Benzene induces apoptosis of murine thymocytes in vivo

苯是一种常见的工业毒物，也普遍存在于环境中，大量接触可导致再障、免疫功能障碍和白血病等。本文研究了苯对小鼠胸腺细胞的影响。实验观察到苯处理的小鼠胸腺明显萎缩，且与苯呈剂量和时间依赖性。与对照组相比，苯致毒小鼠胸腺细胞有以下特点：（１）透射电镜显示细胞皱缩，细胞膜空泡化，核凝缩，呈典型的细胞凋亡特征；（２）ＤＮＡ琼脂糖凝胶电泳呈特征性梯状图谱，片段大小为１８０ｂｐ或其倍数；（３）ＤＮＡ裂解百分率与苯呈剂量和时间依赖性。实验表明苯可以引起小鼠胸腺细胞凋亡，提示苯可通过诱导淋巴细胞凋亡而引起免疫机能障碍。     

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis

The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolically 35S-labeled human neutrophils when used at low concentrations (< 100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) approaches, we found that VAA-I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA-I was also found to reverse the delaying effect of GM-CSF on neutrophil apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In contrast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself and does not alter the GM-CSF-induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA-1077, and z-VAD-FMK, only the latter (inhibitor of caspases-1, -3, -4, and -7) was found to inhibit VAA-I-induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 +/- 1.3 to 54 +/- 3.2% (n=4). Furthermore, we confirm that caspases are involved in VAA-I-induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase-3-dependent. Such degradation was reversed by the z-VAD-FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA-I is a caspase-dependent mechanism that does not involve tyrosine phosphorylation events, G-proteins, PKCs, and PLA2. In addition, we conclude that at least caspase-3 is involved. Correlation between VAA-I-induced neutrophil apoptosis and VAA-I-induced inhibition of de novo protein synthesis is discussed.     

Modulation of interleukin-15-induced human neutrophil responses by the plant lectrin Viscum album agglutinin-I.

The plant lectin Viscum album agglutinin-I (VAA-I) and the interleukin-15 (IL-15) cytokine are two molecules with potential therapeutic properties known to modulate neutrophil functions when used separately. This study was conducted in order to better understand the mode of action of VAA-I and to elucidate how VAA-I could modulate IL-15-induced neutrophil responses. We found that VAA-I cannot induce phosphorylation events in human neutrophils. However, it enhances phagocytosis by itself without altering IL-15-induced phagocytosis. VAA-I was found to reverse the ability of IL-15 to delay neutrophil apoptosis and this was correlated with an inhibition of IL-15-induced de novo protein synthesis. In addition, we also found that IL-15 cannot reverse or attenuate the caspase-induced gelsolin fragmentation observed during apoptosis as assessed by immunoblotting. We conclude that VAA-I can be used to modulate some, but not all, IL-15-induced neutrophil responses and that it acts independent of phosphorylation events.     

Anti-inflammatory effect of Viscum album agglutinin-I (VAA-I): induction of apoptosis in activated neutrophils and inhibition of lipopolysaccharide-induced neutrophilic inflammation in vivo

Viscum album agglutinin-I (VAA-I) is a plant lectin which possesses antitumoral properties. This lectin is also known for its immunostimulatory effects when used at low concentrations (1-100 ng/ml). We have demonstrated recently that VAA-I is a potent inducer of human neutrophil apoptosis in vitro when used at higher concentrations. The role of VAA-I on activated neutrophils has not so far been investigated and its potential proinflammatory properties in vivo are poorly documented. Herein, we demonstrated that VAA-I (1000 ng/ml) induces apoptosis in lipopolysaccharide (LPS)-treated human neutrophils in vitro as well as in murine neutrophils isolated from lipopolysaccharide (LPS)-induced neutrophil influx. Using this model, we found that administration of VAA-I (100 or 1000 ng/ml) did not induce an inflammatory response. However, when used at 1 or 10 ng/ml, VAA-I was found to significantly induce a transitory inflammatory response, based on an increased leucocyte infiltration (>98% neutrophils). Also, we found that VAA-I inhibits LPS-induced neutrophil influx when administered simultaneously with LPS. In such conditions, some characteristic apoptotic neutrophils were observed in the pouch. Unlike LPS, which increased the production of some cytokines, VAA-I (1 or 10 ng/ml) did not increase the production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1Ra, IL-1alpha, IL-beta, IL-8, IL-10 or IL-12 (p70) in human neutrophils. We conclude that VAA-I possesses the ability to induce apoptosis of preactivated neutrophils at a concentration that does not induce a proinflammatory response. Moreover, we conclude that VAA-I can inhibit a LPS-induced proinflammatory response in vivo. These data may provide new clinical perspectives in future mistletoe therapy and favour its potential utilization based on anti-inflammatory activity that at first appears contradictory with its use as immunostimulant.     

脱氧雪腐镰刀菌烯醇对小鼠胸腺细胞凋亡和增殖的影响

目的:探讨脱氧雪腐镰刀菌烯醇(DON)对小鼠胸腺细胞凋亡和增殖的影响。方法:以动物实验、形态学观察、DNA琼脂糖凝胶电泳、流式细胞术方法研究了不同剂量DON对小鼠胸腺细胞凋亡和增殖的影响及其量效关系。结果:FCM检测结果表明,不同浓度DON均能诱导并促进体内小鼠胸腺细胞凋亡,DON0.5mg/kg、1mg/kg、2mg/kg、4mg/kg、8mg/kg组胸腺细胞的平均凋亡百分率分别为6.35%±1.30%、8.30%±1.33%、8.89%±2.15%、10.70%±0.62%和12.54%±2.08%,在0.5mg/kg到8mg/kg的浓度范围内,随DON浓度的增高,胸腺细胞的凋亡率也相应增高,两者呈显著正相关(r=0.788,P＜0.01)。DNA琼脂糖凝胶电泳结果显示,大剂量(8mg/kg和4mg/kg)DON处理组小鼠胸腺细胞出现了凋亡特有的梯状条带。超微结构观察可见DON处理组小鼠胸腺部分细胞出现核染色质固缩凝聚、胞芽等细胞凋亡的超微结构特征表现。大剂量(8mg/kg和4mg/kg)DON可明显降低小鼠胸腺细胞增殖活性(用PI表示,P＜0.01)。结论:脱氧雪腐镰刀菌烯醇可剂量依赖地诱导并促进小鼠胸腺细胞的凋亡和抑制其增殖。     

PRAME induces apoptosis and inhibits proliferation of leukemic cells in vitro and in vivo

PRAME is a germinal tissue-specific gene that is expressed at high levels in haematological malignancies, but the physiological functions of PRAME in leukemia cells are unknown. It has reported that PRAME was found to be predominantly expressed in acute leukemias and high PRAME expression is correlated with a favorable prognosis in childhood acute leukemias, which suggested that PRAME could be involved in the regulation of cell death or apoptosis. In the present study, we tested a hypothesis that the PRAME gene plays a role in the regulation of apoptosis and proliferation of leukemia cells. We observed that KG-1 cells transient overexpressing the PRAME gene (when transfected with pcDNA3.1-PRAME plasmid) significantly induces apoptosis and decreases proliferation in vitro, and repression of PRAME expression by a short interfering RNA exhibited a increased proliferation in K562 cells in vitro and increases tumorigenicity of K562 leukemic cells in nude mice. Our results suggest that the leukemias expressing high levels of PRAME has favorable prognosis. PRAME may be as an attractive target for potential immunotherapy for acute leukemic.     

双歧杆菌表面分子对LPS在小鼠体内调节胸腺细胞凋亡的观察

目的研究双歧杆菌表面分子细胞壁肽聚糖（ＷＰＧ）、脂磷壁酸（ＬＴＡ）对ＬＰＳ体内诱导小鼠胸腺细胞凋亡的调节。方法用ＤＮＡ凝胶电泳、ＴＵＮＥＬ法检测ＷＰＧ、ＬＴＡ对ＬＰＳ体内诱导小鼠胸腺细胞凋亡的影响,并分别用生物活性法和Ｇｒｉｅｓｓ反应测定ＷＰＧ、ＬＴＡ、ＬＰＳ体外诱生ＴＮＦ－α、ＮＯ２－的含量。结果ＷＰＧ、ＬＴＡ可显著抑制ＬＰＳ体内诱导的小鼠胸腺细胞的凋亡。ＷＰＧ、ＬＴＡ单独刺激巨噬细胞时所诱生的ＴＮＦ－α、ＮＯ的量显著低于ＬＰＳ刺激时所产生的这两种活性介质的量,而ＷＰＧ、ＬＴＡ与ＬＰＳ共同应用时可显著降低ＬＰＳ诱导巨噬细胞产生的ＴＮＦ－α、ＮＯ的量;诱生型一氧化氮合成酶抑制剂Ｓ－甲基异硫脲硫酸盐体外可抑制巨噬细胞产生的ＮＯ的量,在体内可部分抑制ＬＰＳ诱导的小鼠胸腺细胞的凋亡。结论ＷＰＧ、ＬＴＡ与ＬＰＳ共同应用时,可抑制ＬＰＳ诱导的巨噬细胞产生的ＴＮＦ－α、ＮＯ的量,从而下调ＬＰＳ体内诱导小鼠胸腺细胞的凋亡     

Protein kinase A or C modulates the apoptosis induced by lectin II isolated from Korean mistletoe, Viscum album var. Coloratum, in the human leukemic HL-60 cells

Mistletoe lectins (MLs) are increasingly used as an anticancer drug in the treatment of human tumors. The cytotoxic activity of MLs against tumor cells is due to programmed cell death (apoptosis). The up-or down-regulation of protein kinas A (PKA) or C (PKC) is known to be associated with the regulation of drug-induced apoptosis. Previously, we isolated cytotoxic MLII from the extract of Korean mistletoe (Viscum album var. Coloratum) and characterized its biochemical properties. The present study was designed to investigate the role of PKA and PKC in ML II-induced apoptosis. Exposure of human leukemia HL-60 cells to various doses of ML II resulted in apoptosis. However, the treatment of these cells with dibutyl-cyclic AMP (DB-cAMP), PKA activator, or 12-O-tertadecanoyl phorbol 13-acetate (TPA), PKC activator, suppressed ML II-induced apoptosis. Furthermore, KT5720 and staurospoline, PKA and PKC inhibitors, respectively, reversed the suppression by DB-cAMP and TPA in the ML II-induced apoptosis of HL-60 cells. These results     

Anti-alpha 4 integrin antibody induces apoptosis in murine thymocytes and staphylococcal enterotoxin B-activated lymph node T cells.

We have shown that an antibody (9C10) to the alpha 4 integrin induces apoptosis in murine immature CD4+ CD8+ thymocytes and in activated (but not resting) mature lymph node T cells. In both cases, apoptosis is blocked by the highly selective protein kinase C (PKC) inhibitor Ro31.8425, suggesting that 9C10 induces signalling through the alpha 4 integrin resulting in PKC activation leading to apoptosis. Overall, our results indicate the potential role of the alpha 4 integrin-mediated interactions in apoptosis induction during T-cell development and following mature T-cell activation.     

The differentiation of murine thymocytes in vivo and in vitro.

The differentiation of lymphoid cells in the early foetal mouse thymus was studied in vivo and in organ culture. The lymphoid precursors found in the 13 and 14 day foetal thymus constituted about 50% of the total thymus cell population. These T precursors were large blast-like cells which already expressed the Thy-1 antigen but were mostly TL negative. Both in vivo and in vitro the blasts were rapidly replaced by a population of typical small lymphocytes which were strongly Thy-1 and TL positive but Ig negative. In organ cultures grown under optimal conditions, large bimodal increases in cell numbers occurred. An initial population of about 2 x 10(4) T precursors per thymus lobe gave rise to nearly 10(6) predominantly Thy-1 and TL positive small lymphocytes by the 6--8th day in vitro. After this time lymphocyte numbers decreased until about the 10th day when they again increased to form a second peak of small lymphocytes on the 12--13th day. These cells were also predominantly Thy-1 positive but the majority were now TL negative. No Ig positive B lymphocytes were detected either by immunofluorescence or by autoradiography using polyvalent anti-MIg sera and no plasma cells were detected by electronmicroscopy. At all times however, minor subpopulations of Thy-1 negative small cells were present. The production of small lymphocytes during the 1st week of culture was critically dependent on culture conditions and particularly on the batch of FCS used. The population developing during the 2nd week required less stringent conditions and was less dependent on FCS. The culture systems described should prove useful in the study of T-lymphocyte differentiation.     

右归丸对糖皮质激素诱导的胸腺细胞凋亡的保护作用

目的：探讨右归丸对激素诱导的小鼠胸腺细胞凋亡的保护作用及机制。方法：采用Annexin V／PI双染法检测糖皮质激素及右归丸干预后小鼠胸腺细胞凋亡比例变化；同时采用RT-PCR法检测凋亡相关基因Bcl-2、BaxmRNA的表达状态。结果：激素处理小鼠的胸腺细胞凋亡比例较正常对照小鼠明显上升（P〈0．01），右归丸干预后小鼠胸腺细胞凋亡率则明显下降（P〈0．01）；同时，激素处理小鼠胸腺细胞Bcl-2的表达下调，Bcl-2／BaxmRNA比值随之下降；而右归丸干预后Bcl-2mR-NA转录水平明显高于激素处理小鼠（P〈0．01），Bax的表达虽无明显改变，Bcl-2／Bax比值较激素处理小鼠明显增高（P〈0．05）。结论：右归丸可明显抑制激素诱导的胸腺细胞凋亡，其机制与逆转激素诱导的Bcl-2／Bax表达失衡密切相关。     

热应激诱导的胸腺细胞凋亡及相关分子表达

目的：探讨体外热应激诱导胸腺细胞凋亡与细胞表达Fas、Fas-L及HSP70的关系。方法：用流式细胞术检测体外热应激后再培养的胸腺细胞不同时间的凋亡率及Fas、Fas-L和HSP70表达阳性率。结果：体外热应激诱导胸腺细胞的凋亡率和Fas及Fas-L表达阳性率均呈时间依赖性增高,体外热应激诱导胸腺细胞Fas/Fas-L的表达与凋良心明显相关,全外热应激后,HSP70表达明显增高,在6h达到高峰,此后缓慢下降。结论：HPS70是热应激胸腺细胞损伤的标志,Fas与Fas-L是介导热应激诱导胸腺细胞凋亡的重要分子。     

The efficiency of Viscum album ssp. album and Hypericum perforatum on human immune cells in vitro

Viscum album L. ssp. album and Hypericum perforatum L. are used for the treatment of different diseases. In this study, the effects of these herbals on immune cells were assessed in vitro. The phagocytosis, candidacidal activity of neutrophils and adhesion function of epithelial cells were investigated. Also, the expression of the surface markers of lymphocytes was analyzed by flow cytometry. It was observed that V. album ssp. album increased phagocytic activity and candidacidal activity of neutrophils and decreased adhesion function of epithelial cells. We also observed that in human peripheral blood mononuclear cells stimulated by Viscum album L. ssp. album the levels of CD4(+)CD25(+) and CD8(+)CD25(+) T cells, CD69 expressions in the activated T lymphocytes and CD3(-)CD16(+)CD56(+) NK cells increased compared to the cells that were not stimulated by this herbal. Whereas CD4(+)CD25(+), CD8(+)CD25(+) T cells, CD 69 expression and CD3(-)CD16(+)CD56(+) Natural killer cells did not show any significant differences with the presence of Hypericum perforatum L. compared to the control group. Hypericum perforatum L. increased candidacidal activity of neutrophils and decreased adhesion function of epithelial cells. In the light of these findings, it is considered that these extracts may be used as an adjuvant treatment option for immune activation in immunosuppressed patients.     

Subpopulations of CD4- CD8- murine thymocytes: differences in proliferation rate in vivo and proliferative responses in vitro

Cell sorting and cytotoxic depletion procedures were used to subdivide the population of CD4- CD8- ("double-negative") thymocytes from adult CBA mice on the basis of expression of Ly-1, HSA (the "heat-stable antigen" M1/69 or B2A2), Pgp-1 glycoprotein, Thy-1, MEL-14 and the PC61 antigenic determinant on the IL2 receptor (IL2R). The level of dividing cells within these subsets was assessed by brief in vivo administration of [3H]-thymidine, followed by radioautography, or by flow cytometric cell cycle analysis after DNA staining. The capacity of the subsets to proliferate in culture, in response to stimulation with concanavalin A (Con A), or with phorbol myristate acetate (PMA) and the calcium ionophore ionomycin, was assessed in high cloning efficiency single-cell culture systems. In general, the proliferative response in culture was inversely related to the rate of cell division in vivo. Response of the double-negative subsets to Con A correlated with expression of the T cell antigen receptor complex; although a high cloning efficiency was obtained from the receptor-positive fractions, very few of the clones were cytotoxic. In particular, a major Ly-1+ HSA- Pgp-1+ double-negative subset, as well as minor Ly-1- HSA- Pgp-1+ subsets, contained very few cells in cycle in vivo, but showed a high cloning efficiency in both culture systems. Conversely, the other major double-negative subset, Ly-1- HSA+ Pgp-1-, included most of the cells in cycle, but showed a reduced cloning efficiency in response to PMA and ionomycin and failed to respond to Con A. The dividing cells within the Ly-1- HSA+ Pgp-1- group were strongly enriched in the IL2R- rather than in the IL2R+ subset, suggesting IL2 was not the growth factor maintaining their proliferation in vivo.     

The Efficiency of Viscum album ssp. album and Hypericum perforatum on Human Immune Cells In Vitro

Viscum album L. ssp. album and Hypericum perforatum L. are used for the treatment of different diseases. In this study, the effects of these herbals on immune cells were assessed in vitro. The phagocytosis, candidacidal activity of neutrophils and adhesion function of epithelial cells were investigated. Also, the expression of the surface markers of lymphocytes was analyzed by flow cytometry. It was observed that V. album ssp. album increased phagocytic activity and candidacidal activity of neutrophils and decreased adhesion function of epithelial cells. We also observed that in human peripheral blood mononuclear cells stimulated by Viscum album L. ssp. album the levels of CD4⁺CD25⁺ and CD8⁺CD25⁺ T cells, CD69 expressions in the activated T lymphocytes and CD3^-CD16⁺CD56⁺ NK cells increased compared to the cells that were not stimulated by this herbal. Whereas CD4⁺CD25⁺, CD8⁺CD25⁺ T cells, CD 69 expression and CD3^-CD16⁺CD56⁺ Natural killer cells did not show any significant differences with the presence of Hypericum perforatum L. compared to the control group. Hypericum perforatum L. increased candidacidal activity of neutrophils and decreased adhesion function of epithelial cells. In the light of these findings, it is considered that these extracts may be used as an adjuvant treatment option for immune activation in immunosuppressed patients.     

Butyric acid-induced apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells.

The purpose of this study was to examine the effect of butyric acid, an extracellular metabolite from periodontopathic bacteria, on apoptosis induction in murine thymocytes, splenic T cells, and human Jurkat T cells. Butyric acid significantly suppressed T-cell viability in both a concentration- and time-dependent fashion. The results of DNA fragmentation assay indicated that butyric acid rapidly induced apoptosis in thymocytes (with 1.25 mM butyric acid and 6 h after treatment) and in splenic T cells and Jurkat cells (with 2.5 mM butyric acid and 16 h after treatment). Incubation of thymocytes or Jurkat cells with 5 mM butyric acid for 21 h resulted in the typical ladder pattern of DNA fragmentation. Furthermore, Jurkat cells treated with 5 mM butyric acid showed the characteristic pattern of apoptotic cells such as chromatin condensation and hypodiploid nuclei. Experiments with fractionated subpopulations of splenic T cells revealed that DNA fragmentation was predominantly observed in CD4+ T cells. Butyric acid-induced apoptosis of thymocytes was decreased by the protein kinase inhibitors H7 and staurosporine. These inhibitors were less effective with similarly treated splenic T cells and Jurkat cells. These data suggest that butyric acid, one of the volatile fatty acids produced by periodontopathic bacteria and one that easily penetrates the oral mucosa, can modulate the immunoregulatory cell population in periodontal tissue by inducing T-cell death through apoptosis.     

5-azacytidine, a chemotherapeutic drug, induces TRAIL-mediated apoptosis in mouse thymocytes in vivo

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL.     

西格列汀抑制膀胱癌细胞增殖和诱导细胞凋亡

目的观察西格列汀对膀胱癌细胞增殖和凋亡的影响。方法西格列汀处理膀胱癌细胞T24及5637后,MTS检测细胞的增殖活性;Western blot检测细胞内组织蛋白酶B以及凋亡相关蛋白的表达变化。结果西格列汀显著抑制了膀胱癌细胞的增殖,经西格列汀处理后,T24及5637细胞内组织蛋白酶B的蛋白表达水平明显下降,凋亡相关蛋白PARP的剪切体表达水平升高。结论西格列汀抑制膀胱癌细胞增殖和诱导癌细胞凋亡,同时降低细胞内组织蛋白酶B的蛋白表达。     

白色念珠菌诱导小鼠胸腺细胞凋亡通路的探究

目的：探讨白色念珠菌诱导小鼠胸腺细胞凋亡的通路。方法：经小鼠尾静脉注射白色念珠菌后，用ELISA检测小鼠血清TNF-α水平；采用荧光分光光度计检测小鼠胸腺凋亡细胞caspase-3在不同时问组的活性变化；流式细胞仪检测小鼠胸腺细胞hax、p53、bcl-2基因产物水平。结果：TNF-α水平显著升高，caspase-3的活性明显增强，bax、p53基因表达上调。结论：白色念珠菌可能通过刺激机体TNF-α水平升高进而激活caspase-3，同时白色念珠菌上调bax、p53基因表达协同激活caspase-3，从而导致小鼠胸腺细胞凋亡。