Detection of clonal T-cell receptor-gamma gene rearrangement by PCR/temporal temperature gradient gel electrophoresis

Limited combinatorial and junctional diversity in TCR-gamma gene rearrangement can result in amplification products that are difficult to interpret when analyzed by conventional gel electrophoresis methods that separate DNA based on size (polymerase chain reaction [PCR]/polyacrylamide gel electrophoresis [PAGE]). We describe a simple approach to the detection of clonal TCR-gamma gene rearrangement using temporal temperature gradient gel electrophoresis (TTGE) that uses a gradual and uniform increase in the temperature of a constant denaturing gel to resolve different DNA molecules based on base pair composition. We tested 42 clinical specimens (30 blood specimens and 12 formalin-fixed paraffin-embedded tissues) for T-cell clonality by PCR/PAGE and PCR/TTGE. Concordant results were obtained in only 22 specimens (52%). Of the 20 discordant cases, 18 samples were positive by TTGE and negative by PAGE. For all of the discordant cases, the TTGE yielded results that correlated better with the clinical data than did the PAGE method. We conclude that PCR/TTGE is more accurate and easier to perform than current methods for detecting clonal populations of T cells.     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

Multiparametric assessment of bursal lymphocyte apoptosis.

When bursal lymphocytes are placed in cell culture, they undergo an apoptotic form of cell death that can be inhibited by phorbol esters and protein synthesis inhibitors. The goal of the current study was to evaluate the time course of this process and the inhibition of this process using several different assays to detect apoptosis: (1) terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) of lymphocyte DNA strand breaks with dUTP-FITC; (2) propidium iodide (PI) staining of lymphocyte chromatin; (3) chloromethyl-x-rosamine (CMX-Ros) binding to lymphocyte mitochondria; (4) merocyanine-540 (MC-540) binding to the lymphocyte plasma membrane; (5) flow cytometric analysis of light scatter from lymphocytes; (6) analysis of genomic DNA from lymphocytes by agarose gel electrophoresis; and (7) cellular caspase-3 activity of lymphocytes. When bursal lymphocyte apoptosis was analyzed as a function of time, or inhibited by phorbol esters or cycloheximide, all of these assays corroborated the apoptotic process. However, treatment of lymphocytes with a cytotoxic level of the proteinase inhibitor, n-ethylmaleimide (NEM) resulted in a putative, necrotic form of cell death that revealed discrepancies among the various assays in the detection of apoptotic cells. Specifically, the CMX-Ros and MC-540 assays erroneously detected the necrotic cells as being apoptotic cells following NEM treatment. These findings indicate the need for additional assays and appropriate treatment controls to verify the apoptotic process when using the CMX-Ros and MC-540 assays.     

已烯雌酚诱导T细胞肿瘤Jurkat细胞系凋亡中转录因子Oct-1的表达

研究己烯雌酚对Ｔ细胞肿瘤的细胞凋亡诱导作用,以及凋亡过程中转录因子Ｏｃｔ－１的表达,方法采用ＤＮＡ梯形片段化,荧光染色流式细胞技术分析细胞凋亡,以电泳泳 动度迁移率法检测Ｏｃｔ－１的表达。结果己烯雌酚可诱导Ｔ细胞出现细菌凋亡,并有典型的ＤＮＡ梯形片段化,１０μｇ己烯雌酚诱导１２ｈ后,凋亡过程中细胞数达３０％以上。己烯雌酚诱导Ｔ细胞凋亡过程与Ｏｃｔ－１转录因子的表达有关。结论己烯雌酚可诱导Ｔ细胞肿瘤     

Immune deficiency following thermal trauma is associated with apoptotic cell death

Thermal injury-associated specific immune deficiency occurs despite indicators of systemic activation of the lymphoid compartment. We investigated the possibility that postburn immune failure and T cell activation are causally related through activation-induced (apoptotic) cell death. The relationship between the cellular immune response and cell mortality was examined in cultures of peripheral blood mononuclear cells (PBMC) from 14 immunosuppressed patients with extensive burns (35–90% total body surface area). Impaired cellular immunity coincided with significantly reduced cell viability as ascertained by propidium iodide staining and dye reduction assays. Following stimulation with the mitogenic lectin, phytohemagglutinin (PHA), the majority of DNA in patient cultures was fragmented, suggesting the occurrence of apoptotic cell death. Even without stimulation a portion of patient cells was apoptotic as indicated by oligonucleosomal bands on agarose gel electrophoresis. Exogenous interleukin-2 or phorbol ester markedly reduced constitutive as well as PHAinduced DNA fragmentation.In situ demonstration of DNA strand breaks in freshly isolated patient PBMC, by a TdT-based labeling technique, confirmed that a larger fraction (up to 60%) of circulating lymphocytes was undergoing apoptosis on the periphery. These novel observations suggest that apoptosis may play a major role in thermal injury-related cellular immunodeficiency.     

Enterovirus 71 infection induces apoptosis in Vero cells

The effects of Enterovirus 71 (HEV71) infection on African green monkey kidney cells (Vero) were investigated. It was found that the infected cells showed progressive cellular morphological changes characteristic in apoptotic cells within 10 hours post-infection. The number of apoptotic cells correlated significantly with the number of HEV71 antigen positive cells when cells were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and stained for HEV71 antigen. Approximately 11, 26, 45 and 50% of the infected cells were apoptotic at 12, 24, 48 and 72 hours post-infection, respectively. Internucleosomal DNA fragmentation, characteristic in the late stage of apoptosis was noted beginning on day 2 post-infection. The DNA fragmentation, however, was absent in cells treated with the heat- and ultraviolet light-inactivated virus inocula. These results demonstrate the capacity of HEV71 to induce apoptosis in the infected cells. The induction, however, requires high level of HEV71 infectivity and the presence of live virus particles, suggesting the need for the presence of specific viral proteins for apoptosis to occur.     

Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.     

hTERT基因反义核酸对顺铂诱导Jurkat细胞凋亡的影响

目的:用人类端粒酶逆转录酶(hTERT)基因的反义寡核苷酸(ASODN)抑制Jurkat细胞端粒酶活性后, 观察化疗药物(顺铂、柔红霉素、长春新碱、足叶乙甙)对Jurkat细胞凋亡的影响。方法:采用台盼蓝拒染法观察hTERT ASODN与化疗药物(顺铂、柔红霉素、长春新碱、足叶乙甙)联合作用对Jurkat细胞系生长的影响;姬姆萨染色法观察凋亡细胞的形态变化;琼脂糖凝胶电泳分析细胞凋亡的DNA断裂情况;通过流式细胞仪对细胞凋亡峰进行定量分析。结果: hTERT ASODN作用于Jurkat细胞24 h再加入柔红霉素、长春新碱、足叶乙甙, 对细胞生长的抑制分别与单用柔红霉素、长春新碱、足叶乙甙及hTERT正义寡核苷酸(sense oligodeoxynucleotide, SODN)联合柔红霉素、长春新碱、足叶乙甙组相比, 无显著差异(P>0.05) 。hTERT ASODN作用于Jurkat细胞24 h再加入顺铂, 分别与SODN联合顺铂作用组、单用顺铂作用组相比, 对细胞抑制明显增强(P<0.05)。hTERT ASODN作用于Jurkat细胞24 h再加入顺铂作用48 h, 细胞出现典型的凋亡形态学改变, 经琼脂糖凝胶电泳即可见到DNA梯形条带, 而SODN与顺铂联合作用组及单独应用顺铂均未见到DNA梯形条带。hTERT ASODN与2.5 μmol/L顺铂联合作用于Jurkat细胞48h的凋亡细胞百分率(25.18±3.63)%分别同SODN与顺铂联合作用组(10.07±2.12)%、单用顺铂作用组(9.73±2.43)%进行比较有显著差异(P<0.01)。 结论:hTERT基因反义寡核苷酸能促进顺铂诱导Jurkat细胞凋亡。     

A cysteine protease from<Emphasis Type="Italic"> Taenia solium</Emphasis> metacestodes induce apoptosis in human CD4+ T-cells

Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.     

Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp

To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.     

己烯雌酚诱导T细胞肿瘤Jurkat细胞系凋亡中转录因子Oct-1的表达(英文)

目的研究己烯雌酚对 T细胞肿瘤的细胞凋亡诱导作用 ,以及调亡过程中转录因子 Oct- 1的表达。方法采用DNA梯形片段化 ,荧光染色流式细胞技术分析细胞凋亡 ,以电泳泳动度迁移率法 (EMSA)检测 Oct- 1的表达。结果己烯雌酚 (5 .0 mg/L )可诱导 T细胞出现细胞凋亡 ,并有典型的 DNA梯形片段化 ,10μg己烯雌酚诱导 12 h后 ,凋亡过程中细胞数达 30 %以上。己烯雌酚诱导 T细胞凋亡过程与 Oct- 1转录因子的表达有关。结论己烯雌酚可诱导 T细胞肿瘤凋亡 ,并与转录因子 Oct- 1的表达有关。     

槲皮素对U937细胞系抑制增殖和诱导凋亡作用的研究

目的 探讨黄酮类化合物槲皮素（Que）对人类单核细胞白血病U937细胞系的抑制增殖和诱导凋亡的作用。方法 应用MTT法检测不同浓度槲皮素对U937细胞的增殖抑制作用；AO／PI荧光染色后倒置荧光显微镜下观察细胞形态学变化；琼脂糖凝胶电泳测定细胞DNA的片段化；应用流式细胞仪检测细胞凋亡率及细胞周期分布。结果槲皮素能明显抑制U937细胞增殖，并存在剂量-效应关系和时间-效应关系；诱导U937细胞出现凋亡所具有的形态学和生化特征；随着槲皮素浓度升高，凋亡细胞和坏死细胞比例增加；将细胞特异性地阻滞在S期，出现凋亡峰。结论 槲皮素能抑制U937细胞增殖，诱导细胞凋亡，并具有细胞周期特异性。     

Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons.

In the rat cerebellar slice preparation, exposure to hypoxia elicited by a 30 min exposure to artificial cerebrospinal fluid continuously gassed with 95% N(2): 5% CO(2) induced a characteristic type of toxicity of Purkinje cells (PCs) resembling excitotoxic-mediated dark cell degeneration (DCD). Morphologically, PCs exhibited marked rounded appearance with cytoplasmic darkening, nuclear condensation and cytoplasmic vacuoles. Using gel electrophoresis, genomic DNA obtained from the cerebellar slice exhibited fragmentation. However, PCs failed to exhibit apoptotic bodies or evidence of phagocytosis, spherical- or crescent-shaped chromatin aggregations or TUNEL-positive staining. Ultrastructural analyses of granule cells revealed the presence of apoptotic bodies and discrete spherical collection of chromatin clumping as well as phagocytosis suggesting that the oligonucleosomal-sized DNA fragments primarily were derived from granule cells. PC-elicited toxicity was attenuated significantly in the presence of the competitive AMPA and NMDA antagonists CNQX and APV, respectively. The present study extends the involvement of excitotoxic processes in mediating hypoxic-induced toxicity of PCs in postnatal rats and suggests, in contrast to DCD elicited by direct application of excitotoxic agents, that DCD associated with acute hypoxic insults in PCs does not resemble classical apoptosis.     

冬凌草甲素增强巨噬细胞对凋亡的U937细胞的吞噬作用

目的：研究具有诱导肿瘤细胞凋亡活性的冬凌草甲素，促进巨噬细胞对因凋亡的肿瘤细胞的吞噬作用。 方法： DNA凝胶电泳检测UV照射（2.4 J/cm2, 4 min）的人组织淋巴瘤U937细胞凋亡；Giemsa染色，Hoechst 33258染色，镜下检测计数吞噬作用。 结果： UV照射（2.4 J/cm2, 4 min）诱导U937细胞发生凋亡，琼脂糖凝胶电泳可见凋亡典型的DNA梯带。2.7 μmol·L^-1的冬凌草甲素具有增强U937分化的巨噬细胞对UV照射诱导凋亡的U937细胞的吞噬作用，并呈时间剂量依赖性，但对非特异性荧光颗粒的吞噬效果较弱。加入抗TNFα和抗IL-1β的抗体，培养12 h, 吞噬增强作用明显受抑制。冬凌草甲素在人外周血来源的巨噬细胞吞噬凋亡的U937细胞过程中同样发挥增强吞噬的效果。 结论： 冬凌草甲素可特异地增强巨噬细胞对凋亡的U937细胞的吞噬作用，其吞噬机制是通过诱导巨噬细胞TNFα和IL-1β的释放。     

Isolation of human rotaviruses in primary cultures of monkey kidney cells.

We succeeded in isolating human rotaviruses from the feces of gastroenteritis patients by using roller cultures of primary cynomolgus monkey kidney cells with trypsin in the maintenance medium but without concentration and trypsin treatment of the inocula at each passage level. These cells were found to be more sensitive than MA-104 cells (derived from fetal rhesus monkey kidney) for the propagation of human rotaviruses. Polyacrylamide gel electrophoresis of the genome RNA revealed that there were small differences in the migration pattern of the segments among all the strains isolated from 1976 to 1981. The cultivation of human rotaviruses in primary cell cultures might aid in developing a liver rotavirus vaccine.     

同型半胱氨酸对培养的人脐静脉内皮细胞凋亡的影响

目的:研究同型半胱氨酸(Hcy)对培养的人脐静脉内皮细胞(hUVEC)凋亡的影响。方法:原代培养hUVEC,通过Hoechst 33258荧光染色观察细胞核形态改变,琼脂糖凝胶电泳检测 DNA片段,流式细胞术检测Annexin V/PI联合标记的细胞凋亡率, Western blot检测P53、Bax的表达及比色法测定caspase 3活性。 结果: 同型半胱氨酸诱导培养的hUVEC出现明显的凋亡形态学改变。琼脂糖凝胶电泳检测可见明显的"DNA ladder"图谱。Hcy诱导凋亡细胞数明显增加。同时促进Bax、P53的表达,使caspase 3活性显著增强。结论: 同型半胱氨酸诱导培养的hUVEC凋亡。     

Apoptosis in cultured rat hepatocytes: the effects of tumour necrosis factor alpha and interferon gamma.

We investigated the cytotoxic effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF alpha and a combination of TNF alpha and IFN gamma exerted a cytotoxic effect, whereas IFN gamma showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF alpha or IFN gamma, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF alpha and IFN gamma was fragmented, and a set of bands of the '200 bp ladder', which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF alpha killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of IFN gamma, which by itself was also weakly cytotoxic.     

The tunel assay in the diagnosis of graft-versus-host disease: caveats for interpretation

Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.     

IGF—IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡的病理学研究

目的：探讨IGF-IR基因的反义硫代磷酸型寡核苷酸对胶质瘤细胞的形态学影响。方法：根据IGF-IRcDNA序列设计正义,反义寡核苷酸片段,并对其部分碱基进行硫代磷酸修饰。体外培养的胶质瘤细胞分别经正义寡核苷酸和反义寡核苷酸处理,应用倒置显微镜活细胞观察,HE染色光镜观察,透射电镜及DNA琼脂糖凝胶电泳等方法研究IGF-IR反义硫代磷酸型寡核苷酸诱导胶质瘤细胞凋亡作用。结果：经反义寡核苷酸转染的胶质瘤细胞中,呈现典型的凋亡形态学改变,凋亡细胞最早期表现为细胞体积,容量减少,染色质凝聚,继之染色质集聚于核膜下成7新月状或块状;细胞核内染色质可完全固缩成团呈“黑洞”样或萎缩的核破裂形成一些较小的膜包绕的球体位于胞质内,最后出泡形成凋亡小体,DNA琼脂糖凝胶电泳分析经反义寡核苷酸处理的胶质瘤细胞DNA降解片段,可见有明显的小分子量DNA梯状条带,而野生型和正义寡核苷酸处理的胶质瘤细胞未见DNA梯状条带。结论：IGF-IR所介导失发泌环路IGF-I/IGF-IR。在胶质瘤细胞增殖和维持恶性表型中起重要作用。IGF-IR反义硫代磷酸型寡核苷酸能诱导胶质瘤细胞凋亡。     

Correlation of apoptosis with comet formation induced by tea polyphenols in human leukemia cells.

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.