重症肌无力患者CD4、CD8细胞表面Fas分子的变化

为探讨Fas分子、CD4、CD8分子在重症肌无力发生中的作用 ,从胸腺及外周血分离单个核细胞 ,用荧光标记的单克隆抗体 (Fas FITC、CD4 PE、CD8 Cy )和流式细胞仪检测细胞表面Fas、CD4、CD8表达情况。结果发现 ,(1)MG患者外周血单个核细胞 (PBMC )CD4 + CD8 细胞明显低于对照组 (P <0 0 2 ) ;(2 )MG患者Fas分子在PBMC的表达率明显低于对照组 (P<0 0 2 ) ,Fas+ CD4 + 细胞也低于对照组 (P <0 0 5 ) ;(3)在Fas+ 的胸腺细胞中 ,MG患者的双阳性细胞显著低于对照组 (P <0 0 1) ,而CD4 CD8+ 细胞则高于对照组 (P <0 0 5 )。说明重症肌无力患者淋巴细胞存在Fas表达异常 ,表现为外周CD4 + 细胞活化后的凋亡障碍和胸腺CD4 + CD8+ 细胞凋亡障碍 ,可能与重症肌无力患者自身反应性T细胞的形成及疾病的发生有关     

胃癌患者PBMCs中CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响

目的:探讨胃癌患者外周血单个核细胞（PBMCs）中的CD4+CD25+T细胞体外增殖及对CD4+CD25-T细胞增殖的影响。 方法:以免疫磁性分离方法 （MACS）分选出胃癌患者外周血单个核细胞中的CD4+CD25+T及CD4+CD25-T细胞后，用流式细胞仪分析细胞的纯度及活力；再以小鼠抗人CD3单抗、小鼠抗人CD28单抗及rh IL-2作为共刺激因子，观察与CD4+CD25-T细胞共培养时，CD4+CD25+T细胞对CD4+CD25-T细胞增殖的抑制效应。 结果:（1）分选后健康对照组及胃癌患者PBMC 中CD4+CD25+ T细胞纯度分别为83.8%±1.84%、84.13%±2.77％，两者相比，无显著差异（P＞0.05）;(2)经MACS 分选后正常对照组与胃癌患者CD4+CD25+ T细胞活力分别为98.52%±0.72%、97.80%±0.95%，两者相比，无显著差异（P＞0.05）；(3)无论是健康对照还是胃癌患者的CD4+CD25+T均具有明显抑制效应性T细胞如CD4+CD25-T细胞的增殖，随着CD4+CD25+T细胞数的增加，这种抑制增殖的能力也相应增加，当CD4+CD25+∶〖KG-*2〗CD4+CD25-T达 1∶〖KG-*2〗1时，抑制率最大达到50％。 结论:MACS分选法能够分选出高纯度及活力的CD4+CD25+T细胞，分选后CD4+CD25+T细胞在体外均能抑制CD4+CD25-T细胞增殖，且这种抑制效应呈一定效靶比关系。     

淋巴细胞表面CD40分子的表达和作用

CD40和CD40L(CD40 ligand,CD154)都属于肿瘤超家族成员。CD40-CD40L(CD40 ligand,CD154)的相互作用一直是与APC和T细胞的活化相关联,并且在肿瘤免疫、自身免疫性疾病、炎症反应等中起重要的作用。目前,有研究发现,CD40不仅表达在APC、内皮细胞以及某些肿瘤细胞上,也会在T细胞中有表达。CD40+CD8T细胞的研究主要集中在细胞信号通路和过继免疫治疗方面,CD40+CD4T细胞主要集中在自身免疫性疾病的研究。本文将对CD40在T细胞表面的表达及其作用做一综述。     

Th17细胞:一种新的效应CD4+T细胞亚群

当CD4^＋T细胞被激活以后，通过不同的分化途径获得特定的生物学功能。根据产生细胞因子和生物功能的不同，传统上将CD4^＋T细胞分为Th1和Th2细胞亚群。Th1细胞产生IFN-1和IL-2，通过活化巨噬细胞清除胞内病原微生物并介导迟发型变态反应；Th2细胞产生IL-4、IL-5和IL—13，介导由嗜酸性粒细胞引起的炎性反应，清除细胞外病原微生物并参与变态反应。IFN-γ和IL-4相互拮抗，调控着Th1和Th2细胞的扩增和功能。     

观察佛波脂和二甲基亚砜联合诱导人肺腺癌GLC分化过程中的形态和超微结构

目的 探讨佛波脂(TPA)和二甲基亚砜(DMSO)联合诱导分化剂对人肺腺癌GLC细胞形态的影响。方法 采用细胞计数，光镜、扫描电镜与透射电镜观察，经过TPA和DMSO联合诱导的人肺腺癌GLC细胞增殖以及形态和超微结构的变化。结果 经过联合诱导分化剂作用后，细胞增殖受到抑制，细胞体积增大，呈现扁平铺展状态，细胞核质比例变小，核仁数量减少。细胞表面微绒毛减少，细胞边缘丝状伪足减少，片状伪足增多，核形态较规则，核内异染色质减少，常染色质增多，细胞质中的细胞器数量增多，结构趋于正常。结论 TPA和DMSO联合诱导能够一定程度改变GLC细胞恶性形态结构特征并对GLC细胞具有一定的诱导分化作用。     

间充质干细胞对不同刺激条件下Th1/Th2细胞比值的影响

目的探讨间充质干细胞(MSC)对不同刺激作用下T淋巴细胞向Th1及Th2亚群转化的影响。方法将MSC植入24孔板中,在第3天加入经佛波脂(PMA)及离子霉素(ionomyc in)刺激的T淋巴细胞,或者按照不同比例将MSC加入双向混合淋巴细胞培养体系中,用流式细胞术分析各组T淋巴细胞亚群和内因子IL-4、IFN-γ的分泌变化情况,从而间接了解Th1及Th2亚群的改变。结果对于共刺激作用下单独培养的T细胞,MSC能轻度抑制其Th1细胞的转化;而在混合淋巴细胞培养体系(MLC)中,MSC能够明显抑制其中的CD8 T细胞亚群和Th1细胞,轻度提高Th2细胞比率。结论MSC在体外能抑制CD8 T细胞(CTL)和Th1细胞,升高Th2细胞。所以在临床上可能有减轻移植后急性移植物抗宿主病(aGVHD)的发生,而保留移植物抗白血病(GVL)的潜力。     

脱氢表雄酮对鼠成骨细胞及CD4+T细胞协同刺激分子表达的影响

目的探讨脱氢表雄酮（DHEA）对成骨细胞（osteoblasts，OB）及CD4^＋T细胞表达协同刺激分子的调控作用。方法颅骨酶解法培养鼠OB，体外模拟雌激素撤退；免疫磁珠细胞分选（rnagnetic cell soaing，MACS）法分离CD4^＋T细胞；将OB或CD4^＋T细胞分为对照组、E2及DHEA处理组，并以LPS刺激；以流式细胞术分析OB表面CD80、CD86以及CD4^＋T细胞表面CD28、CTLA-4的表达。结果经E2及DHEA处理后，OB表达CD80、CD86显著增加（P〈0．05，P〈0．01）；CSA可降低对照组及DHEA处理组OBCDS0、CD86的表达（P〈0．01）。除DHEA组CD28^＋T细胞百分比增加外（P〈0．05），其余各组CD4^＋T细胞CD28和CTLA-4的表达无显著改变（P〉0．05）。结论DHEA可上调鼠OB协同刺激分子CD80、CD86表达，该作用可被CsA阻滞；DHEA还上调CD4^＋T细胞CD28的表达，提示可改善骨．免疫调节网络。     

PMA特异性抑制K562细胞BCR/ABL和FynmRNA的表达

目的: 研究12-肉豆蔻酸-13-乙酸佛波酯(PMA)对K562细胞BCR/ABL与Fyn mRNA表达的影响及两者之间的关系。方法: 1~250 μg/L PMA刺激K562细胞 24 h后,应用实时荧光定量PCR技术检测各组BCR/ABL和Fyn mRNA的水平。结果: PMA明显抑制BCR/ABL和Fyn mRNA的水平,该下调作用均具有显著的量效关系,且BCR/ABL和Fyn的mRNA表达水平下调表现出明显的相关性。比较刺激Molt-4细胞株的结果, PMA抑制作用具有细胞选择性。K562细胞株经诱导后出现伪足样的形态改变。结论: PMA通过抑制BCR/ABL融合基因的转录下调Fyn转录水平。     

Induction of CD3δεω) by phorbol 12-myristate 13-acetate

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3ω chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Tiα, CD3γ and CD3ζ chains two-to threefold and the synthesis of Tiβ and CD35δεω complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount of total assembled TcR complexes increased approximately threefold and the maturation of the TcR was not affected as determined by analysis of oligosaccharide side chain processing in the Golgi apparatus. Activation of Jurkat cells with anti-CD3 monoclonal antibody, calcium ionophore, or mitogenic lectins did not affect the synthesis of the TcR components. In other cells studied (the human leukemic T cell line CEM, a panel of variants of the Jurkat T cell line and peripheral blood mononuclear cells) PMA also increased the synthesis of the TcR components. However, for all cell lines studied the amount of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3ω in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3δεω complexes was immunoprecipitated as compared to the amount obtained from untreated Jurkat cells, and these observations indicate that the CEM cell line may be a qualified candidate for purification of CD3ω.     

PMA对THP-1细胞和RA患者单核细胞及培养上清中CD147表达的影响

目的：研究豆蔻佛波醇乙酯（PMA）刺激前后,体外培养的人单核细胞系THP-1细胞和类风湿关节炎（RA）患者外周血单核细胞（human peripheral blood monocytes,HPBM）上及培养上清中CD147的表达.方法：以贴壁法分离RA患者HPBM.用PMA刺激体外培养的RA患者HPBM和THP-1细胞,采用流式细胞术动态检测THP-1细胞、RA患者HPBM膜表面CD147的表达.用双抗体夹心ELISA法检测培养上清中CD147分子的含量.结果：PMA刺激前,THP-1细胞膜及RA患者HPBM表面CD147分子的表达均较高,培养上清中均可检测到CD147分子.PMA刺激后,THP-1细胞培养上清中CD147的含量升高,THP-1细胞上CD147的表达先升高后降低,最后进入稳定期;RA患者的HPBM上CD147的表达下降,培养上清中CD147的含量升高,于刺激后2 d进入稳定期.结论：THP-1细胞、RA患者HPBM膜表面CD147分子可从细胞上脱落或由细胞直接分泌到培养液中.PMA可上调可溶性CD147的表达.     

PMA-activation of peripheral blood and tonsillar B lymphocytes induces large adhesive cells reminiscent of large extrafollicular (monocytoid) B cells

Extrafollicular (EF) B lymphocytes differ in size and morphology depending on the lymphatic organ involved and the kind of inflammatory reaction. On reevaluating EF B cells in various sites and conditions we discriminated three forms: a small (lymphoid) and intermediate (centrocytoid), and a large (monocytoid) variant. Immunohistochemically, these variants could be discriminated by their differential expression of adhesion molecules CD62L (L-selectin) and CD11c: small EF B cells were strongly L-selectin⁺ and CD11c^–; intermediate cells were moderately CD62L⁺ and CD11c^–; large cells were faintly CD62L⁺ or ^– but expressed CD11c. In 72 h cultures of normal peripheral and tonsillar B cells, cross-linking surface immunoglobulin in the presence of interleukin-2 or interleukin-4 led to formation of clusters in vitro together with an increase in cell size and a slight up-regulation of CD11c, as determined by flow cytometry. Stimulation with phorbol 12-myristate 13-acetate (PMA), however, gave rise to large, plastic adherent cells which also showed strong homotypic adhesion, expressed CD62L at minimal levels and CD11c at comparably highest levels and altogether mimicked the large cell variant of EF B cells. We conclude that EF B cells are subjected to cytokine-induced metamorphosis and that differences in cell size and morphology reflect their state of activation and activation-associated adhesion properties. Our data suggest that EF B cells in all anatomical sites are functionally closely related cells which — possibly mediated by CD11c/CD18 — may become sessile and proliferate locally once activated by appropriate signals.     

Daxx抑制K562细胞向巨核细胞分化

目的:观察过表达死亡结构域相关蛋白(Daxx)对K562细胞活力和向巨核细胞分化的影响。方法:建立稳定过表达Daxx的K562细胞,荧光显微镜观察、实时荧光定量PCR和Western blot检测Daxx的过表达效果,CCK-8法检测过表达后细胞活力的变化;佛波酯(PMA)诱导K562细胞向巨核细胞系分化,Western blot检测在K562细胞向巨核细胞分化过程中Daxx和p-ERK的表达变化,流式细胞术检测CD41和CD61的表达变化;PMA处理过表达Daxx的K562细胞,NBT还原实验检测细胞分化情况,流式细胞术检测过表达Daxx后CD41和CD61的表达变化,Western blot检测p-ERK的蛋白水平。结果:建立了稳定的过表达Daxx的K562细胞,过表达Daxx抑制K562细胞的活力。PMA诱导K562细胞向巨核细胞分化,CD41和CD61表达水平增高,同时p-ERK的蛋白水平升高,Daxx表达水平下降。过表达Daxx可以抑制K562细胞向巨核细胞分化,CD41和CD61表达降低,同时p-ERK的蛋白水平降低。结论:过表达Daxx可以抑制K562细胞生长及向巨核细胞分化,同时抑制ERK的磷酸化。     

CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals

We investigated whether development of mutations leads to loss of CD8 T-cell recognition in HIV-1 infection and is possibly linked to alterations in HIV-1-specific CD4(+) T-cell responses in 2 HIV-infected individuals. In patient, H434 full genome sequencing of HIV-1 biological clones at early and late time points during disease progression showed development of fixed mutations in 16 predicted HIV-specific CTL epitopes. Loss of T-cell recognition and reactivity against wild-type and mutant epitopes was observed primarily for the HLA-B27-restricted KK10 epitope and HLA-A2-restricted SL9 epitope. Similarly, in patient H671, decreasing numbers of HLA-A3-restricted CD8(+) T cells specific for the wild-type RK9 epitope was observed after CTL escape. Only in patient H434 loss of CTL responses was paralleled by a decrease in HIV-specific IL-2(+) CD4(+) T-helper responses. This suggests that loss of T-cell reactivity may not be directly linked to HIV-specific CD4(+) T-cell responses but that increased viremia after CTL escape may influence CD4(+) T-helper responses.     

Optimized THP-1 differentiation is required for the detection of responses to weak stimuli

Objectives: The differentiation of THP-1 monocytes into macrophages is mainly conducted at a phorbol 12-myristate 13-acetate (PMA) concentration of 10–400 ng/ml. However, this concentration might be high enough to upregulate the expressions of some genes in differentiated macrophages, which could overwhelm gene expression increases induced by other stimuli. The present study was performed to optimize the PMA concentration required to differentiate monocytes whilst minimizing gene upregulation. Methods: THP-1 cells were treated with 2.5–100 ng/ml PMA and analyzed for the extent of cell adherence, the surface marker of macrophages, and stable differentiation without undesirable gene upregulation. The stably differentiated THP-1 cells at the minimum PMA concentration were treated with 10 ng/ml LPS or 125 nM amyloid beta (Aβ_1-42). Results: The treatment of THP-1 with 5 ng/ml PMA was found to be sufficient to induce stable differentiation without undesirable gene upregulation. These macrophages differentiated at 5 ng/ml responded well to secondary weak stimuli like 10 ng/ml LPS or 125 nM of amyloid beta (Aβ_1-42). Conclusions: This finding suggests that THP-1 cells are well differentiated by 5 ng/ml PMA, and that the resulting differentiated macrophages respond well to secondary weak stimuli without being overwhelmed by undesirable gene upregulation induced by PMA. Received 10 March 2006; returned for revision 9 August 2006; accepted by G. Wallace 11 August 2006     

Bladder cancer immunogenicity: expression of CD80 and CD86 is insufficient to allow primary CD4+ T cell activation in vitro

Transitional cell carcinomas (TCC) of the urinary bladder are known to express proteins which can yield potentially immunogenic peptide epitopes for expression in the context of cell surface class I or class II MHC antigens. However, additional costimulatory ligands must also be expressed before such a cell might directly induce full activation and proliferation of resting, antigen-specific T lymphocytes. Intravesical therapy might be used to manipulate T cell costimulation in order to promote specific rejection of TCC cells. This in vitro study examined the potential of such a strategy by transfection of the prototypical TCC line J82 with the important costimulatory molecules CD80 (B7-1) and CD86 (B7-2). Untransfected J82 cells expressed class I and II MHC antigens, a range of cell adhesion molecules, though did not induce T cell proliferation in a robust, allogeneic co-culture system. Transfected J82 cells expressed CD80 or CD86 at levels comparable to an antigen-presenting B cell line. Furthermore, functional surface expression of CD80 and CD86 was demonstrated in a mitogen-dependent assay of costimulation. However, neither CD80+ nor CD86+ transfectant J82 cells could induce significant proliferation of antigen-specific CD4+ T cells. Further analysis showed that bystander J82 cells could inhibit independent T cell activation in an effect dependent on direct cell contact. This inhibitory effect was associated with increased cell death in the responding lymphocyte population and is concordant with surface expression of CD95L by the J82 cell line.     

Stimulation of the expression and the enzyme activity of aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 on human renal cell carcinoma cells and renal tubular epithelial cells by T cell-derived cytokines, such as IL-4 and IL-13.

Aminopeptidase N (APN) and dipeptidylpeptidase IV (DPIV) are transmembrane type II molecules widely distributed in mammalian tissues. In recent years, the interest in cell surface peptidases has increased considerably because, among other things, several reports indicate roles of ectopeptidases in tumour cell metastasis. Investigations into the regulation of APN and DPIV on tumour cells are rare. We report, for the first time, that IL-4 and IL-13 can up-regulate protein expression as well as enzymatic activity of both the peptidases on renal carcinoma cells and renal tubular epithelial cells in culture. The analysis of mRNA by competitive polymerase chain reaction (PCR) confirmed our results with respect to the APN increase at the level of gene expression. IL-1 beta and tumour necrosis factor-alpha (TNF-alpha) augmented the IL-4-induced effect with respect to APN but not to DPIV. A 5-day incubation with interferon-gamma (IFN-gamma) increased protein expression, especially of APN and, to a lesser extent, also of DPIV, whereas no significant increase in enzymatic activity could be observed. Small concentrations of transforming growth factor-beta 1 (TGF-beta 1) inhibit the expression and enzyme activity of DPIV. IL-6, IL-7, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any effect on APN and DPIV. For a prospective therapeutic regimen with T cell-derived cytokines it has to be considered that--besides their effect on tumour cell growth--cytokines might affect surface ectopeptidases involved in tumour cell adhesion processes. The inhibition of APN and DPIV could be a new approach to suppression of cancer spread.