Ku80分子表达对肝癌细胞DNA双链损伤水平的影响

目的 观察Ku80分子表达对肝癌细胞DNA双链损伤水平的影响.方法 Western blot检测40例肝癌和癌周组织中γ-H2AX和Ku80表达水平;将Ku80基因转染到SMMC7721细胞;免疫荧光和Western blot检测y-H2AX焦点分布和蛋白表达.结果 γ-H2AX在肝癌组织中的表达与癌周比较明显增高(31/40,77.5％),而Ku80表达明显减低(30/40,75％),γ-H2AX表达上调与Ku80表达下调相关(P＜0.05);筛选获得稳定高表达Ku80的克隆细胞;免疫荧光结果显示,Ku80 高表达克隆γ-H2AX焦点数目与对照组比较明显减低(P＜0.01);Western blot显示,Ku80高表达克隆γ-H2AX表达与对照组比较明显减低.结论 Ku80表达下调与肝癌细胞的DNA双链损伤相关,Ku80分子表达可减低肝癌细胞DNA双链损伤水平.     

共刺激人外周血淋巴细胞对大肠癌细胞的杀伤作用

目的探讨CD28单克隆抗体与CD80共刺激活化的人外周血淋巴细胞(PBLs)在体外对大肠癌细胞株HT-29杀伤强度及杀伤作用机制,为大肠癌的过继免疫治疗提供参考。方法获得PBLs；共刺激；检测活化细胞的体外淋巴细胞毒作用,并用电子显微镜观察效应细胞杀伤的大肠癌细胞超微结构,用流式细胞仪检测大肠癌细胞的凋亡指数。结果共刺激PBLs对肿瘤细胞杀伤作用较强,达到(69．35±1．17)%。电镜结果显示,效应细胞作用12h肿瘤细胞出现坏死,部分可见凋亡。流式细胞仪检测效应细胞凋亡指数为24 h 19．6%,48 h效应细胞凋亡指数为12．1%。结论活化的淋巴细胞杀伤大肠癌细胞是通过坏死及凋亡两条途径来实现的。     

三氧化二砷对前列腺癌DU-145细胞周期阻滞及凋亡的影响

目的探讨三氧化二砷(As2O3)对前列腺癌非雄激素依赖细胞系DU-145细胞的增殖抑制作用及对细胞周期和凋亡的影响.方法应用体外细胞生长抑制试验(MTT比色法)研究As2O3对DU-145细胞生长的影响,流式细胞仪检测细胞周期分布情况及凋亡情况,琼脂糖凝胶电泳检测细胞DNA的变化.结果As2O3对DU-145细胞的增殖有明显的抑制作用,随着药物浓度增加和作用时间延长,凋亡细胞明显增加,出现G2/M期阻滞和DNA的断裂.结论As2O3可明显抑制前列腺癌DU-145细胞的增殖,诱导细胞凋亡,抑制细胞周期.     

肠道部分缺血再灌注损伤诱发外周血淋巴细胞凋亡

目的：观察肠道部分缺血再灌注(I/R)损伤过程中外周血淋巴细胞凋亡及其在植物血凝素(PHA)刺激下增殖能力的变化。方法：大耳白兔55只,分为肠部分I/R组、假手术对照组、正常对照组,用自行设计的血流阻断器阻断SMA血流50%,维持4h,制作肠道部分缺血再灌注损伤动物模型。采用流式细胞术(AnnexinV/PI双染法)测定外周血淋巴细胞凋亡变化,采用免疫组织化学方法观察外周血淋巴细胞caspase-3蛋白的表达,采用MTT比色法测定外周血淋巴细胞增殖能力。结果：肠道缺血期外周血淋巴细胞凋亡率明显升高,肠道再灌注期后外周血淋巴细胞凋亡比例骤然减少;外周血淋巴细胞caspase-3表达与凋亡改变呈相同趋势;肠部分I/R损伤后外周血淋巴细胞增殖能力明显降低。结论：肠道部分I/R损伤后,外周血T-淋巴细胞增殖功能明显受抑,外周血淋巴细胞凋亡变化与细胞内caspase-3的表达有关。     

线粒体融合素基因-2对乳腺癌细胞增殖的影响及其机制

目的 观察外源性线粒体融合素基因-2(Mfn2)对乳腺癌细胞(MCF-7)增殖的影响,探讨其抑制乳腺癌细胞产生增殖的机制.方法 将含有Mfn2cDNA的质粒在阳离子聚合物的介导下在体外转染MCF-7,蛋白印记法(Western blot)检测绿色荧光蛋白(GFP)、p21waf1、CDK2蛋白的表达,细胞计数测定Mfn2对MCF-7细胞增殖的影响,DNA直方图分析法测定Mfn2对MCF-7周期分布的影响,用Cyclins/DNA双参数流式细胞术对Cyclin A进行标记,Cellquest软件分析.结果 转染Mfn2基因的MCF-7细胞可以稳定高表达GFP蛋白;细胞计数测定表明转染Mfn2cDNA后,MCF-7细胞增殖明显受抑,DNA直方图分析法测定显示细胞停滞于S期;转染质粒组S期细胞占42.70%,转染空质粒组和空白对照组分别为17.15%、19.58%(P《0.05);用Cyclins/DNA双参数流式细胞术检测转染质粒组Cyclin A较对照两组明显升高;Western blot显示转染质粒组p21高表达,磷酸化CDK2低表达.结论 转染Mfn2基因可以明显抑制MCF-7细胞的增殖,并使细胞停滞于S期.     

DNA修复蛋白磷酸化组蛋白H2AX在胃癌中的表达及其意义

DNA双链断裂(DSBs)是真核生物后果最严重的DNA损伤之一[1].如果DSBs不能及时而准确地修复,可能导致基因突变、基因组不稳定、细胞凋亡甚至癌症的发生[2].磷酸化H2AX(γ-H2AX)是启动DSBs修复的关键因子,其在维持基因组稳定性方面具有重要作用[3].已有研究结果表明γ-H2AX在多种肿瘤中异常表达,但在胃癌中的研究不多本研究旨在观察γ-H2AX在胃癌的表达,并探讨其临床意义. 一、材料和方法 1.一般资料:收集2006年1月至2008年10月于广西医科大学胃肠腺体外科进行手术切除的胃癌标本30例,其中男16例,女14例;年龄≤60岁18例,年龄＞60岁12例,平均年龄53岁.所有患者术前未接受化疗、放疗、免疫治疗以及其他针对肿瘤的治疗.按照国际抗癌联盟1997年TNM分期标准进行肿瘤分期.γ-H2AX抗体、β-肌动蛋白(β-actin)抗体购自美国Abcam公司;SP即用型试剂盒购自北京中杉金桥生物公司.     

激素抵抗性前列腺癌细胞增殖凋亡及Caspase-3活性的观察

目的 探讨激素抵抗性前列腺癌细胞增殖、凋亡状态和Caspase-3活性及意义.方法 应用DNA电泳、流式细胞术 (FCM) 等技术检测了12例正常前列腺 (NP)、31例激素性前列腺癌(HRPC)和31例激素依赖性前列腺痛(HDPC) 组织DNA条带、DNA指数(DI)、增殖指数(PI)、细胞周期和Caspase-3活性,评价激素抵抗性前列腺癌组织增殖凋亡状况和Caspase-3活性.结果 HRPC DI、细胞异倍体率明显高于HDPC和NP组织,且G1/Go期细胞减少,S期和G2/M期细胞增多(P＜0.05),其中SPF明显升高(P＜0.01).NP、HRPC及HDPC组织前列腺癌组织G1期前端均出现了一个称之为凋亡峰的亚倍体峰(亚G1期),但HRPC细胞的凋亡峰明显低于HDPC和NP组织.HRPC组织细胞增殖指数(PI)明显高于HDPC和NP组织,HRPC凋亡峰值(AI)明显降低,PCa组织PI/AI明显增高,差异均有统计学意义(P＜0.01).NP、HRPC和HDPC组织Caspase-3均处于活性状态,但HRPC组织细胞内caspase-3活性明显低于HDPC和NP组织(P＜0.01).结论 与HDPC相比,HRPC细胞表现为异倍体率增高、增殖过度和细胞凋亡减少,且呈现明显的增殖凋亡失衡状态.细胞凋亡Caspase家族的Caspase-3活性下降可能与HRPC发生和进展过程相关.     

肿瘤坏死因子-α损伤的肺动脉内皮细胞对肺动脉平滑肌细胞增殖的影响及内皮细胞热应激反应的干扰作用

目的探讨肿瘤坏死因子-α(TNF-a)损伤的肺动脉内皮细胞对肺动脉平滑肌细胞(PASMC)增殖的影响及内皮细胞热应激反应的干扰作用。方法以终浓度为500、1000、1500u/ml的TNF-a孵育融合成层的肺动脉内皮细胞或经过热应激反应的内皮细胞1h,然后用不含血清的DMEM培养基培养24h,收集上清培养液制成条件培养液(EC-CMⅠ、EC-CMⅡ)用于孵育PASMC(Ⅰ组、Ⅱ组),同时用正常条件下肺动脉内皮细胞条件培养液(EC-CMⅢ)孵育PASMC(Ⅲ组)。应用流式细胞法测定EC-CMⅠ、EC-CMⅡ和EC-CMⅢ培养PASMC 24h后的细胞内DNA含量,根据曲线下面积计算处于各细胞周期的细胞百分数,并与未加EC-CM培养的PASMC(Ⅳ组)进行比较。结果与IV组比较,Ⅲ组PASMC的G0/G1期细胞百分比明显增加(P＜0.05),S期和G2/M期细胞百分比明显降低(P＜0.05),而Ⅰ组和Ⅱ组G0/G1期细胞百分比明显降低(P＜0.05)、S期和G2/M期细胞百分比明显增加(P＜0.05)。与Ⅰ组相比,Ⅱ组PASMC的G0/G1期细胞百分比明显增加(P＜0.05),S期和G2/M期细胞百分比明显降低(P＜0.05)。PASMC的DNA含量与TNF-α浓度呈明显正相关。结论正常状态下肺动脉内皮细胞能抑制PASMC的增殖,TNF-α刺激后内皮细胞可促进PASMC增殖,肺动脉内皮细胞热应激反应可降低该增殖作用。     

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.     

大鼠脂肪来源干细胞对紫外线造成的软骨细胞DNA损伤的修复作用研究

目的探讨大鼠脂肪来源干细胞(adipose-derived stem cells,ADSCs)对紫外线照射造成的软骨细胞DNA损伤的修复作用。方法取3~4周龄SD大鼠(体质量100~120 g)腹股沟脂肪,利用Ⅰ型胶原酶消化法体外分离培养ADSCs并传代;取第3代细胞采用流式细胞仪检测其表面相关标记,行成骨及成脂诱导鉴定其多向分化潜能。另取SD大鼠关节软骨,利用酶消化法分离培养软骨细胞并传代,并行甲苯胺蓝染色鉴定。取第3代软骨细胞,采用40 J/m~2剂量紫外线照射;照射后细胞分别以正常培养基培养(照射组)、以含ADSCs培养上清的培养基培养(ADSCs上清组)或与ADSCs共培养(ADSCs组)24h。以不进行紫外线照射的正常第3代软骨细胞作为对照(对照组)。采用MTS法检测细胞增殖情况,免疫荧光染色、Western blot法检测软骨细胞DNA损伤标志蛋白磷酸化组蛋白2A变异体(phosphorylatedhistone family 2A variant,γH2AX)的表达情况。结果经流式细胞仪检测及成骨、成脂诱导鉴定,所培养细胞为ADSCs。对照组、照射组及ADSCs上清组吸光度(A)值分别为2.20±0.10、1.34±0.04、1.57±0.06,照射组及ADSCs上清组显著低于对照组,照射组显著低于ADSCs上清组,比较差异均有统计学意义(P0.05)。免疫荧光染色示,照射组、ADSCs上清组及ADSCs组γH2AX蛋白荧光强度明显高于对照组,但ADSCs上清组及ADSCs组γH2AX蛋白荧光强度明显弱于照射组,ADSCs组和ADSCs上清组则无明显区别。Western blot法检测示,照射组、ADSCs上清组及ADSCs组γH2AX蛋白相对表达量均明显高于对照组,但ADSCs上清组及ADSCs组显著低于照射组,差异均有统计学意义(P0.05);ADSCs组和ADSCs上清组比较差异无统计学意义(P0.05)。结论大鼠ADSCs有助于恢复紫外线照射后的软骨细胞的增殖,降低DNA损伤标志蛋白γH2AX的表达,对紫外线照射所致软骨细胞损伤具有修复作用。     

Investigation of T and natural killer cell function with monoclonal antibodies

The relationship of T cell and natural killer (NK) cell antigens was examined using two monoclonal anti-T cell antibodies. The first--anti-HuLy-m1 (identical to OKTlla), reactive with all E-RFC+ cells and 50% null cells including NK cells--could partially block (greater than 50%) NK activity. The second--anti-HuLy-m2, reactive with T cells, some B cells, and null cells--could stimulate and not block NK activity. The enhancement of NK cell function by anti-HuLy-m2 was not related to interferon stimulation, and neither antibody altered effector:target cell binding. When used together neither augmentation nor blocking effects were observed, indicating different sites of reaction with NK cells. To further explore a possible association between T cell and NK antigens, anti-HuLy-m1 and -m2 were used to investigate certain T cell functions. Neither antibody was mitogenic for peripheral blood lymphocytes (PBLs) or blocked PHA stimulation of PBLs. In addition anti-HuLy-m1 did not inhibit a mixed lymphocyte culture (MLC) but could inhibit cytotoxic T lymphocytes (CTLs) almost as effectively as OKT3. However, anti-HuLy-m2 had no effect in an MLC and showed only minimal inhibition of a CTL assay. These studies demonstrate a relationship of the membrane structure of T cells and NK cells at an antigenic and functional level.     

The ability of sperm selection techniques to remove single- or double-strand DNA damage

A wide variety of techniques for the preparation of sperm are currently available, of which the most commonly employed are density-gradient centrifugation (DGC) and swim-up (SUP). To date, these methods appear to be effective in selecting functional sperm for assisted reproduction techniques (ART), but they may have negative effects on sperm DNA. In this study, the ability of these semen processing techniques to eliminate spermatozoa containing single- and double-strand DNA damage was assessed by the two-tailed comet assay and the sperm chromatin dispersion test in 157 semen samples from patients seeking assisted reproduction treatment. Our results indicated that SUP and DGC are equally efficient in eliminating spermatozoa containing double-strand DNA damage and sperm with highly damaged (degraded) DNA, as characterized by the presence of both single- and double-strand DNA breaks. However, DGC is more efficient than SUP in selecting spermatozoa that are free from single-strand DNA damage. Future studies should characterise the importance of the various types of DNA damage and examine the sperm processing protocols used in each laboratory to determine their ability to eliminate DNA damage and hence, prevent the potential transmission of genetic mutations via ART.     

Evidence of impaired T cell function in hemodialysis patients: potential role for secondary hyperparathyroidism

Previous studies in our laboratory showed that the T cell is a target for parathyroid hormone (PTH) action. It is theoretically possible, therefore, that chronic exposure of the T cells to high blood levels of PTH in patients with chronic renal failure may adversely affect T cell function. We examined in both normal subjects and dialysis patients several aspects of T cell function, including (1) T cell proliferation in response to phytohemagglutinin (PHA) mitogen with and without PTH and with and without exogenous interleukin 2 (IL-2); (2) the IL-2 production induced by PHA with and without PTH, and (3) resting levels of cytosolic calcium--[Ca2+]i--and the increment in [Ca2+]i in response to anti-CD3 antibody. Although PHA significantly (p less than 0.01) stimulated proliferation of T cells from both normal subjects and dialysis patients, the magnitude of the stimulation was significantly (p less than 0.01) smaller in the latter group. In both normal subjects and dialysis patients, exogenous IL-2 alone stimulated T cell proliferation, and the magnitude of the stimulation was similar to that produced by PHA. Also, IL-2 augmented PHA-induced proliferation of T cells from normal subjects, but failed to do so in T cells from dialysis patients. PHA did not augment IL-2 production by T cells from dialysis patients, and PTH did not correct this defect. The resting levels of [Ca2+]i in T cells from dialysis patients were significantly (p less than 0.01) higher, and the increments in [Ca2+]i in response to anti-CD3 antibody were significantly (p less than 0.01) lower than in T cells from normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adriamycin induces H2AX phosphorylation in human spermatozoa

Aim： To investigate whether adriamycin induces DNA damage and the formation of γH2AX （the phosphorylated form of histone H2AX） foci in mature spermatozoa. Methods： Human spermatozoa were treated with adriamycin at different concentrations, γH2AX was analyzed by immunofluorescent staining and flow cytometry and doublestrand breaks （DSB） were detected by the comet assay. Results： The neutral comet assay revealed that the treatment with adriamycin at 2 μg/mL for different times （0.5, 2, 8 and 24 h）, or for 8 h at different concentrations （0,4, 2 and 10 μg/mL）, induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of γH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP 1 with γH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase （PI3K） family, abolished the co-appearance of these two proteins with γH2AX. Conclusion： Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/ repair proteins as somatic cells.     

Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent

BACKGROUND: Genetic mutations found in pancreatic cancer (K-ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the phosphatidylinositol 3-kinase (PI3K)- and/or the p44/42-mitogen-activated protein kinase [p44/42-MAPK or extracellular signal-regulated kinase (ERK)] signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. METHODS: Proliferation was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% fetal calf serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) was also added. AKT phosphorylation (indicating PI3K activity) and ERK phosphorylation (ERK activation) were determined by Western blot. Cell viability was determined by MTT assay. Cell cycle progression and apoptosis were determined by flow cytometry. A two-tailed t test was used for statistical analysis of the data (significance P < 0.05). RESULTS: LY294002 inhibited the PI3K pathway without affecting ERK activation in response to serum. PD98059 inhibited the ERK pathway specifically. In both BxPC-3 and Panc-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G(1) cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. CONCLUSIONS: PI3K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. ERK plays a lesser role in mitogen-induced proliferation. Pharmacological inhibition of PI3K may decrease proliferation, increase apoptosis, and potentially confer therapeutic benefit in pancreatic cancer.     

Spermiogenic germ cell phase-specific DNA damage following cyclophosphamide exposure

The production of genetically competent spermatozoa is essential for normal embryo development. The chemotherapeutic drug cyclophosphamide creates cross-links and DNA strand breaks in many cell types, including germ cells. This study assessed the phase specificity of the susceptibility of spermiogenic germ cells to genetic damage induced by cyclophosphamide. Adult male rats were given cyclophosphamide using one of four schedules: 1) high dose/acute- day 1, 100 mg/kg; 2) low dose/subchronic, 4 days-days 1-4, 6.0 mg/kg/d; 3) high dose/subchronic, 4 days-day 1, 100 mg/kg, and days 2-4, 50 mg/kg/d; and 4) low dose/chronic-daily, 6.0 mg/kg/d for 14-28 days. To capture cauda epididymal spermatozoa exposed to cyclophosphamide during late, mid-, and early spermiogenesis, animals were sacrificed on days 14, 21, and 28, respectively. Spermatozoa were analyzed for DNA strand breaks using the comet assay. No dramatic increases in damage were seen after high-dose/acute exposure to cyclophosphamide. Subchronic exposure showed a dose-related increase in DNA damage; maximal damage, as demonstrated by comet tail parameters, was seen after 21 days, reflecting an increased susceptibility of step 9-14 spermatids. Low-dose chronic exposure to cyclophosphamide induced DNA damage, which reached a plateau by day 21. The magnitude of damage at all time points after low-dose chronic exposure was much greater than that following low-dose exposure for 4 days, indicating an accumulation of damage over time. Thus, the DNA damage induced by cyclophosphamide is germ cell phase-specific. The most damaging effects of cyclophosphamide occurred during a key point of sperm chromatin remodeling (histone hyperacetylation and transition protein deposition). We speculate that strand breaks disrupt chromatin remodeling, hence affecting chromatin structure and embryo development.     

Cimetidine-induced augmentation of human lymphocyte blastogenesis by mitogen, bacterial antigen, and alloantigen

The effect of Cimetidine, a histamine-type 2 receptor antagonist, was evaluated on the in vitro proliferative response of normal human peripheral blood lymphocytes (PBLs). Cimetidine (10(-3) to 10(-8) M) increased mitogen-induced blastogenesis by 22% (phytohemagglutinin (PHA) and by 27% (pokeweed) over nondrug-treated control values (P less than 005 for PHA and pokeweed). Preincubation of PBLs with Cimetidine further augmented blastogenesis as much as 2- to 3-fold (P less than 0.005 for both mitogens). Multiple testing of the same normal subject demonstrated consistent reproducibility of increased proliferation by Cimetidine. Similar statistically significant amplifications of the proliferative res-ponse were observed when bacterial antigen (streptokinase-streptodornase) or alloantigen was used to induce blastogenesis. Optimally effective concentrations of Cimetidine ranged from 10(-5) to 10(-7) M, which corresponds to expected clinical serum levels. These observations suggest that a histamine-type 2 receptor antagonist is capable of modulating the proliferative response of PBLs in the absence of exogenously added histamine. The immunoregulatory implication of this Cimetidine-induced proliferative augmentation is discussed in relation to clinical transplantation and cancer immunotherapy.     

Production of synergistic but nonidentical mechanisms of immunosuppression by rapamycin and cyclosporine.

We report that the mechanism of rapamycin (RAP) inhibition is synergistic, but nonidentical, to the mechanism of CsA inhibition. Like CsA, RAP inhibits T cell proliferation following mitogen (PHA) and/or alloantigen (MLR) stimulation. RAP levels of 100, 33, 11, 3.6, 1.2, and less than 1 ng/ml reduced PHA stimulation by 81%, 84%, 81%, 83%, 62%, and 33%, respectively, without cytotoxicity. The RAP concentration required to achieve 50% proliferative inhibition of either mitogen (PHA) or MLR assays revealed an interindividual variability of 5 pg/ml RAP (2 individuals), 1 ng/ml (3 individuals), and 100 ng/ml (2 individuals). Unlike CsA, RAP proliferative inhibition was not restricted to the G0 phase of the cell cycle. Addition of 100, 10, or 1 ng/ml RAP at the onset (G0), or 24 hr following cultivation (G1) similarly inhibited DNA synthesis by 42%, 42%, and 41% compared with 44%, 48%, and 47%, respectively. PWM-stimulated B cell proliferation was primarily RAP-sensitive during the G0 phase of the cell cycle. RAP at 100, 10, and 1 ng/ml inhibited B cell proliferation 46%, 51%, and 50% when added during G0 but only 15%, 20%, and 20% when added during G1. Generation of a cyclosporine-sensitive cytoplasmic activation signal, activator of DNA replication (ADR), was reduced by RAP. RAP reduction did not correlate directly with T cell proliferative inhibition (as does CsA). RAP-induced proliferative inhibition of 40% and 80% resulted in ADR inhibition of 16% and 33%. Proliferative inhibition was synergistically increased when CsA and RAP were used in combination, whereas ADR inhibition was only additively enhanced. Mechanistic disparity between RAP and CsA may potentiate clinical immunosuppression when RAP and CsA are used together.     

Detection of single- and double-strand DNA breaks after traumatic brain injury in rats: comparison of in situ labeling techniques using DNA polymerase I, the Klenow fragment of DNA polymerase I, and terminal deoxynucleotidyl transferase

DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3+/-15.8, dentate gyrus 58.6+/-12.8, CA1 = 15.8+/-5.9, CA3 = 12.8+/-4.2 cells/x 400 field, mean +/- SEM, all p < 0.05 versus naive). Cells with DNA breaks were detected by Klenow as early as 30 min and were maximal at 24 h (cortex = 56.3+/-14.3, dentate gyrus 78.0+/-16.7, CA1 = 25.8+/-4.7, CA3 = 29.3+/-15.1 cells/x 400 field, all p < 0.05 versus naive). Cells with DNA breaks were not detected by TUNEL until 2 h and were maximal at 24 h (cortex = 47.7+/-21.4, dentate gyrus 63.0+/-11.9, CA1 = 5.6+/-5.4, CA3 = 6.9+/-3.7 cells/x 400 field, cortex and dentate gyrus p < 0.05 versus naive). Dual-label immunofluorescence revealed that PANT-positive cells were predominately neurons. These data demonstrate that TBI results in extensive DNA damage, which includes both single- and double-strand breaks in injured cortex and hippocampus. The presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.     

Human anti-pig cell-mediated cytotoxicity in vitro involves non-T as well as T cell components

Abstract: Human anti-pig cell-mediated cytotoxicity was studied in vitro. Unprimed human peripheral blood lymphocytes (PBL) were able to lyse pig targets but not human targets when the assay was performed in human serum (HS). Much less, but still detectable, lysis was obtained using fetal calf serum (FCS). Human PBLs cultured for 6 days in FCS without stimulating cells showed substantial lysis of pig targets. This lysis was increased by the addition of human IL-2. Unseparated human PBLs stimulated for 6 days with pig cells in serum-free media lysed pig targets expressing the same or different SLA antigens as the stimulating cells. This lysis could not be significantly inhibited by anti-CD3 or anti-CD8 blocking antibodies during the effector phase of the assay. T cell-enriched human PBLs treated with anti-CD 16 and anti-CD56 antibodies plus complement also lysed pig targets. These effector cells were significantly inhibited by anti-CD3 and anti-CD8 antibodies but not by anti-CD4 antibodies. Furthermore, these primed T cell effectors could only lyse pig targets that shared the same MHC class I antigens as the sensitizing stimulators.
These results suggest that human anti-pig cell-mediated cytotoxicity in vitro has at least three different components in bulk culture: 1) an ADCC component depending on natural antibodies from human serum, 2) an NK and/or a LAK cell component that is enriched by in vitro culture and interleukin-2 (IL-2), and (3) an allospecific T cell component, involving CD3+, CD8+, class I-specific effector cells.