Implication of different effector mechanisms by cord blood–derived and peripheral blood–derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines

Background aimsCytokine-induced killer (CIK) cells ex vivo–expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56⁺ CIK cells (CD3⁺CD56⁺ and CD3⁻CD56⁺) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56⁺ PB and CB-CIK cells to kill B-ALL cell lines have not been studied.MethodsCB and PB-CIK cells were differentiated. CD56⁺ CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines.ResultsWe found that cytotoxic granule proteins were expressed at higher levels in CD56⁺ PB-CIK than in CD56⁺ CB-CIK cells. However, CD56⁺ CB-CIK cells expressed more tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) compared with CD56⁺ PB-CIK cells. We observed that CD56⁺ CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56⁺ PB-CIK cells that used only the NKG2D pathway. In contrast, CD56⁺ PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56⁺ CB-CIK cells used only the NKG2D pathway.ConclusionsOur results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.     

Autologous bone marrow–derived CD133 cells with core decompression as a novel treatment method for femoral head osteonecrosis: a pilot study

Background

Avascular necrosis (AVN) of femoral head is a progressive bone disease due to ischemia of femoral head; patients experience pain and they can not do normal activity. There is not an effective way to treat the cause of this disease. In recent studies, treatment of this disease using pluripotent stem cell–derived mesenchyme is safe and effective, but this method needs more investigation. In this study, the safety and efficacy of CD133+ cells were evaluated as a novel method of stem cell therapy to treat AVN.

Impairment of Vα24-Jα18+Vβ11+ natural killer T cells in adult acute lymphoblastic leukemia patients

Type I natural killer T (NKT) cells are attractive candidates for cancer immunotherapy. In this study, we examined the characteristics of type I NKT cells in patients with adult B-cell acute lymphoblastic leukemia (ALL). We first identified type I NKT cells as Vα24-Jα18 and Vβ11 double-positive CD3⁺ lymphocytes. Using this method, we found that the adult B-cell ALL patients presented significantly lower level of type I NKT cells than the age- and sex-matching control subjects. The expression of IL-21 by type I NKT cells was then examined using intracellular flow cytometry, which showed that with α-GalCer stimulation, the adult B-cell ALL patients presented significantly lower level of IL-21⁺ type I NKT cells than control subjects. By both flow cytometry and ELISA, we found that the vast majority of IL-21-expressing type I NKT cells expressed IL-21R, which was also reduced in adult B-cell ALL patients. Using an in vitro co-culture system, we demonstrated that IL-21R⁺, but not IL-21R^-, type I NKT cells could promote the IFN-γ, granzyme B, and perforin expression by CD8 T cells in an IL-21-dependent fashion. This type I NKT cell-mediated stimulatory effect was reduced in adult B-cell ALL patients than in control subjects. In addition, we observed a positive correlation between the frequency of IL-21R⁺ type I NKT cells and the frequencies of IFN-γ-, granzyme B-, and perforin-expressing circulating CD8 T cells in adult B-cell ALL patients directly ex vivo. Overall, this study identified an IL-21-related impairment in type I NKT cells from adult B-cell ALL patients.     

The in vitro growth of a cord blood–derived cell population enriched for CD34+ cells is influenced by its cell cycle status and treatment with hydroxyurea

Objective

Cell cycle plays a fundamental role in the physiology of hematopoietic stem and progenitor cells. In the present study we used a negative selection system to obtain an immature cell population—enriched for cord blood–derived CD34⁺ cells—and we determined its proliferation, expansion and differentiation patterns as a function of the cell cycle status. The effects of hydroxyurea (HU) were also assessed.

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

YKL-40、CD4+/CD8+比值与腺病毒肺炎患儿炎性因子和并发喘息的关系研究

摘要 目的：探讨血清壳多糖酶3样蛋白1（YKL-40）、外周血CD4⁺/CD8⁺比值与腺病毒肺炎（AP）患儿炎性因子和并发喘息的关系。方法：选取2019年10月～2022年10月湖北省妇幼保健院收治的97例AP患儿为AP组,根据是否并发喘息分别为喘息组和无喘息组,另选取同期50例体检健康儿童为对照组。收集AP患儿的临床资料,采用酶联免疫吸附法检测血清YKL-40和炎性因子[白细胞介素（IL）-6、IL-8、肿瘤坏死因子-α（TNFα）]水平,流式细胞术检测外周血CD4⁺、CD8⁺比例并计算CD4⁺/CD8⁺比值。采用Spearman相关性分析AP患儿血清YKL-40、CD4⁺/CD8⁺比值与炎性因子水平的相关性,多因素Logistic回归分析AP患儿并发喘息的影响因素。结果：与对照组比较,AP组血清YKL-40、外周血CD8⁺比例升高,CD4⁺比例、CD4⁺/CD8⁺比值降低（P＜0.05）。AP组血清IL-6、IL-8、TNF-α水平高于对照组（P＜0.05）。Spearman相关性分析显示,AP患儿血清YKL-40与IL-6、IL-8、TNF-α水平呈正相关,外周血CD4⁺/CD8⁺比值与IL-6、IL-8、TNF-α水平呈负相关（P＜0.05）。97例AP患儿住院期间喘息发生率为50.52%（49/97）。多因素Logistic回归分析显示,呼吸衰竭、小气道病变、特应性体质和血清IL-6、IL-8、TNF-α、YKL-40升高为AP患儿并发喘息的独立危险因素,外周血CD4⁺/CD8⁺比值升高为独立保护因素（P＜0.05）。结论：AP患儿血清YKL-40水平升高和外周血CD4⁺/CD8⁺比值降低,与炎性因子水平升高和并发喘息密切相关。     

人慢性胃炎粘膜内CD3+细胞,S-100+树突状细胞和nNOS表达的意义

采用免疫细胞化学方法探讨胃粘膜内CD3+细胞,S-100+树突状细胞和nNOS的表达与慢性胃炎的关系及意义.检测标本均取自胃窦部活检的胃粘膜组织.结果显示CD3+细胞主要分布于粘膜上皮、腺上皮和固有膜内,而S-100+树突状细胞则主要位于固有膜内,正常组与浅表性胃炎组和萎缩性胃炎组,浅表性胃炎组与萎缩性胃炎组细胞数量有显著性差异( P<0.01),nNOS阳性反应主要位于粘膜上皮和腺上皮的基底部,但各组之间nNOS的表达程度不同,特别是萎缩性胃炎与浅表性胃炎有显著性差异(P<0.01),我们认为,对CD3+ 细胞,S-100 +树突状细胞和nNOS的检测,不仅有助于判断胃炎的病变程度和临床疗效,而且也为胃炎的治疗提供新的启示.     

2型糖尿病并发肺结核患者PCT、HMGB1、CD4+/CD8+比值与继发肺部感染的关系

摘要 目的：探讨2型糖尿病并发肺结核患者降钙素原（PCT）、高迁移率族蛋白1（HMGB1）、CD4⁺/CD8⁺比值与继发肺部感染的关系。方法：选择2019年1月至2022年6月四川大学华西医院呼吸与危重症医学科收治的97例2型糖尿病并发肺结核患者,根据入院治疗时是否继发肺部感染分为肺部感染组（53例）及非肺部感染组（44例）。检测两组血清PCT、HMGB1水平以及外周血CD4⁺/CD8⁺比值。单因素和多因素Logistic回归分析2型糖尿病并发肺结核患者继发肺部感染的因素。受试者工作特征（ROC）曲线分析PCT、HMGB1和CD4⁺/CD8⁺比值预测2型糖尿病并发肺结核患者继发肺部感染的价值。结果：肺部感染组血清PCT、HMGB1水平高于非肺部感染组（P＜0.05）,外周血CD4⁺/CD8⁺比值低于非肺部感染组（P＜0.05）。糖化血红蛋白及血清PCT、HMGB1水平升高是2型糖尿病并发肺结核患者继发肺部感染的危险因素（P＜0.05）,高CD4⁺/CD8⁺比值是保护因素（P＜0.05）。PCT、HMGB1、CD4⁺/CD8⁺比值预测2型糖尿病并发肺结核患者继发肺部感染的曲线下面积为0.719、0.761、0.738,联合PCT、HMGB1和CD4⁺/CD8⁺比值预测的曲线下面积为0.878,高于各指标单独预测。结论：2型糖尿病并发肺结核患者血清PCT、HMGB1水平增高,外周血CD4⁺/CD8⁺比值降低,均与继发肺部感染有关,PCT、HMGB1联合CD4⁺/CD8⁺比值可辅助预测2型糖尿病并发肺结核患者继发肺部感染的风险。     

益生菌联合布地奈德对支气管哮喘患儿的疗效及对外周血CD4+和CD8+细胞水平的影响

研究益生菌联合布地奈德对支气管哮喘患儿的疗效及对外周血CD4⁺和CD8⁺细胞水平的影响, 为该类患者的治疗提供参考。

选择2019年6月至2020年5月我院收治的92例支气管哮喘患儿为研究对象, 按照随机数字表法分为观察组(益生菌联合布地奈德治疗)和对照组(布地奈德治疗)各46例, 比较两组患儿疗效、临床症状消失时间、治疗前及治疗4周后T淋巴细胞(CD4⁺、CD8⁺)和肺功能[用力肺活量(FVC)、第1秒用力呼气容积(FEV1)、最大呼气峰流速(PEF)]水平。

治疗4周后, 两组患儿总有效率比较差异无统计学意义(93.48%vs 89.13%, χ²=0.137, P=0.711)。观察组患儿咳嗽消失时间、气促消失时间、哮鸣音消失时间、憋喘消失时间均显著低于对照组(均P < 0.05)。治疗4周后, 观察组患儿CD4⁺细胞水平显著低于治疗前及同期对照组, CD8⁺细胞、肺功能(FVC、FEV1、PEF)水平显著高于治疗前及同期对照组(均P < 0.05)。

益生菌联合布地奈德对支气管哮喘的疗效显著, 能够促进患儿临床症状恢复, 改善患儿肺功能, 下调外周血CD4⁺细胞水平, 上调CD8⁺细胞水平。

     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

可溶性Jagged-1/Fc嵌合蛋白诱导淋巴结细胞向CD4+CD25+ T细胞分化

直接用可溶性Jagged-1/Fc嵌合蛋白(Jagged-1/Fc)在体外诱导小鼠淋巴结细胞向CD4 CD25 T细胞分化.通过荧光标记单克隆抗体染色结合流式细胞术,观察不同剂量Jagged-1/Fc在不同时间对淋巴结细胞向CD4 CD25 T细胞分化的影响,观察Jagged-1/Fc诱导T细胞内细胞因子的变化;藉ELISA法检测Jagged-1/Fc诱导分化的T细胞分泌TGF-β1、IL-4和IL-10的水平.结果显示,超过500.0μg/L剂量的Jagged-1/Fc使CD4 CD25 T细胞百分比明显增高,诱导时间需要4～6天,抗Jagged-1单抗能抵消Jagged-1/Fc的诱导作用,用DAPT阻断Notch信号通路的活化也能抑制Jagged-1/Fc的诱导作用,Jagged-1/Fc诱导分化的T细胞培养上清中IL-4和IL-10的水平明显增高,TGF-β1无明显变化,胞内IL-4,IL-10,IL-2和TNF-α的水平也呈增高趋势.上述结果表明,可溶性Jagged-1/Fc嵌合蛋白在体外可诱导小鼠淋巴结细胞向CD4 CD25 调节性T细胞分化.     

Enhancement of proliferation of human umbilical cord blood–derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB–HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34⁺ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.     

传染性单核细胞增多症患儿NLR、CD4+/CD8+比值、ADA与EBV-DNA载量的相关性分析及对肝损害的影响

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血中性粒细胞/淋巴细胞比值（NLR）、CD4⁺/CD8⁺比值、腺苷脱氨酶（ADA）与EB病毒（EBV）-脱氧核糖核酸（DNA）载量的相关性,分析其对IM患儿肝损害的影响。方法：选择2019年1月至2022年4月我院儿科收治的102例IM患儿（IM组）,另选择同期我科收治的95例EB病毒检测阴性的发热患儿（非IM组）和体检健康的73例健康儿童（对照组）。根据是否发生肝损害将IM患儿分为肝损害组（61例）和非肝损害组（41例）。比较外周血NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量,Pearson法分析NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量的相关性。多因素Logistic回归分析IM患儿发生肝损害的影响因素。结果：IM组ADA高于非IM组和对照组（P＜0.05）,且非IM组高于对照组（P＜0.05）,NLR、CD4⁺/CD8⁺比值低于非IM组和对照组（P＜0.05）,且非IM组低于对照组（P＜0.05）,IM组EBV-DNA载量高于非IM组（P＜0.05）。IM患儿ADA与EBV-DNA载量呈正相关（r=0.493,P＜0.05）,NLR、CD4⁺/CD8⁺比值与EBV-DNA载量呈负相关（r=-0.419、-472,P＜0.05）。肝损害组ADA、EBV-DNA载量高于非肝损害组（P＜0.05）,NLR、CD4⁺/CD8+⁺比值低于非肝损害组（P＜0.05）。肝脏肿大、高EBV-DNA载量、高ADA是IM患儿肝损害的危险因素（P＜0.05）,高NLR、高CD4⁺/ CD8⁺比值是保护因素（P＜0.05）。结论：IM患儿ADA增高,NLR、CD4⁺/CD8⁺比值降低,与EBV-DNA载量增加以及肝损害有关。     

视频脑电图联合PEWS评分、NSE、CD4+/CD8+比值对病毒性脑炎患儿病情评估及预后预测的临床价值

摘要 目的：探讨视频脑电图（VEEG）联合儿童早期预警评分（PEWS）、神经元特异性烯醇化酶（NSE）、CD4⁺/CD8⁺比值对病毒性脑炎患儿病情评估及预后预测的价值。方法：选择2020年3月至2022年3月南京医科大学附属儿童医院收治的152例病毒性脑炎患儿，根据病情严重程度将患儿分为重症组（67例）和轻症组（85例），另选择72例无神经系统损伤住院患儿为对照组。治疗2周后，根据儿童格拉斯哥预后量表（CGOS）将其分为预后良好组（4~5级，89例）与预后不良组（1~3级，63例）。所有研究对象均接受PEWS测评和VEEG检查，检测血清NSE水平，计算CD4⁺/CD8⁺比值。采用单因素和多因素Logistic回归分析影响病毒性脑炎患儿预后的因素。采用受试者工作特征（ROC）曲线分析PEWS、VEEG 及血清NSE、CD4⁺/CD8⁺比值预测病毒性脑炎患儿预后的价值。结果：重症组PEWS、VEEG重度异常比例及血清NSE水平高于轻症组和对照组，CD4⁺/CD8⁺比值低于轻症组和对照组（P＜0.05）；轻症组PEWS、VEEG重度异常比例及血清NSE水平高于对照组，CD4⁺/CD8⁺比值低于对照组（P＜0.05）。预后不良组PEWS、VEEG重度异常比例、血清NSE水平高于预后良好组，CD4⁺/CD8⁺比值低于预后良好组（P＜0.05）。多因素Logistic回归分析结果显示，持续惊厥、高PEWS、VEEG重度异常、血清NSE水平升高是病毒性脑炎患儿预后不良的危险因素，CD4⁺/CD8⁺比值升高是保护因素（P＜0.05）。联合PEWS、VEEG 及血清NSE、CD4⁺/CD8⁺比值预测病毒性脑炎患儿预后曲线下面积为0.859，高于各指标单独预测。结论：病毒性脑炎患儿高PEWS、VEEG重度异常、血清NSE水平升高、CD4⁺/CD8⁺比值降低，与病情加重和预后不良有关，联合以上四项指标辅助预测病毒性脑炎患儿预后的价值较高。     

小鼠血管壁CD34+干细胞的分离、培养及鉴定

血管壁干细胞(vascular wall-resident stem cells, VW-SCs)在维持血管正常功能以及损伤血管的修复过程中发挥着关键性的作用,对其功能特性的研究将有助于干细胞的调控及应用。本文旨在建立稳定的VW-SCs分离、培养及分选鉴定技术,为进一步深入研究VW-SCs在生理和疾病状态下的增殖、迁移和分化等机制提供丰富、可靠的细胞来源。首先利用组织块贴壁法获取小鼠主动脉外膜以及肠系膜动脉血管壁细胞,传代培养到细胞数量至少达到1×10⁷后经磁珠分选和流式细胞术鉴定CD34⁺的VW-SCs;其次采用细胞免疫荧光染色检测干细胞标志物(CD34、Flk-1、c-kit、Sca-1)以及平滑肌标志物(SM22、SM MHC)、内皮标志物(CD31)和细胞核内分裂增殖相关蛋白(Ki-67);血管壁CD34+干细胞的定向分化采用EBM-2内皮分化诱导培养基和FM-2成纤维细胞培养基分别培养7天和3天;利用细胞免疫荧光染色和q-PCR检测内皮细胞标志物(CD31, VWF)和成纤维细胞标志物(Vimentin, PDGFRα)的表达。此外...     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34⁺富集细胞在SCF+IL-3+IL-6+FL+Tpo细胞因子组合下的体外扩增特性,发现：CD34⁺富集细胞具有很高的扩增潜力,在本实验条件下其总细胞持续扩增了8周,扩增倍数达31270.9±8640.5倍;而MNC在培养至第4周扩增就已呈现下降趋势,最大仅扩增了53.3±6.2倍。对比集落和CD34⁺细胞的扩增发现,MNC的集落密度和CD34⁺细胞含量由第0天至第7天有一个上升的过程,而CD34⁺富集细胞在培养过程中,集落密度和CD34⁺细胞含量却始终呈下降趋势。在体外培养过程中,CD34⁺富集细胞的CFU-GM和CD34⁺细胞最大分别扩增了185.7±14.1和191.7±188.8倍,明显高于MNC的12.4±3.2和50.6±33.2倍;而CD34⁺富集细胞和MNC的BFU-E则只实现了少量扩增,分别为7.2±5.2和10.1±3.4倍。结果显示,从CD34⁺富集细胞出发扩增造血干/祖细胞,可以得到更多的CD34⁺细胞和CFU-GM集落形成细胞。      

CD4+CD25+Foxp3hi细胞在夏氏疟原虫感染DBA/2小鼠作用研究

利用调节性T细胞消除的致死型夏氏疟原虫(Plasmodium chabaudi chabaudi AS,P.c chabaudi AS)感染鼠疟模型,探讨DBA/2小鼠对P.c chabaudi AS感染易感性的原因。DBA/2小鼠对P.c chabaudi AS易感,伴随原虫血症增加CD4+CD25+Foxp3+细胞数量明显增加,且以CD4+CD25+Foxp3hi增加更为明显。原虫血症达峰值时CD4+CD25+Foxp3hi细胞数量亦达到峰值。相比,Treg消除鼠的原虫出现时间和疟血症峰值时间均明显延迟,且在疟血症达峰值前(5～8 d)原虫血症水平明显低于对照组。与之相应,CD4+CD25+Foxp3hi细胞数量明显处于低水平。同时,Treg消除鼠生存期明显延长。由此提示,P.c chabaudi AS感染导致Foxp3表达增加,扩增的CD4+CD25+Foxp3hi细胞有利于疟原虫复制和逃避宿主免疫应答,进而影响疟疾感染的进程和最终结局。     

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i+ ,K i+ -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na⁺,K⁺-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na⁺,K⁺-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of ⁸⁶Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na⁺,K⁺-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na⁺]_i/[K⁺]_i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na⁺,K⁺-ATPase-mediated Na⁺ and K⁺ fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio.