Annexin V-FITC/PI双标记与Hoechst33342/PI双标记流式细胞术检测细胞凋亡的比较

目的比较annexin V-FITC/PI双标记与Hoechst33342/PI双标记流式细胞术检测细胞凋亡的优缺点。方法用DEX诱导胸腺细胞凋亡和H2O2诱导K562细胞系细胞凋亡,应用Hoechst33342/PI和annexin V-FITC/PI双色标记流式细胞术检测体系检测细胞凋亡情况。结果 Annexin V-FITC/PI双标记法能准确地反映2种模型中的细胞凋亡情况,与其检测原理一致;而Hoechst33342/PI双标记法虽能准确反映DEX诱导胸腺细胞的凋亡情况,但在检测H2O2诱导的K562细胞凋亡时,其早期凋亡细胞的分布与其检测原理明显不符。结论与annexin-V-FITC/PI双标记法相比,Hoechst33342/PI双标记法有一定的局限性。     

2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响

 目的：探讨2型登革病毒（DENV2）感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法：用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果：DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论：登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。     

胰岛素样生长因子1对β样淀粉蛋白引起的神经元凋亡和tau蛋白磷酸化的作用(英文)

本研究的目的是阐明胰岛素样生长因子1(IGF-1)对β样淀粉蛋白(Aβ)引起的神经元凋亡的保护作用,以及tau蛋白磷酸化的作用。用MTT(四甲基偶氮唑盐)方法检测细胞活性,用流式细胞学结合Annexin V-FITC和PI(碘化丙锭)双染的方法检测早期凋亡和晚期凋亡/坏死,用Hoechst 33342染色观察凋亡细胞形态学,用免疫细胞化学的方法检测tau蛋白磷酸化。IGF-1阻止了Aβ25-35引起的培养的大鼠海马神经元的毒性,MTT值显著增加,从54.51%增至61.8%,Hoechst 33342阳性细胞的百分比从30.77%减少到22.81%。Aβ25-35孵育使Annexin V单标记细胞(Annexin V+/PI-)以及Annexin V/PI双标记细胞(An-nexin V+/PI +)的百分比显著增加(分别为3.41%和19.47%),应用100 ng/ml的IGF-1可显著减少Annexin V单标记细胞和Annexin V/PI双标记细胞的百分比分别至2.98%和15.16%。Aβ25-35可增加tau蛋白磷酸化,AT8阳性细胞占41.84%,而IGF-1则可抑制这一效应。我们的结果表明IGF-1可保护神经元,降低Aβ的细胞毒性,减少早期和晚期凋亡/坏死细胞的比例,抑制tau蛋白磷酸化,这可能是IGF-1神经保护作用的细胞机制。     

唑来膦酸抑制U937细胞增殖及诱导凋亡

目的:研究唑来膦酸(ZOL)对人类急性髓系白血病细胞U937的增殖抑制及促凋亡作用。方法:CCK-8法检测不同时间ZOL对U937细胞的生长抑制率;流式细胞术检测ZOL对U937细胞周期的影响;Annexin V-PI法及Hoechst 33342法检测ZOL作用前后细胞凋亡情况变化,JC-1检测ZOL对U937细胞线粒体膜电位变化的影响;克隆形成实验检测U937细胞克隆形成能力;Western blot法检测ZOL对U937细胞周期和凋亡相关蛋白的变化。结果:CCK-8结果显示ZOL可以抑制U937细胞的活力,并呈时间-剂量依赖性;Annexin V-PI及Hoechst33342结果显示ZOL可以促进U937细胞凋亡,且呈时间-剂量依赖性;JC-1结果显示ZOL可以明显降低U937细胞线粒体膜电位;PI法证实ZOL将U937细胞周期阻滞在S期,克隆形成实验证实0.2 mmol/L ZOL可以完全抑制U937细胞的克隆形成能力;Western blot结果显示ZOL作用于U937细胞48 h后细胞周期相关蛋白p21表达显著增强,促凋亡蛋白Bax表达增强,抑凋亡蛋白Bcl-2表达明显减弱。结论:ZOL抑制U937细胞的增殖和克隆形成主要是由于抑制了细胞周期相关蛋白表达,同时ZOL可以促进U937细胞凋亡,这种作用主要是通过调节线粒体凋亡途径相关蛋白来实现的。     

幽门螺杆菌空泡毒素VacA促进胃上皮细胞NLRP3炎性小体激活

目的探究H. pylori空泡毒素VacA对胃上皮细胞NLRP3炎性小体激活的影响。方法采用WT H. pylori26695和△VacA H. pylori26695感染AGS细胞,Western blotting、q RT-PCR、ELISA检测NLRP3、Pro-Caspase-1、Caspase-1、pro-IL-1β和IL-1β的表达。siRNA-NC、siRNA-NLRP3转染AGS后,分别用PBS、WT H. pylori26695和△VacA H. pylori26695刺激,qRT-PCR和细胞因子ELISA检测试剂盒分别检测pro-IL-1β的mRNA表达水平和IL-1β的蛋白表达水平。收集慢性胃炎患者标本,鉴别出H. pylori阳性患者的VacA基因型(s1m1型和s1m2型),通过q RT-PCR检测NLRP3、caspase-1、pro-IL-1β的mRNA表达水平及采用细胞因子ELISA试剂盒检测IL-1β蛋白表达水平。C57BL/6小鼠腹腔注射DMSO或者DMSO+Mcc950,H. pylori菌液灌胃,qRT-PCR和ELISA检测胃组织IL-1β的表达。结果 H.pylori感染AGS细胞,与对照组相比,WT H. pylori26695组IL-1β的mRNA和蛋白表达水平均明显升高(P0.01);△VacA H. pylori26695组与WT H. pylori26695组相比,IL-1β的m RNA和蛋白表达水平均明显下降(P0.01)。与siRNA-NC组相比,采用WT H. pylori26695和△VacA H. pylori 26695刺激转染siRNA-NLRP3的AGS细胞pro-IL-1β的m RNA水平和IL-1β蛋白表达水平均明显下调(P0.01)。H. pylori阳性患者较阴性患者NLRP3、Caspase-1和IL-1β的mRNA表达水平明显升高(P0.01);与VacA s1m2型患者相比,VacA s1m1型患者NLRP3、Caspase-1和IL-1β的mRNA表达水平显著升高(P0.01),同时IL-1β成熟分泌增加。H. pylori慢性胃炎小鼠动物模型中,与对照组相比,Mcc950组中在WT H. pylori26695和△VacA H. pylori26695灌胃后,pro-IL-1β的mRNA水平和IL-1β蛋白水平表达均明显下调(P0.01)。结论H. pylori空泡毒素VacA能够激活胃上皮细胞NLRP3炎性小体,促进IL-1β的成熟分泌。     

地塞米松对大鼠肝细胞BRL-3A增殖和凋亡的影响

目的探讨地塞米松对体外培养大鼠肝细胞BRL-3A增殖和凋亡的影响。方法用不同浓度的地塞米松处理BRL-3A细胞,于12、24、48、72、120 h后用MTT检测地塞米松对BRL-3A细胞活性的影响,同时分别用Annexin V-FITC双染法、PI单染色法、实时定量-聚合酶链反应(Real-time PCR)等方法检测地塞米松对BRL-3A细胞周期和凋亡的影响。结果 MTT检测表明,地塞米松能够抑制BRL-3A细胞的活性;Annexin V-FITC双染法分析显示,地塞米松具有显著促进BRL-3A细胞凋亡的效果;PI单染色法检测细胞周期分布情况表明,地塞米松处理后的细胞与对照组相比增殖能力减弱;用Real-time PCR检测促细胞凋亡基因Caspase-3、Caspase-8、Caspase-9和促增殖基因Ccnd1和Jun的mRNA表达情况,结果显示,用地塞米松处理后的细胞中Caspase-3、Caspase-8和Caspase-9均表达上调,而Ccnd1和Jun均表达下调,说明地塞米松能促进BRL-3A细胞的凋亡。结论地塞米松可以促进BRL-3A细胞的凋亡,并抑制其增殖。     

肿瘤坏死因子a促进人冠状动脉内皮细胞的凋亡

背景：研究表明冠状动脉内皮细胞凋亡参与了动脉粥样硬化的发生、发展过程。 目的：观察肿瘤坏死因子a对人冠状动脉内皮细胞的诱导损伤作用。 方法：取对数生长期的人冠状动脉内皮细胞c-12221,分别加入含0(对照),200,400,600 mg/L肿瘤坏死因子a的培养液,采用MTT检测细胞增殖率变化,Hoechst 33258/PI双染观察凋亡细胞形态变化,Annexin V-FITC和PI双染流式检测细胞凋亡率变化,高内涵活细胞成像系统检测细胞线粒体膜电位变化。 结果与结论：肿瘤坏死因子a呈剂量依赖性抑制人冠状动脉内皮细胞的增殖。Hoechst 33258/PI染色观察可见凋亡细胞染色质凝集、细胞核碎裂成碎片等典型细胞凋亡的特征性变化,不同质量浓度肿瘤坏死因子a组细胞凋亡率高于对照组(P < 0.05),线粒体膜电位低于对照组(P < 0.05),且呈剂量依赖性。表明肿瘤坏死因子a呈剂量依赖性促进人冠状动脉内皮细胞凋亡,抑制其增殖,作用机制与线粒体凋亡通路有关。     

胰岛素样生长因子I通过改善线粒体功能保护新生大鼠心肌细胞免于凋亡

 目的：探讨线粒体机制在胰岛素样生长因I(IGF-I)保护心肌细胞中的作用。方法：体外培养新生大鼠心肌细胞,过氧化氢处理诱导凋亡,JC-1线粒体膜电位检测法和透射电镜观察心肌细胞线粒体膜电位和形态的改变,Annexin V-FITC/PI双染色法、caspase-3活性测定、DNA-ladder分析和Hoechst 33258染色方法观察心肌细胞凋亡的情况。结果：过氧化氢可诱导心肌细胞凋亡,siRNA下调Kruppel 样因子9（KLF9）48 h后,心肌细胞线粒体膜电位下降率明显降低,由对照组的(24.0±1.6)%,降为IGF-I处理组的(18.3±1.2)%和KLF9下调组的(15.2±1.2)%;线粒体形态明显改善;DNA片段化改善;caspase-3活性降低,与对照组相比IGF-I处理组降低(1.30±0.28)倍,KLF9下调组降低(1.31±0.43)倍;Annexin V-FITC/PI双染法显示细胞凋亡率对照组为（42.5±1.8）%,IGF-I处理组为（22.4±4.2）%,KLF9下调组为（32.5±3.5）%;Hoechst 33258染色结果显示凋亡小体减少,KLF9下调组与IGF-I的抗心肌细胞凋亡效果相似。结论：IGF-I通过下调KLF9表达改善线粒体功能,保护心肌细胞免于凋亡。     

hPOT1基因过表达对HeLa细胞细胞周期和凋亡的影响

目的观察人POT1(protection of telomeres1)基因过表达对HeLa细胞细胞周期和细胞凋亡的影响。方法利用本课题组构建的hPOTl基因真核表达重组质粒pcDNA3-hPOT1,经脂质体介导瞬时转染HeLa细胞;通过RT-PCR和EMSA法(电泳迁移率改变分析)检测外源基因的表达效果,流式细胞术分析细胞周期,Hoechst33342荧光染色检测细胞凋亡。结果pcDNA3-hPOT1重组质粒转染HeLa细胞48h后,mRNA和蛋白质分析表明,外源性hPOT1基因能在HeLa细胞中有效表达,HeLa细胞阻滞于细胞周期s期,而对凋亡无明显影响。结论hPOT1基因可能参与了高等真核细胞细胞周期调控过程,但与细胞凋亡无密切关系。     

幽门螺杆菌VacA通过激活NF-kB诱导巨噬细胞分泌及凋亡

目的 研究幽门螺杆菌(Helicobacterpylori)空泡毒素(VacA)单一毒力决定簇对THP-1巨噬细胞分泌和凋亡的影响,以及核因子KB(nuclear factor KB,NF-KB)在调节VacA诱导的THP-1巨噬细胞功能中的作用.方法 将pDsRed-Monomer-Cl/vacA转染THP-1巨噬细胞,ELISA法检测巨噬细胞培养上清IL-1β、TNF-α含量;Griess试剂分析培养上清的一氧化氮(NO)水平;荧光探针DCFH-DA检测培养上清的活性氧(ROS);流式细胞仪检测细胞的凋亡率;凝胶阻滞试验(electro-phoretic mobility gel shift assay,EMSA)检测NF-kB的核转位.结果 重组质粒转染后6 h,重组质粒组培养上清中TNF-α、IL-1β含量明显高于阴性对照组(P<0.05);且分别于转染后6 h或24 h到达峰值;转染后6 h或12 h,重组质粒组培养上清中NO、ROS含量明显高于阴性对照组(P<0.05),且于转染后24 h达到高峰.转染后16 h,重组质粒组的凋亡率明显升高,与阴性对照组相比,差异有统计学意义(P<0.05).重组质粒组加NF-kB的特异性抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)后,IL-1β、TNF-α、NO、ROS的分泌量和细胞的凋亡率明显降低,与重组质粒组相比差异有统计学意义(P<0.05).EMSA试验显示,转染后3h细胞核内有活化的NF-kB,且于转染后12h活性最强.结论 VacA蛋白瞬时高表达上调THP-1巨噬细胞分泌IL-1β、TNF-α、NO和ROS;VacA蛋白瞬时高表达诱导THP-1巨噬细胞凋亡;NF-kB可能参与调节VacA诱导的THP-1巨噬细胞的分泌和凋亡.     

Antigenic diversity among Helicobacter pylori vacuolating toxins

Helicobacter pylori vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from H. pylori strain 60190 and generated a panel of 10 immunoglobulin G1kappa anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple H. pylori strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different H. pylori strains.     

Application of Bead-ELISA method to detect Helicobacter pylori VacA

Helicobacter pylori is an etiological agent of gastritis, gastric ulcer and gastric cancer. In order to clarify the significance of vacuolating cytotoxin (VacA) for the pathogenesis of Helicobacter pylori infection, we established and applied the sandwich bead enzyme-linked immunosorbent assay (Bead-ELISA) for quantitative determination of VacA in the culture mediums of H. pylori and other species of Helicobacter. The minimum concentration of VacA in culture medium detected by Bead-ELISA was 25 pg VacA/ml and its sensitivity was found to be quite high compared to vacuolation assay and Western blot analysis, e.g. the minimum concentrations of VacA in culture medium required for detection by vacuolation assay and Western blotting were 11 ng/ml and 38 ng/ml, respectively. All the H. pylori strains used were found to produce VacA in the culture medium by Bead ELISA, even though some strains were negative by Western blot and vacuolation assay. The results obtained by Bead-ELISA was consistent with those by PCR amplification of a 785 bp vacA fragments. A toxin immunologically similar to VacA produced by other strains of Helicobacter such as H. muridarum (ATCC49282), H. mustelae (F10) and H. felis (ATCC49179) could not be detected by Bead-ELISA as well as Western Blot.     

The intermediate region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay

Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.     

A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype

Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Delta49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Delta49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Delta49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.     

Resistance of primary murine CD4+ T cells to Helicobacter pylori vacuolating cytotoxin

Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of gastric cancer and peptic ulcer disease. H. pylori secretes a toxin, VacA, that targets human gastric epithelial cells and T lymphocytes and enhances the ability of H. pylori to colonize the stomach in a mouse model. To examine how VacA contributes to H. pylori colonization of the mouse stomach, we investigated whether murine T lymphocytes were susceptible to VacA activity. VacA inhibited interleukin-2 (IL-2) production by a murine T-cell line (LBRM-33), similar to its effects on a human T-cell line (Jurkat), but did not inhibit IL-2 production by primary murine splenocytes or CD4+ T cells. VacA inhibited activation-induced proliferation of primary human CD4+ T cells but did not inhibit the proliferation of primary murine CD4+ T cells. Flow cytometry studies indicated that the levels of VacA binding to primary murine CD4+ T cells were significantly lower than levels of VacA binding to human CD4+ T cells. This suggests that the resistance of primary murine CD4+ T cells to VacA is attributable, at least in part, to impaired VacA binding to these cells.     

Vacuolating Cytotoxin of Helicobacter pylori Induces Apoptosis in the Human Gastric Epithelial Cell Line AGS

Helicobacter pylori induces cell death by apoptosis. However, the apoptosis-inducing factor is still unknown. The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS. The supernatant from the vacA wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not. That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA. Furthermore, a recombinant VacA produced in Escherichia coli was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation. These findings demonstrate that the vacuolating cytototoxin of H. pylori is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.     

Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells.

Many Helicobacter pylori strains produce a cytotoxin (VacA) that induces vacuolation in epithelial cells. In this study, binding and internalization of the cytotoxin by HeLa or AGS (human gastric adenocarcinoma) cells were characterized by indirect fluorescence microscopy. Cells incubated with the cytotoxin at 4 degrees C displayed a uniform fluorescent plasma membrane signal. Preincubation of the cytotoxin with either rabbit antiserum to approximately 90-kDa H. pylori VacA or sera from H. pylori-infected persons inhibited its binding to cells and blocked its capacity to induce cytoplasmic vacuolation. Recombinant VacA fragments (approximately 34 and approximately 58 kDa), corresponding to two proteolytic cleavage products of approximately 90-kDa VacA, each bound to the plasma membrane of HeLa cells. Antiserum reactive with the approximately 58-kDa VacA fragment inhibited the binding of native H. pylori cytotoxin to cells and inhibited cytotoxin activity, whereas antiserum to the approximately 34-kDa fragment had no effect. When incubated with cells at 37 degrees C for > or = 3 h, the H. pylori cytotoxin localized intracellularly in a perinuclear location but did not localize within cytotoxin-induced vacuoles. When cells with previously bound cytotoxin were incubated with anticytotoxin serum at 4 degrees C and then shifted to 37 degrees C, vacuolation was completely inhibited. Bound cytotoxin became inaccessible to the neutralizing effects of antiserum after 60 to 120 min of incubation with cells at 37 degrees C. These data suggest a model in which (i) VacA binds to cells primarily via amino acid sequences in its 58-kDa fragment, (ii) VacA internalization occurs slowly in a temperature-dependent process, and (iii) VacA interacts with an intracellular target.     

Serological assays for identification of human gastric colonization by Helicobacter pylori strains expressing VacA m1 or m2

The Helicobacter pylori vacA gene encodes a secreted protein (VacA) that alters the function of gastric epithelial cells and T lymphocytes. H. pylori strains containing particular vacA alleles are associated with differential risk of disease. Because the VacA midregion may exist as one of two major types, m1 or m2, serologic responses may potentially be used to differentiate between patients colonized with vacA m1- or vacA m2-positive H. pylori strains. In this study, we examined the utility of specific antigens from the m regions of VacA as allele-specific diagnostic antigens. We report that serological responses to P44M1, an H. pylori m1-specific antigen, are observed predominantly in patients colonized with m1-positive strains, whereas responses to VacA m2 antigens, P48M2 and P55M2, are observed in patients colonized with either m1- or m2-positive strains. In an Asian-American population, serologic responses to VacA m region-specific antigens were not able to predict the risk of development of gastric cancer.     

Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.