Mitochondrial dysfunctions in circulating T lymphocytes from human immunodeficiency virus-1 carriers [see comments]

In several models of lymphocyte apoptosis, two alterations of mitochondrial function precede advanced DNA fragmentation: (1) a reduction of mitochondrial transmembrane potential (delta psi m) and (2) an increase in mitochondrial generation of superoxide anion. Here we show that two fluorochromes allow for the identification of analogous mitochondrial perturbations in circulating T lymphocytes from human immunodeficiency virus (HIV)-1+ donors. The first among these fluorochromes, the cationic lipophilic dye DiOC6(3), measures delta psi m; the second marker, hydroethidine (HE), is nonfluorescent, unless it is oxidized by superoxide anions to the product ethidium (Eth). CD4+ or CD8+ cells from clinically asymptomatic HIV-1 carriers contain a significantly elevated percentage of cells endowed with enhanced HE --> Eth conversion and/or reduced DiOC6(3) uptake as compared with normal controls. Phenotypic characterization of (HE --> Eth)high cells from HIV+ donors shows that these cells possess a low delta psi m, thus demonstrating a functional alteration of mitochondria. In addition, (HE --> Eth)high cells display a reduced incorporation of the cardiolipin- specific dye nonyl-acridine orange (NAO), showing a structural defect of the cardiolipin-containing inner mitochondrial membrane. Control experiments involving rotenone, an inhibitor of the respiratory chain complex I, indicate that the reactive oxygen species responsible for HE --> Eth conversion is generated during mitochondrial electron transport. In synthesis, it appears that mitochondrial alterations occur in a significant percentage of circulating T lymphocytes from HIV- 1 carriers. The extent of delta psi m reduction, as determined ex vivo, correlates with the frequency of cells undergoing DNA fragmentation after overnight in vitro culture. These observations may be important for the understanding and for the direct ex vivo quantitation of HIV- triggered lymphocyte destruction.     

Platelet adhesion to fibronectin in flow: the importance of von Willebrand factor and glycoprotein Ib

We describe glycoprotein (GP) Ib as a mediator of adhesion to fibronectin, specifically in flow. A monoclonal antibody (MoAb) directed to the von Willebrand factor (vWF)-binding site on this receptor or the absence of this receptor on the platelet membrane, in the case of a patient with the Bernard-Soulier syndrome, reduced platelet coverage to fibronectin to approximately 30% of the control value. A MoAb directed to the GP Ib-binding site on vWF showed a similar effect. With washed platelets in the absence of plasma vWF, the inhibitory effect of the anti-GP Ib antibody was the same as with whole blood. No inhibition with the anti-GP Ib antibody was observed when we used blood from patients with severe von Willebrand disease (vWD) or from a patient with vWD type I (platelet low). Addition of vWF to vWD blood resulted in restoration of adhesion. Immunoelectron microscopy on platelets adhering to fibronectin showed that GP Ib was homogeneously distributed over the entire surface of the platelet. vWF was present at the central zone and the edges of the platelet and at the basal interface between the platelet and the fibronectin surface. No direct binding of vWF to fibronectin could be demonstrated. These data indicate that GP Ib-mediated adhesion to fibronectin fully depends on vWF and that normal levels of plasma or platelet vWF are sufficient for optimal adhesion to fibronectin. The data suggest that the presence of platelets during perfusion is a prerequisite for vWF to support platelet adhesion to fibronectin.     

Effects of Intrapericardial Sotalol and Flecainide on Transmural Atrial Electrophysiology and Atrial Fibrillation

Introduction: Intrapericardial (IPC) delivery of antiarrhythmic agents is an appealing idea to increase the therapeutic width and reduce side effects of drugs, particularly in the thin atria. The aim of this study was to determine the effects of IPC versus intravenous (IV) d,l-sotalol and flecainide infusion on transmural atrial electrophysiology and sustained atrial fibrillation (AF) in the goat.
Methods and Results: Effects of IPC and IV sotalol and flecainide infusion on epi- and endocardial atrial electrophysiology, ECG, and tissue drug concentrations were studied in goats without and with persistent AF (>24 hours). Epicardial atrial refractory period (AERP, bcl 400 ms) increased after 120 minutes of 1 mg/kg/hour IPC sotalol with 61 ± 8 ms (P = 0.02), whereas the endocardial AERP was not affected. One mg/kg/hour IPC flecainide increased the epicardial pacing threshold and the epicardial AERP with 4 ± 0.5 mA (P = 0.003) and 33 ± 11 ms (P = 0.05), respectively. Endocardial values were unchanged. Marked ST-elevations in the precordial ECG leads were observed after IPC flecainide. In the AF group, IPC drugs did not prolong AF cycle length to a greater extent than IV delivery. The number of cardioversions was not different between the two delivery routes. A steep transmural drug concentration gradient after IPC sotalol and flecainide was observed in all heart chambers.
Conclusion: IPC sotalol and flecainide infusion in goats markedly affects epicardial atrial electrophysiology. IPC delivery, however, does not prolong AFCL or terminate AF to a greater extent than IV infusion. This suggests that the perpetuation of AF is not dominated by the epicardial and sub epicardial atrial layers.     

HLA associations with leukemia

Frequencies of 35 HLA A, B, C, and DR antigens were determined in 1,834 leukemic Caucasoids to evaluate possible associations between HLA and leukemia. In comparison with the frequencies of HLA antigens in published controls, the frequency of Cw3 was significantly higher in patients with acute lymphoblastic leukemia (relative risk = 2.64, P less than 0.0002), acute myelogenous leukemia (relative risk = 1.92, P less than 0.0007), and chronic myelogenous leukemia (relative risk = 2.07, P less than 0.002; P values adjusted for multiple comparisons). The frequency of Cw4 was elevated in patients with acute lymphoblastic leukemia (relative risk = 2.01, P less than 0.0003), acute myelogenous leukemia (relative risk = 2.06, P less than 0.0002), and chronic myelogenous leukemia (relative risk = 2.14, P less than 0.0008). The frequency of Aw19 was significantly decreased in patients with acute myelogenous leukemia (relative risk = 0.68, P less than 0.01) and chronic myelogenous leukemia (relative risk = 0.59, P less than 0.005). None of the other 32 HLA antigens investigated had a statistically significant association with leukemia. The data suggest that Cw3 and Cw4 may be markers for leukemia susceptibility genes, while Aw19 may be a marker for decreased susceptibility to leukemia.     

Follicle-stimulating hormone and insulin regulation of 17 beta-estradiol secretion and granulosa cell proliferation within immature rat ovaries maintained in perifusion culture

The present study examined the effects of FSH and insulin (IN) on 17 beta-estradiol (E2) secretion and granulosa cell proliferation. For these studies, immature rat ovaries were maintained in perifusion culture and continuously exposed to FSH (0-800 ng RP-1 eq/ml) and /or IN (0.2 U/ml). At specific times, the ovaries were removed from perifusion, and the perifusate was assayed for E2 by RIA. The ovaries were then incubated with [3H]thymidine ([3H]T) in order to estimate granulosa cel mitotic activity. FSH increased E2 secretion in a dose-dependent manner (P less than 0.05), but did not enhance [3H]T incorporation (P greater than 0.05) after 48 h of perifusion. Conversely, IN increased [3H]T incorporation (P less than 0.05) without stimulating E2 secretion (P greater than 0.05) after 48 h of perifusion. FSH alone or in combination with IN stimulated [3H]T incorporation at 24 h of perifusion compared to both zero hour control (P less than 0.05) and IN treatments (P less than 0.05). The inability of FSH to stimulate [3H]T incorporation after 48 h did not appear to be due to increased E2, since IN stimulated [3H]T incorporation in the presence of 100 ng E2/ml. Further, continuous exposure to FSH was required to maintain E2 secretion, demonstrating that after 24 h, FSH loses its capacity to stimulate granulosa cell [3H]T incorporation, but not E2 secretion. Finally, when ovaries are pretreated with FSH for 48 h and then exposed to FSH and/or IN, [3H]T incorporation was stimulated, and E2 secretion inhibited. These data suggest that 1) initially, FSH acts to stimulate mitosis then promotes granulosa cell E2 secretion; and 2) once granulosa cells begin to secrete E2 they are still capable of mitosis, but their mitotic activity is no longer directed by FSH.     

Safe and effective direct implantation of a new stent through 5 F. guiding catheters with delivery from the radial artery: initial results of a prospective registry

OBJECTIVES: To evaluate the safety and efficacy of the direct implantation of a new stent via the radial artery through a 5 F. guiding catheter. Background advances in the design of stents and stent delivery systems have facilitated the performance of direct stenting and the use of thinner guiding catheters. METHODS: This registry enrolled prospectively 125 patients (147 lesions, 20.4% AHA/ACC class B2/C) who underwent elective percutaneous coronary revascularization procedures for stable or unstable angina between November 2000 and March 2001. RESULTS: Cannulation of the radial artery was attempted in 92.7% and was successful in 91.0% of cases. Direct stenting was successful in 88.7% of lesions and procedural success was 99.3%. In-hospital major adverse cardiac events occurred in 1.6% of cases (one death, one semi-urgent coronary artery bypass operation). The final rate of successful stent implantation through 5 F. guiding catheters was 96.7%. There were no access-site-related complications. Failure to cross the lesion occurred in 10% of attempts. At a mean follow-up of 7 ± 2.8 months after discharge from hospital, 79% of patients had remained free of angina, and 89% had remained free of ischemic events. CONCLUSIONS: Direct stenting with a new stent design was safe, effective, and could be accomplished through 5 F. guiding catheters with favorable long-term clinical outcomes. (Int J Cardiovasc Intervent 2003; 5: 72-76)     

参芪健胃颗粒抗炎效应

目的：观察参芪健胃颗粒对急性炎症模型的抗炎作用。方法：实验于2006—02／05在河南中医学院动物实验中心完成。①将50只昆明小鼠随机均分为5组：大、中、小剂量的参芪健胃颗粒组（2．4，1．2，0．6g/kg），三九胃泰颗粒组（10g／kg）及生理盐水组。给药体积0．02mL/g。灌服给药1次/d，连续给药5d，于最后1次给药后1h，每鼠右耳涂二甲苯，4h后处死小鼠，沿耳廓基线解剖位置仔细剪下全耳，对齐双耳，修剪，使每耳大小一致，迅速用分析天平称重，并计算肿胀度（肿胀度=右耳重-左耳重）。②将40只SD大鼠随机均分为5组：大、中、小剂量的参芪健胃颗粒组（16，8，4g／kg），三九胃泰颗粒组（6．7g／kg）及同体积生理盐水组。给药体积2mL/kg。灌服给药1次/d，连续给药5d，于最后1次给药后立即在大鼠足趾肿胀仪上测大鼠正常左后足跖体积，给药后30min立即给每鼠左后足跖皮下注射0．1mL新配制的10％新鲜鸡蛋清液，分别于给蛋清后30，60，120，240，360min再次在大鼠足趾肿胀仪上测大鼠左后足跖体积，并计算肿胀率[足跖肿胀率=（给药后不同时问足跖体积-同侧正常足跖体积），同侧正常足跖体积]。结果：50只小鼠和40只大鼠均进入结果分析。①大、中剂量的参芪健胃颗粒组和三九胃泰颗粒组耳壳肿胀度显著小于生理盐水组（P〈0．01），小剂量参芪健胃颗粒组小于生理盐水组（P〈0．05）。②与生理盐水组比，大剂量参芪健胃颗粒组和三九胃泰颗粒组在给药后30～60min均可明显抑制大鼠蛋清性足跖肿胀，使足跖肿胀率明显减小（P〈0．05），在给药后120-360min均可显著抑制大鼠蛋清性足跖肿胀，使足跖肿胀度明显减小（P〈0．01）；中剂量参芪健胃颗粒组在给药后30-120min可明显抑制大鼠蛋清性足跖肿胀，使足跖肿胀率明显减少（P〈0．05），在给药后240-360min可显著抑制大鼠蛋清性足跖肿胀，使足跖肿胀率显著减少（P〈0．01）；小剂量参芪健胃颗粒在给药后30～360min仅有抑制大鼠蛋清性足跖肿胀的趋势。结论：参芪健胃颗粒可显著抑制小鼠耳壳肿胀和大鼠足趾肿胀程度，有较好的抗炎作用。     

剪碎法及匀浆法制备兔肌肉VX2肿瘤单细胞悬液的方法比较

目的:比较剪碎法和匀浆法制备兔肌肉VX2肿瘤单细胞悬液的效果。方法:实验于2006-10-25/2007-04-01在安徽医科大学第一附属医院中心实验室进行。切取兔肌肉VX2肿瘤组织块,分别采用剪碎法和匀浆法制备兔肌肉VX2肿瘤单细胞悬液。经苏木精-伊红染色,光学显微镜观察细胞形态学差异,并行AnnexinV/PI双染法,流式细胞仪分析细胞凋亡率和细胞坏死率的差异。结果:①细胞形态:匀浆法细胞产率高,细胞形态完整,细胞分布均匀,细胞碎片及细胞团较少;而剪碎法所得细胞悬液在光镜视野中有较多的杂质和细胞团,完整的细胞相对较少。②流式细胞仪分析结果:匀浆法制备的单细胞悬液左下象限正常细胞分布较集中,右下象限凋亡细胞较少,左上象限坏死细胞及细胞碎片较少;所得细胞凋亡率和细胞坏死率值明显低于剪碎法[细胞凋亡率:(8.90±1.24)%,(15.75±1.53)%;细胞坏死率(35.22±1.56)%,(40.16±1.68)%,P均<0.01]。结论:用匀浆法制备兔肌肉VX2肿瘤单细胞悬液优于剪碎法。     

应用细胞培养、流式细胞技术和Western Blot检测氮杂糖化合物SZL在体外抑制人宫颈癌HeLa细胞增殖与诱导凋亡的效应

目的:存在于细胞表面的糖复合物参与细胞的通讯、增殖、迁移、分化等生理过程,在机体中具有复杂的生物学调控作用。探讨氮杂糖化合物SZL在体外对人宫颈癌HeLa细胞生长、DNA损伤、线粒体膜电位以及细胞凋亡的干预。方法:实验于2004-12/2006-12在北京大学医学部细胞生物学系完成。①实验材料:人子宫颈癌HeLa细胞株由北京大学医学部细胞生物学系杜晓娟副教授提供。SZL由北京大学天然药物及仿生药物重点实验室叶新山教授合成。②实验方法:取对数生长期HeLa细胞,消化后制成细胞悬液,按1.5×103/孔的密度接种,培养24h细胞完全贴壁后,SZL组加入5,10,25,50,100μmol/L的SZL,空白对照组加入DMEM培养液。③实验评估:SZL作用24,48,72h后,采用酸性磷酸酶法检测细胞生长抑制情况,瑞氏染色镜下观察细胞形态;AnnexinV-PI染色后,流式细胞仪检测细胞凋亡率;Rhodamine123标记活细胞后,流式细胞仪检测线粒体膜电位的变化;WesternBlot法检测SZL作用后凋亡相关蛋白的表达;单细胞凝胶电泳检测SZL作用后细胞DNA损伤情况。结果:①细胞生长抑制:5,10,25,50,100μmol/LSZL作用24,48,72h后的半数抑制浓度分别为73.6,43.2,33.6μmol/L,显著抑制Hela细胞的增殖。镜下空白对照组HeLa细胞分布均匀,胞质透明清亮,呈铺展状态生长;50μmol/LSZL作用24h后,HeLa细胞密度变稀,体积变小,胞核染色质呈聚集状态。②细胞凋亡:50,100μmol/LSZL作用24h后细胞凋亡率分别为(3.51±0.41)%,(58.46±8.45)%;在24～48h过程中,凋亡的细胞逐渐坏死,SZL作用48h后细胞凋亡率分别为(10.51±2.20)%,(17.29±7.52)%。空白对照组24,48h后细胞凋亡率均为1%。③线粒体跨膜电位的变化:50μmol/LSZL作用24,48h后,HeLa细胞的线粒体膜电位平均荧光强度均明显低于空白对照组(P<0.01)。④凋亡相关蛋白的表达:25,50,100μmol/LSZL作用HeLa细胞48h后,凋亡相关蛋白pro-caspase3、Bcl-2表达降低,细胞色素c含量升高。⑤细胞DNA损伤:50μmol/LSZL作用24h后,受损细胞DNA在电场中迁出,呈现彗星状拖尾。结论:氮杂糖类化合物SZL能够抑制HeLa细胞的增殖,诱导细胞发生DNA损伤,通过干预线粒体途径实现细胞凋亡。     

人工全膝关节置换术中关节周围注射镇痛药物对术后痛的效果

目的:关节周围注射混合镇痛药物是比较新颖的术后镇痛方法。运用随机对照的前瞻性方法观察人工全膝关节置换术中膝关节周围注射布比卡因、吗啡、肾上腺素等混合药物的止痛效果。方法:①实验对象:于2006-09/2007-05上海市第六人民医院关节外科病房选取60例择期、单侧人工全膝关节置换患者,患者知情同意参加本实验,随机分为研究组30例和对照组30例。②实验方法:研究组患者术中关节周围注射镇痛药物(0.5%布比卡因30mL,肾上腺素1:200000,吗啡10mg,生理盐水30mL),对照组患者没有运用镇痛药物。所有患者术后都运用镇痛泵(曲马多500mg,氯诺昔康16mg,稀释到100mL,2mL/h),持续运用48h。③实验评估:术前、术后分别记录静止和活动视觉模拟疼痛评分和膝关节活动度。结果:60例患者均进入结果分析。①视觉模拟疼痛评分:研究组术后6,12,24h静止视觉模拟疼痛评分低于对照组(P均<0.01),研究组术后6,12,24h运动视觉模拟疼痛评分也低于对照组(P均<0.01)。两组术后36,48,72h静止和运动视觉模拟疼痛评分比较差异无显著性(P>0.05)。②手术前后镇痛药物使用情况:研究组术后有6例需长期服用奇曼丁(曲马多),对照组术后有18例需长期服用奇曼丁,两组相比有统计学差异。同时,对照组术后有5例需肌注吗啡(10g/L)。③膝关节活动度:研究组术后第1,2,3天膝关节活动度高于对照组(P均<0.01)。两组术后第1,2周膝关节活动度比较差异无显著性。④并发症:没有任何因为注射而引起的伤口并发症、心脏或中枢神经系统的毒性被发现。结论:人工全膝关节置换术中应用关节周围注射镇痛药物可以在术后早期减少静止和活动状态疼痛评分并且减少术后镇痛药的使用、改善术后早期关节活动度。