell Growth Determination Kit. Insulin production was detected using enzyme-linked immunosorbent assay. Three different FM containing dimethylsulfoxide (DMSO) or glycerol and sucrose were used for cryoprotection of FIPIC. RESULTS: Isolation and purification of FIPIC resulted in 95% +/- 1.3% viability and 97% +/- 1.4% purity. Cryopreservation with FM I (containing DMEM, FCS, DMSO) yielded 98.4% and FM III (containing DMEM, FCS, glycerol) 93.1% viability, whereas only 85.6% were alive when cryoprotection is performed with FM II (containing DMSO, BM). Glucose stimulation revealed a loss of 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P < .05) when FIPIC were stored in FM II. DISCUSSION: Low concentrations of DMSO or the use of glycerol and sucrose seem to be equivalent to cryopreserve FIPIC.     

创伤患者外周血淋巴细胞自噬变化及其与糖代谢的关系

目的 观察创伤患者外周血淋巴细胞的自噬变化规律以及与创伤后血精变化的关系.方法 根据损伤严重度评分(ISS)或简明损伤定级标准(MS)将创伤患者分为轻伤组、重伤组、严重伤组,同时设立健康对照组,分别于创伤后1、3、5、10 d采集外周血,采用密度梯度离心结合贴壁分离法分离创伤患者外周血淋巴细胞,应用激光共聚焦显微镜和透射电镜观察细胞自噬现象;将分离得到的淋巴细胞进行MDC染色并裂解,应用荧光分光光度计(激发光波长380 nm、发射光波长525 nm)测定荧光强度进行定量分析;于创伤后1、3、5 d检测患者血糖水平,分析创伤后自噬与血糖的相关性.结果 与健康对照组比较,创伤患者外周血淋巴细胞自噬水平明显升高,有显著变化(P＜0.01).结论 创伤后外周血淋巴细胞自噬水平与创伤严重程度、创伤后血糖成正相关.     

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase) suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100 nM ouabain to cultures of peripheral blood lymphocytes activated with 5 microg/ml phytohemagglutinin (PHA) did not modify the increased expression of the Fas receptor or its ligand FasL induced by the mitogen. However, treatment with ouabain potentiated apoptosis induced by an anti-Fas agonist antibody. A synergy between ouabain and PHA was also observed with regard to plasma membrane depolarization. PHA per se did not induce dissipation of mitochondrial membrane potential but when cells were also exposed to ouabain a marked depolarization could be observed, and this was a late event. It is possible that the inhibitory effect of ouabain on activated peripheral blood lymphocytes involves the potentiation of some of the steps of the apoptotic process and reflects an exacerbation of the mechanism of activation-induced cell death.