激发型4-1BB单抗联合凋亡肿瘤细胞负载的树突状细胞在恶性肿瘤免疫治疗中的作用

目的：研究激发型4-1BB单抗（2A）联合凋亡肿瘤细胞负载的DC（AP-DC）疫苗在小鼠B细胞淋巴瘤免疫治疗中的作用.方法：凋亡小鼠B细胞淋巴瘤细胞A20负载的DC用来制备AP-DC.A20荷瘤小鼠分别被注射AP-DC、2A单抗或二者联合.观察肿瘤生长情况及小鼠生存期.流式细胞术检测免疫治疗后荷瘤鼠脾脏T细胞的表型和胞浆内细胞因子;3H-TdR掺入试验检测T细胞体外增殖能力;ELISA法检测荷瘤鼠血清和脾脏T细胞培养上清中IL-2、IFN-γ和IL-10含量.结果：应用激发型4-1BB单抗2A或AP-DC疫苗对荷瘤小鼠开展免疫治疗,可抑制肿瘤生长,延长荷瘤鼠生存期,获得12.5%或25%的肿瘤完全缓解率.但二者联合应用,治疗效果更好,可获得62.5%的肿瘤完全缓解率,并且荷瘤鼠可长期生存.联合治疗后荷瘤鼠脾脏T细胞体外增殖更为显著,CD4^＋IFN-γ^＋细胞的比例也明显增加.IL-2和IFN-γ的分泌水平在联合治疗荷瘤鼠的血清和脾脏T细胞的培养上清中升高更明显,而IL-10的分泌则降低.结论：激发型4-1BB单抗和AP-DC在肿瘤免疫治疗中有协同作用.     

致敏树突状细胞活化细胞毒性T细胞抗肿瘤作用的研究

目的：研究树突状细胞（DCs）激活的细胞毒性T细胞的抗肿瘤及预防肿瘤发生的作用。 方法： 细胞因子诱生人PBMC未成熟DCs，加入肿瘤细胞抗原提取物致敏DCs产生成熟DCs；通过细胞形态、表面标记鉴定成熟DCs，MTT法测成熟DCs活化的细胞毒性T细胞(CTL)的体外杀伤活性；裸鼠体内注射活化CTL观察其抑制移植瘤生长及发生的作用。 结果： 经过7 d培养，获得大量形态典型、具有强烈刺激增殖能力、高表达CD80(63.5%)、CD83（67.6%）和CD3/ HLA-DR(83.2%)的DCs。其活化的CTL在20∶1效靶比时对抗原来源细胞株自身的杀伤率达75%以上，对同系细胞株的杀伤活性为35%-45%，对其它种系肿瘤细胞仅有微弱杀伤力（P<0.01）。CTL对裸鼠结肠癌HT-29移植瘤有特异性的生长抑制和预防生成作用（P<0.05）。CTL治疗组肿瘤组织中PCNA表达水平显著低于对照组（P<0.05）。 结论： 肿瘤细胞抗原活化的DC诱导CTL对肿瘤有特异性的杀伤作用，体内应用可特异性抑制移植结肠癌的生长或预防小鼠结肠癌移植瘤的发生。     

淤胆血清“病理微环境”诱导胚胎干细胞向肝细胞分化

目的:探讨淤胆血清"病理微环境"培养体系诱导胚胎干细胞(ESC)向肝细胞分化的可行性。方法:将小鼠ESC细胞系E14在无白血病抑制因子培养基中培养,使其自发分化为拟胚体,加入FGF-4和HGF初步诱导,然后置于5％淤胆鼠血清"病理微环境"筛选培养液中继续培养2周,然后进行细胞形态学观察,白蛋白和CK8/18免疫组化染色,白蛋白与转甲状腺蛋白RT-PCR检测,细胞糖原染色及尿素合成功能分析。结果: 经初步诱导分化的ESC置于5％淤胆血清"病理微环境"筛选培养液中培养,初期细胞生长受抑制,2周后分化为肝细胞样细胞,细胞呈现良好的均质性;免疫组化染色显示白蛋白和CK8/18表达;RT-PCR显示有白蛋白、转甲状腺蛋白等的mRNA转录;细胞有糖原和尿素合成功能。结论:采用含淤胆血清的病理微环境培养体系从经FGF-4和HGF初步诱导的胚胎干细胞中有效筛选出了具有功能的肝细胞,细胞有较好的均质性,为临床肝细胞替代治疗、获取丰富供体细胞来源提供了新思路。     

雌激素处理DCs在诱导小鼠同种皮肤移植免疫耐受中的作用

目的：研究雌二醇（17β-estradiol, E₂）处理的供体骨髓来源树突细胞（dendritic cells, DCs）在诱导小鼠同种异基因皮肤移植免疫耐受中的作用。方法：体外培养小鼠骨髓来源DCs，然后用E₂处理。受体小鼠在皮肤移植前经尾静脉分别输注经E₂处理的供体小鼠DCs、供体小鼠成熟DCs以及不成熟DCs，输注PBS为对照组。1周后对受体小鼠进行同种异基因皮肤移植，手术后观察皮肤存活情况，应用流式细胞术检测手术前后受体小鼠外周血中CD4+CD25+调节性T细胞百分率的变化。结果：在同种异基因皮肤移植中，受体小鼠尾静脉输注经E2处理的DCs组与对照组和不成熟DCs组比较，移植皮肤存活时间显著延长，较不成熟DCs组存活时间平均延长10.6 d（P<0.01）；与对照组和不成熟DCs组比较，E₂处理组外周血中CD4+CD25+ 调节性T细胞百分率明显升高（P<0.01）。结论：在同种异基因小鼠皮肤移植模型中，E₂处理的供体小鼠DCs可以延长移植皮肤存活时间。     

管花苷B对抗H2O2诱导的PC12细胞凋亡

目的：观察肉苁蓉提取物管花苷B对H₂O₂诱导的PC12细胞损伤的影响。方法：用MTT法检测细胞存活率，以激光共聚焦显微镜荧光染色法检测细胞内活性氧的产生和线粒体膜电位的变化，DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生，并用荧光酶标仪测定caspase-3的活性。结果：100 μmol·L^-1 H₂O₂处理细胞24 h显著降低细胞的存活率；诱导细胞发生凋亡，凋亡率达48.0％；细胞内活性氧水平及caspase-3的活性显著升高；而线粒体膜电位却明显降低，红/绿荧光强度的比值由正常的5.97降低为0.41左右。而预先给予1、10或100 mg·L^-1浓度的管花苷B处理细胞12 h，可显著提高细胞存活率；并可有效抑制DNA ladder的发生；流式细胞仪检测凋亡率分别降低到30.9%、18.3%和6.2%；激光共聚焦显微镜结果显示管花苷B可明显降低细胞内活性氧的水平；并可逐渐恢复线粒体的高能量状态；caspase-3的活性不断降低，并呈现了一定的剂量依赖性。结论：管花苷B能显著地抑制H₂O₂诱导的PC12细胞凋亡，其神经细胞保护作用可能与其降低细胞内活性氧水平，维持线粒体膜电位的高能状态和抑制caspase-3的活性有关。     

Dual function of Langerhans cells in skin TSLP-promoted TFH differentiation in mouse atopic dermatitis

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CD40-mediated lymphotoxin α expression in human B cells is tyrosine kinase dependent

The cytokine lymphotoxin (LT)α is known to play a role in B cell activation. As the engagement of the B cell antigen CD40 is known to lead to B cell proliferation and differentiation, we studied LTα expression in human B cells after CD40 ligation. We demonstrate that anti-CD40 monoclonal antibody (mAb) induces strong LTα mRNA and surface expression in human tonsil B cells. Induction of LTα mRNA and surface expression by CD40 ligation is inhibited by the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein in a dose-dependent manner. The protein kinase C (PKC)-specific inhibitors sphingosine and bisindolylmaleimide caused negligible inhibition of anti-CD40-induced LTα mRNA and surface expression. No inhibition is observed with the protein kinase (PKA) inhibitors H89 and HA1004. Cross-linking of the transmembrane phosphatase CD45 to CD40 by using goat-anti-mouse F(ab')₂ fragments strongly inhibits CD40-mediated LTα expression in human B cells, confirming the role of PTK activation in CD40-mediated induction of LTα expression. Inhibitors of the serine/threonine protein phosphatases PP1 and PP2A, okadaic acid and calyculin induce LTα mRNA expression. In contrast, cyclosporin A, an inhibitor of the serine/threonine phosphatase calcineurin has no effect on anti-CD40-induced LTα expression. These results suggest that induction of LTα expression in B cells following engagement of CD40 involves activation of protein tyrosine kinases.     

Human interdigitating dendritic cells directly stimulate CD40-activated naive B cells

Human interdigitating dendritic cells (IDC) were isolated from tonsils based on their CD40⁺ lineage-negative expression in situ. Isolated IDC displayed a phenotypic profile similar to that of IDC in tonsils and spleen in situ, characterized by high-level expression of major histocompatibility complex class II, the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86), expression of the late DC maturation marker CD83, and no expression of CD1a, CD13, or CD33. IDC also showed weak nonspecific esterase staining and had the ability to induce an allogeneic mixed lymphocyte reaction. In this study, we further show that in the presence of surrogate activated T cells in the form of CD40 ligation and IL-2, IDC enhance the proliferation of naive B cells and induce their differentiation into plasma cells producing IgM. Evidence for the anatomical co-localization of naive B cells and IDC in the T cell area together with the data obtained in vitro implies a role for IDC in the initiation of the extrafollicular reaction.     

Bone marrow mast cell reaction in preleukaemic myelodysplasia and in aplastic anaemia

Summary The relationship of bone marrow mast cell counts to prognosis was investigated in 48 patients with preleukaemic myelodysplasia, in 59 patients with aplastic anemia and in a DMBA induced myelodysplasia/leukaemia rat model. In patients with myelodysplasia terminating in overt leukaemia the number of mast cells per square millimeter was not correlated to duration of the preleukaemic course. Leukaemia development probabilities of patients at risk were not different for low and elevated mast cell counts. In aplastic anaemia, however, a lower bone marrow mast cell count was related to a higher survival probability and longer survival time. In the animal model no significant differences could be found between myelodysplastic, leukaemic, and control animals.     

Immunofluorescent detection of anti-cytoskeleton antibodies using vinblastine-treated mononuclear cells

A reliable and reproducible immunofluorescence method is described for the detection of anti-cytoskeleton antibodies in human sera, based on the use of vinblastine-treated peripheral blood mononuclear cells as substrate. Three immunofluorescence patterns associated with antibodies to microfilaments, intermediate filaments and microtubules are readily identified.