Ca2+ mobilization in physiologically stimulated single T cells gradually increases with peptide concentration (analog signaling)

We have investigated Ca²⁺ mobilization in single T cells stimulated with their physiological ligand, i.e. antigenic peptide bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). Fibroblasts expressing I-E^d class II molecules were pulsed with a peptide derived from the λ2³¹⁵ immunoglobulin light chain. Onto such antigen-pulsed fibroblasts were sedimented cloned Th1 cells loaded with Fura-2. Changes in cytosolic Ca²⁺ concentration in single T cells were continually monitored by use of an imaging system based on fluorometry. Ca²⁺ mobilization was both peptide-specific and MHC-restricted. Within seconds of the initial APC-T cell contact, a Ca²⁺ spike could be observed. The Ca²⁺ response gradually declined over a 25-min period, during which oscillations were noted. Various parameters characterizing the magnitude of the Ca²⁺ response (latency, increase rate, max and mean Ca²⁺ increase, frequency and period of oscillations) all correlated with the amount of peptide used for pulsing the fibroblasts. Thus, Ca²⁺ mobilization in single T cells appears not to be an all or none phenomenon. Rather, activation is incremental (analog signaling), the degree of Ca²⁺ mobilization probably being related to the number of stimulatory peptide-MHC complexes on the surface of the APC. The extent of calcium mobilization and lymphokine production (interleukin (IL)-2, IL-3, interferon-γ) correlated, at least at the population level.     

妊娠肝内胆汁淤积症患者胎盘组织中血管内皮生长因子的表达

目的观察血管内皮生长因子(VEGF)在妊娠肝内胆汁淤积症(ICP)胎盘中的表达.方法采用免疫组织化学方法,检测25例正常妊娠胎盘(对照组)和25例ICP胎盘(ICP组)中VEGF的水平.结果 VEGF在正常妊娠胎盘和ICP胎盘中分布基本一致,分布在滋养细胞、血管及绒毛间质.在ICP组VEGF为轻度表达占76%,中度表达占24%,无重度表达;而在对照组VEGF轻度表达占24%,中度表达占44%,重度表达占32%.ICP组中VEGF表达明显低于对照组(P＜0.01).结论 VEGF在胎盘中主要由绒毛滋养细胞分泌,ICP胎盘中VEGF的减少可能与胎盘血管生成减少及胎盘滋养叶细胞浸入异常有关,在ICP发病中占有一定的地位.     

Effect of anti-interferon-γ and anti-interleukin-2 monoclonal antibody treatment on the development of actively and passively induced experimental allergic encephalomyelitis in the SJL/J mouse

SJL/J mice challenged with myelin basic protein (MBP) in complete Freund's adjuvant (CFA) developed only mild chronic-relapsing experimental allergic encephalomyelitis (EAE) with very low incidence. However, treatment of challenged mice with anti-infeferonγ (IFN-γ) monoclonal antibody (mAb) determined severe disease in all cases. Similarly, in passive EAE, the addition of anti-IFN-γ to the in vitro MBP-activated cells at the time of transfer led to significant disease exacerbation in all recipients. The disease enhancing effect was observed only when the mAb was given at the time of active challenge or of passive transfer, but not at later times. Anti-interleukin-2 (IL-2) antibody had only a marginal effect in the active induction, but drastically reduced the manifestations of passive EAE, even when mixed with a disease-enhancing dose of anti-IFN-γ. These findings support the notion that IL-2 is required for disease induction whereas IFN-γ plays a disease-limiting role early in the development of EAE.     

血管内皮生长因子和E26转录因子在乳腺癌中的表达及意义

目的 试图揭示血管内皮生长因子（VEGF）和E26转录因子（E26 transformation-specificl,ETS-1)在乳腺癌组织中的表达规律,探讨其在血管生成和肿瘤浸润转移中的作用机制。方法 应用原位杂交和免疫组织化学链霉素抗生物系－过氧化物酶复合物法（SP）法,检测48例乳腺癌组织中VEGF和ETS－1的mRNA和蛋白的表达。结果 乳腺癌细胞高表达VEGF mRNA和蛋白,阳性率分别为75％（36／48）,70．8％（34／48）,而血管内皮细胞几乎不表达;ETS－1既表达在乳腺癌细胞,也表达在血管内皮细胞。癌细胞中mRNA和蛋白表达阳性率分别为85．4％（41／48）,79．2％（38／48）;VEGF和ETS－1高表达组的血管密度明显高于低表达组（均P＜0．01）;VEGF和ETS－1的表达与组织学分级和淋巴结转移密切相关,并且高表达组的微血管密度明显高于低表达组（P＜0．01）。结论 VEGF和ETS－1可促进乳腺癌血管形成,同时也促进肿瘤的浸润和转移;检测VEGF和ETS－1的表达可做为乳腺癌恶性度,浸润转移等生物学行为的参考指标。     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.     

淋巴因子激活的NK细胞杀伤肿瘤细胞的免疫电镜观察

用胶体金标记的扫描与透射免疫电镜术观察ＣＤ１６＾＋淋巴因子激活的杀伤细胞杀伤肺腺癌细胞系ＬＴＥＰａ２或人红白血病细胞Ｋ５６２的过程。发现ＣＤ１６＾＋ＬＡＫ细胞能伸出分支的指状突起较深地插入肿瘤细胞浆内，造成靶细胞表面大小深浅不等的隐窝，及陷窝内局部细胞膜损伤，在ＣＤ１６＾＋ＬＡＫ细胞这种指状突起基底部附近的浆内，有大量胞浆颗粒与囊泡聚集。靶细胞被攻击后常发生凋落型死亡。同时可见坏死型死亡。说明ＣＤ     

雄激素抑制对出血创伤后免疫反应影响的研究

目的探讨雄激素抑制对出血创伤后免疫功能的影响与作用机制. 方法建立不同方式雄激素抑制与出血创伤的雄鼠动物模型,出血创伤后24 h检测胸腺细胞凋亡、脾细胞增殖与其产生的白细胞介素(IL)-2、IL-3. 结果出血创伤加速雄鼠胸腺细胞凋亡,干扰胸腺细胞选择与凋亡的正常程序,雄激素抑制对此有保护作用,且不同雄激素抑制措施具有协同效应;出血创伤使雄鼠脾细胞增殖及产生IL-2、IL-3均受到显著抑制,而抑制雄激素可使之提高. 结论抑制雄激素,在中枢免疫器官及外周免疫细胞水平均有保护作用,暂时的雄激素抑制措施是保护出血创伤后雄鼠免疫功能的简单而有效的方法.     

重组人白血病抑制因子对体外移植前鼠胚发育的影响

目的：观察重组人白血病抑制因子（rhLIF)对体外移植前鼠胚发育的影响。方法：将36只小鼠随机分成3组,每组12只。组I（体内对照）小鼠注射人绒毛膜促性腺激素（hCG)116-120h后处死,组Ⅱ及组Ⅲ（体外对照）小鼠注射hCG44-48h后处死,收集组Ⅱ及组Ⅲ的2细胞期鼠胚,组Ⅱ的鼠胚用人输卵管液（HTF）＋10％人血清培养,组Ⅲ的鼠胚用HTF＋10％人血清＋rhLIF(1000U/ml）培养,观察并记录各细胞期鼠胚发育的数目。结果：（1）组Ⅱ和组Ⅲ发育到4细胞期、8细胞期、桑椹胚阶段的鼠胚百分率（分别为93．4％、87．7％、75．0％和94．5％、91．2％、85．4％）相似,差异无显著性（P＞0．05）。（2）组Ⅱ发育到囊胚期、扩张囊胚期和孵出期的鼠胚百分率低于组Ⅲ（分别为48．1％、32．1％、18．4％和82．3％、59．7％、36．3％）,差异有显著性（P＜0．05）。（3）组I和组Ⅲ发育到囊胚期的鼠胚百分率（分别为86．0％和82．3％）相比,差异无显著性（P＞0．05）。结论：rhLIF对鼠胚的早期发育没有显著影响,但能促进移植前晚期鼠胚的生长、分化和孵出。     

抗血管内皮生长因子发夹状核酶基因抑制卵巢癌移植瘤生长的实验研究

目的 采用抗血管内皮生长因子（VEGF）发夹状核酶基因,阻断卵巢癌中VEGF的自分泌和（或）旁分泌通路,以检测抗VEGF发夹状核酶基因对卵巢癌细胞VEGF表达及其肿瘤生长的影响。方法 采用脂质体介导的方法,将自行设计和构建的抗VEGF发夹状核酶基因真核表达载体转染人卵巢癌细胞SKOV3,采用氨基糖甙庆大霉素（G418）筛选获得阳性克隆;RNA斑点杂交检测核酶基因在卵巢癌细胞中的表达;逆转录-聚合酶链反应（RT-PCR）技术检测转染前后卵巢癌细胞中VEGF的表达;透射电子显微镜观察卵巢癌细胞超微结构改变;观察裸鼠皮下成瘤实验检测转染前后细胞的致瘤能力的变化。结果 转染核酶基因后的卵巢癌细胞VEGFmRNA表达明显降低;透射电子显微镜观察出现凋亡细胞;裸鼠致瘤能力减弱,肿瘤组织中VEGF表达及血管形成减少。结论 抗VEGF发夹状核酶基因可显著抑制卵巢癌细胞VEGF表达,通过减少血管形成抑制肿瘤生长,为进一步开展肿瘤血管靶向基因治疗,提供了实验依据。