人白细胞介素4诱导杀伤细胞的研究

人重组白细胞介素４（ｒｈＩＬ－４）在ＰＨＡ协同下，从人外周血单核细胞（ＰＢＭＣ）诱导出明显的ＬＡＫ活性，其对Ｋ５６２、Ｒａｊｉ细胞的杀伤力低于ＩＬ－２诱导者，对ＴＢＬ－Ｅ，ＰＨＡ活化的淋巴母细胞（ＰＨＡ－ｂｌａｓｔｓ）的杀伤力和ＩＬ－２诱导者相似。在ＰＨＡ介导的４小时５１Ｃｒ杀伤试验中，加入ＰＨＡ后，ＩＬ－４－ＬＡＫ对ＰＨＡ－ｂｌａｓｔｓ的杀伤力提高２．３倍，而ＩＬ－２－ＬＡＫ对ＰＨＡ－ｂｌａｓｔｓ的杀伤力无变化，提示ＩＬ－４主要诱导ＣＴＬ样活性，而ＩＬ－２主要诱导ＮＫ样活性，ＩＬ－４诱导效应ＣＴＬ的能力强于ＩＬ－２。我们的实验同时证实，在淋巴细胞活化的早期，ＩＬ－４抑制ＩＬ－２诱导的ＬＡＫ活性，淋巴细胞活化后，ＩＬ－４与ＩＬ－２有协同作用，增强ＩＬ－２诱导的ＬＡＫ活性。     

低密度脂蛋白诱导肾小管上皮细胞增殖的信号通路

为观察低密度脂蛋白（ＬＤＬ）对肾上管上皮细胞的影响及涉及的相关信号传导机制，以人近曲行小管上皮细胞系（ＨＫ－２）为对象，采用３Ｈ－ＴｄＲ掺入与细胞计数测定增殖；特异性底物磷酸化法测定细胞外信号调节激酶（Ｅｎｇ）活性；凝胶迁移率法检测转录活化因于（ＡＰ－１）ＤＮＡ结合活性。发现ＬＤＬ１００～５００ｕｇ／ｍＬ刺激ＨＫ－２细胞７２ｈ可促进其增殖：加入ＬＤＬ２５０ｕｇ／ｍＬ后２～６０ｍｉｎ内可使该细胞ＥＲＫ活性明显增加，１０ｍｉｎ时达高峰；不同剂量（５０～５００ｕｇ／ｍＬ）刺激时，ＥＲＫ活性分别较对照组增加１．５６～２．１９倍。ＬＤＬ促使核内转录因子ＡＰ－１结合活性增加。ＥＩＵＬ特异性阻断剂ＰＤ（５０ｕｍｏｌ／Ｌ）可以分别阻断ＬＤＬ刺激的ＨＫ－２细胞ＥＲＫ活性和ＤＮＡ合成。ＲＭＡ阻断ＰＫＣ信号途径后，ＬＤＬ对于ＥＲＫ的活化效应明显减弱。     

IL—2,IL—4和IL—7体外诱导人外周血淋巴因子激活的杀伤细胞效应…

比较了淋巴因子ＩＬ－２、ＩＬ－４和ＩＬ－７ 在体外诱导健康人外周血淋巴因子激活的杀伤（ＬＡＫ）细胞活性的效应。结果表明，ＩＬ－７可单独，亦可与ＩＬ－２、ＩＬ－４协同诱导ＬＡＫ细胞，而且ＩＬ－７诱导ＬＡＫ细胞的效应不被抗ＩＬ－２、抗ＩＬ－４所抑制。ＩＬ－７单独诱导的ＬＡＫ细胞活性高峰迟于ＩＬ－２或ＩＬ－４所诱导的活性高峰，且与增殖反应曲线一致。抗ＣＤ８抗体明显抑制ＩＬ－７诱导ＬＡＫ细胞的效应，而ＩＬ     

IL-6D24-Linker-PE40重组毒素的分子生物学特性预测

目的：探讨重组毒素ＩＬ－６Ｄ２４－Ｌｉｎｋｅｒ－ＰＥ４０融合蛋白及其接头设计的合理性。方法；应用核酸和蛋白质序列分析软件系统－ＧＯＬＤＫＤＥＹ软件，对ＩＬ－６Ｄ２４－Ｌｉｎｋｅｒ－ＰＥ４０重组毒索融合蛋白柔性、抗原性、亲水性、表位等分子生物学特性进行计算机模拟预测，并做Ｗｅｓｔｅｍ ｂｌｏｔ分析进行验证。结果：ＩＬ－６Ｄ２４－Ｌｉｎｋｅｒ－ＰＥ４０分别保留了ＩＬ－６Ｄ２４和ＰＥ４０的表位特征，没有     

慢性活动性乙型肝炎患者细胞免疫功能检测及其临床意义

测定了例慢性活动乙型肝炎患者外周血单个核细胞（ＰＢＭＣ）产生的白细胞介素２（ＩＬ－２）活性，其中部分病人检测了血清可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）水平，膜白细胞介素２受体（ｍＩＬ－２Ｒ）表达，ＬＡＫ活性及外周血Ｔ淋巴细胞亚群，并分析了它们之间的相关性，结果表明：ＩＬ－２活性，ｍＩＬ－２Ｒ表达，ＬＡＫ活性，ＣＤ４／ＣＤ８比值显著低于正常对照组（Ｐ〈０．００１），而ｓＩＬ－２Ｒ水平，ＣＤ８细胞显     

聚乙二醇修饰重人白细胞介素—2及其生物学特性

采用聚乙二醇活性酯化学修饰重组人白细胞介素－２及修饰后复性制得修饰产物ＰＥＧ－ｒＩＬ－２，ＰＥＧ－ｒＩＬ－２系列研究，经ＳＤＳ－Ｐ；ＡＧＥ和ＣＴＬＬ－２短期细胞２培养结合＾３Ｈ－ＴｄＲ掺入法测定，ＰＥＧ－ｒＩＬ－２分子量为２５ｋＤ，生物活性保留６９．７％；采用ＬＤＨ释放法测定证实ＰＥＧ－ｒＩＬ－２激活的ＬＡＫ细胞对人体肝癌细胞ＢＥＬ－７４０２和Ｋ５６２细胞的杀伤作用和ｒＩＬ－２相近甚至强于ｋＩＬ－     

慢性乙型肝炎,肝硬化患者外周血LAK细胞活性和sIL—2R测定的意义

采用＾３Ｈ－ＴｄＲ释放法测定５１例慢性肝病患者（ＣＰＨ１０例、ＣＡＨ２３例、ＬＣ１８例）外周血ＬＡＫ细胞活性，并用酶联法测定患者血清中ｓＩＬ－２Ｒ含量；与２９例正常对照组比较，发现肝病患者ＬＡＫ活性降低，ＨＢＶＤＮＡ阳性组ＬＡＫ活性较阴性组低（Ｐ〈０．０５），ｓＩＬ－２Ｒ增高，且慢性肝病组ＬＡＫ活性与ｓＩＬ－２Ｒ水平呈负相关，说明ＬＡＫ活性与机体免疫功能状态有关，ＨＢＶ的复制和高浓度的ｓＩＬ－２Ｒ     

肝癌患者LAK,IL—2活性,mIL—2R及sIL—2R表达研究

本文报道肝癌病人的ＬＡＫ、ＩＬ－２活性及ｍＩＬ－２Ｒ表达均明显低于正常人，而ｓＩＬ－２Ｒ表达则显著高于正常人。且ＩＬ－２活性与ＬＡＫ活性呈正相关，与ｓＩＬ－２Ｒ呈负相关；而ｓＩＬ－２Ｒ与ｍＩＬ－２Ｒ呈正相关，与ＬＡＫ活性呈负相关。表明肝癌病人的细胞免疫功能可能处于一种抑制或紊乱状态。同时检测ｓＩＬ－２Ｒ及ｍＩＬ－２Ｒ可作为肝癌病人免疫状态的监测指标。     

创伤后活化T细胞内cAMP代谢,蛋白激酶A活性的变化及意义

研究了创伤小鼠活化Ｔ细胞内ｃＡＭＰ代谢、蛋白激酶Ａ（ＰＫＡ）活性的变化及同Ｔ细胞功能的关系。结果显示，创伤后活化Ｔ细胞内ｃＡＭＰ含量增加，这一变化同创伤后Ｔ细胞白介素２（ＩＬ－２）生成减少，ＩＬ－２受体（ＩＬ－２Ｒ）表达受抑，Ｔ淋巴细胞转化（ＴＬＴ）降低密切相关。创伤后活化Ｔ细胞内腺苷酸环化酶（ＡＣ）、ＰＡＫ活性增加，ｃＡＭＰ－磷酸二酯酶（ｃＡＭＰ－ＰＤＥ）活性降低。ＰＫＡ抑制剂Ｈ－８在体外可明显     

胸腺因子D对LAK细胞活性诱导的影响

本文报道利用ＭＴＴ法和４小时５１Ｃｒ释放法，在有或无ＩＬ－２存在的情况下，研究了ＴＦＤ对正常人ＰＢＭＣ体外增殖、ＬＡＫ活性诱导的影响。结果表明：单纯ＴＦＤ不能促进静止的淋巴细胞增殖，也不能诱导出高活性的ＬＡＫ细胞，但可促使经ＩＬ－２活化的淋巴细胞进一步增殖如先用ＩＬ－２活化淋巴细胞２４小时，再加入ＴＦＤ联合诱导，第４天时，ＬＡＫ活性可达８１．３±６．５％，明显高于单用ＩＬ－２组（Ｐ＜０．０１）。     

重组铜绿假单胞菌外毒素PE38KDEL的克隆、表达与活性检测

目的对铜绿假单胞菌外毒素A的编码序列进行改造,以获得相对分子质量小、活性强的重组毒素PE38KDEL。方法基于铜绿假单胞菌外毒素A的编码序列的高GC含量,通过PCR反应和酶切技术,构建重组毒素PE38KDEL基因,并于大肠杆菌表达,用Westernblot分析表达产物。为检测其生物活性,将其与靶向分子IL4突变体cpIL4(13D)融合,构建融合蛋白表达载体,并于大肠杆菌表达,表达产物经亲和色谱和阴离子交换色谱纯化后,用MTT法检测其对表达IL4受体的黑色素瘤细胞LiBr的细胞毒性。结果成功构建了PE38KDEL基因,并在大肠杆菌BL21(DE3)中得到了高效表达,表达量占细胞全蛋白的21%左右;构建了融合蛋白cpIL4(13D)PE38KDEL,并于大肠杆菌AD494(DE3)中得到高效表达,纯化后融合蛋白的纯度达95%以上,细胞毒实验证明其对黑色素瘤细胞LiBr具有良好的杀伤作用,这说明改造后的PE38KDEL是具有细胞毒效应的。结论重组毒素PE38KDEL的构建,为各种导向药物的构建奠定了基础。     

Overexpressed cell surface interleukin-4 receptor molecules can be successfully targeted for antitumor cytotoxin therapy

A variety of human solid cancer cell lines and primary cell cultures has been reported to overexpress high-affinity receptors (R) for interleukin-4 (IL-4), a pleiotropic immunoregulatory cytokine. The significance of IL-4R expression is not known; however, IL-4 is able to upregulate adhesion molecules, inhibit cell proliferation, and mediate signal transduction in tumor cell lines. To target IL-4R, we produced a chimeric protein composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin [termed IL4(38-37)-PE38KDEL or cpIL4-PE]. The recombinant cpIL4-PE was highly cytotoxic to cancer cells, but not toxic to normal B cells, T cells, monocytes, and CD34+, even though these cells express detectable numbers of IL-4R. The cytotoxicity was specific because excess of recombinant IL-4 neutralized the cpIL4-PE effect. To further develop this molecule, in vivo antitumor activity was tested in animal models of human cancer. This agent showed remarkable antitumor activity in AIDS-Kaposi's sarcoma, glioblastoma multiforme, and breast cancer models in immunodeficient animals. cpIL4-PE caused partial or complete regression of established human tumors. Preclinical efficacy and toxicity studies provided a therapeutic window in which this cancer-targeted agent could be used. On the basis of these studies, we initiated a Phase I clinical trial for the treatment of recurrent glioblastoma multiforme. Our preliminary clinical results suggest that cpIL4-PE has antitumor activity against the deadliest form of brain tumors, without detectable toxicity to normal brain tissues. Thus, IL-4 receptors represent novel targets for cancer cytotoxin therapy.     

靶向EGFR 免疫毒素BI7D12-PE38KDEL 的制备及其体外活性测定

目的:构建基于EGFR 纳米抗体的免疫毒素BI7D12-PE38KDEL,并检测其对EGFR 肿瘤细胞的细胞毒作用。方法:采用分子克隆技术,将靶向EGFR 的纳米抗体7D12 以二价的形式,通过柔性连接肽(G4S)4 与铜绿假单胞菌外毒素的截断形式PE38KDEL 连接,构建原核表达载体pET28a-BI7D12-PE38KDEL。然后将其转化到BL21(DE3)感受态细胞,用IPTG进行诱导表达。通过镍柱亲和层析法纯化目的蛋白,并经Western blot 验证。通过流式细胞技术检测该免疫毒素与EGFR 阳性细胞A549、HT29、MCF-7 和EGFR 阴性细胞CEM、Jurkat 的结合能力后,将其按照一定的浓度梯度进行细胞毒实验研究,72 h后,使用WST-1 试剂检测BI7D12-PE38KDEL 对上述细胞的杀伤效果。结果:通过SDS-PAGE 和Western blot 实验验证,成功的制备了重组免疫毒素BI7D12-PE38KDEL,该毒素大部分以可溶性的方式表达。BI7D12-PE38KDEL 能够与EGFR 阳性的A549、HT29、MCF-7 肿瘤细胞特异性的结合,并且对上述细胞的细胞毒作用与阴性对照组CEM 和Jurkat 相比具有极其显著性差异(P<0.01)。结论:成功地构建了基于EGFR 纳米抗体的重组免疫毒素BI7D12-PE38KDEL,该免疫毒素能特异性的结合并杀伤EGFR 阳性的肿瘤细胞。     

重组人IL-6D24-PE40外毒素融合蛋白的构建及其生物学效应

目的 构建重组人白细胞介素6－绿脓杆菌外毒素融合蛋白IL－6D24－PE40,以选择性杀伤高表达IL－6受体（IL－6R）的肿瘤细胞和白血病细胞。方法 采用重叠延伸的基因融合技术将N－末端缺失24个氨基酸的重组人白细胞介素6（IL－6D24）cDNA与缺失细胞结合区的绿脓杆菌外毒素PE40基因进行融合,构建IL－6D24－PE40融合基因。利用HB101／pBV220表达系统,实现了IL－6D24－PE40融合蛋白在大肠杆菌中的高效表达,经MonoQ柱进行层析纯化,采用MTS法检测细胞毒活性。结果 IL－6D24－PE40融合蛋白在大肠杆菌中的表达水平达到40％－60％。包涵体蛋白经分离、复性及纯化得到纯度＞95％的融合蛋白。Western blot证明,纯化的融合蛋白与IL－6抗体及PEA抗体均发生特异性结合。细胞毒活性检测证实,IL－6D24－PE40融合蛋白能高度特异地选择性杀伤高表达IL－6R的U937细胞,ID50约为250ng/ml,而对不表达IL－6R的CEM细胞无杀伤作用。结论 IL－6D24－PE40融合蛋白能够选择性杀伤高表达IL－6R的靶细胞,有希望成为导向治疗高表达IL－6／IL－6R系统的某些白血病和其他肿瘤的新型导向药物。     

Development of a recombinant interleukin-4-Pseudomonas exotoxin for therapy of glioblastoma.

About 12,000 Americans are diagnosed with malignant astrocytoma each year. Despite surgery, radiotherapy, and chemotherapy, the prognosis of these patients remains poor. Targeted toxins based on the identification of novel antigens or receptors provide a promising new approach to treating cancer. We have identified one such cell surface protein in the form of interleukin (IL)-4 receptors (IL-4R) on human malignant astrocytoma. Normal brain tissues from frontal cortex and temporal lobe cortex do not express IL-4R. To target IL-4R, we generated a chimeric fusion protein composed of IL-4 and Pseudomonas exotoxin (IL4-PE). This toxin is highly cytotoxic to IL-4R-bearing human brain cancer cells. Preclinical toxicologic experiments were performed in mice, rats, and guinea pigs to determine an maximum tolerated dose. Intrathecal administration in cynomolgus monkeys produced high cerebrospinal fluid levels without any central nervous system or other abnormalities. When IL4-PE was injected into the right frontal cortex of rats, localized necrosis was observed at 1,000 but not < or =100 microg/ml doses. Intravenous administration of this biologic to monkeys produced reversible grade 3 or grade 4 elevations of hepatic enzymes in a dose-dependent manner. These results indicate that localized administration can produce nontoxic levels of IL4-PE that may have significant activity against astrocytoma. In vivo experiments with nude mice have demonstrated that IL4-PE has significant antitumor activity against human glioblastoma tumor model. Intratumor administration of IL4-PE has been initiated for the treatment of malignant astrocytoma in a phase I clinical trial.     

Cancer gene therapy utilizing interleukin-13 receptor alpha2 chain

Cancer cells are known to express cell surface molecules such as specific antigens or cytokine receptors, e.g., EGFR, Fas/CD95, gp100, HER-2/neu, IL-13Ralpha2, and MAGE. Among them, interleukin-13 receptor (IL-13R) alpha2 chain is expressed on certain types of cancer cells including glioblastoma, AIDS Kaposi's sarcoma, and head and neck cancer. This protein is one of the receptor components for IL-13, a Th2 cell-derived pleiotropic immune regulatory cytokine. IL-13Ralpha2 chain on these cancer cells can be targeted with a receptor-directed cytotoxin termed IL13-PE to induce specific cancer cell killing, however, this molecule does not mediate cytotoxicity to cells that do not express or express low levels of IL-13Ralpha2. In order to achieve a broad therapeutic window for IL13-PE, plasmid-mediated gene transfer of IL-13Ralpha2 in cancer cells was employed in vitro and in vivo. Cancer cells transfected with IL-13Ralpha2 demonstrated increased binding to IL-13 and sensitivity to IL13-PE in vitro. In vivo intratumoral gene transfer of IL-13Ralpha2 profoundly enhanced the antitumor activity of IL13-PE, providing complete elimination of established tumor in some xenografts. In this review article, current findings from IL-13Ralpha2 gene transfer in a variety of human cancer models in nude mice are summarized. In addition, safety issues and possible future directions utilizing this therapeutic approach are discussed.     

Interleukin-13 fusion cytotoxin arrests Schistosoma mansoni egg-induced pulmonary granuloma formation in mice

Schistosoma mansoni egg-induced lung pathology requires the actions of interleukin (IL)-4 and IL-13. Because receptors for IL-4 and IL-13 share chains, we examined the effect of a fusion protein comprised of IL-13 and Pseudomonas exotoxin (IL13-PE) on the development of pulmonary granulomas in mice. At day 8 after an intravenous injection of live S. mansoni eggs, whole lung samples from IL13-PE-treated mice exhibited significantly lower IL-4 and IL-13 gene expression, smaller granulomas, decreased collagen levels, and increased IL-13 receptor alpha2 gene expression compared to controls. The therapeutic effects of IL13-PE were also observed at day 16 despite the termination of IL13-PE treatment at day 8. These studies demonstrate that targeting IL-4- and IL-13- responsive cells with IL13-PE effectively arrests S. mansoni egg granuloma formation.     

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.     

B cell receptors for interleukin 2: demonstration of IL2 internalization and of complementary effects of lipopolysaccharide and phorbol diester on receptor expression

We have previously shown (J. Exp. Med. 1984. 160: 1170) that murine B cells activated with lipopolysaccharide (LPS) and rabbit anti-mouse Ig antibodies together (LPS-RaMIg blasts) express high-affinity interleukin 2 receptors (IL 2R) and respond to IL 2. This is not the case for B cells activated with either LPS alone (LPS blasts) or anti-Ig alone. In the present study IL 2R function and expression were further investigated by using this model. First, it was found that LPS-RaMIg blasts internalize IL 2 in a time- and temperature-dependent manner very similar to that occurring in CTLL (T lineage) cells. LPS blasts, however, did not internalize IL 2. LPS blasts were found to express 12 times less binding sites for anti-IL 2R monoclonal antibody (PC 61 monovalent Fab) as compared to LPS-RaMIg blasts and at least 30 times less IL 2 binding sites of high as well as of lower affinity. Second, with regard to the requirements for receptor expression, it was observed that either anti-Ig or phorbol diester (phorbol-12-myristate 13-acetate) can induce IL 2R and IL 2 responsiveness (proliferation assay) in LPS blasts but not in fresh B cells. Taken together these results provide further evidence for the similarity of IL 2R function in activated B and T cells, confirm that surface-Ig cross-linkage and phorbol-12-myristate 13-acetate have similar effects on B cells and suggest that LPS on the one hand, and phorbol-12-myristate 13-acetate or anti-Ig on the other provide complementary signals necessary for IL 2R expression.     

Administration of IL-2-PE40 via osmotic pumps prevents adjuvant induced arthritis in rats. Improved therapeutic index of IL-2-PE40 administered by continuous infusion

IL-2-PE40 is a chimeric cytotoxin composed of interleukin 2 (IL-2) fused to a truncated form of Pseudomonas exotoxin (PE) that lacks its binding domain. IL-2-PE40 has been shown to exhibit therapeutic potency in several models in vivo when administered i.p. twice a day. Here we show that the continuous administration of IL-2-PE40 by an osmotic pump specifically prevents the development of adjuvant induced arthritis in rats with an improved therapeutic efficacy as compared to daily repeated i.p. injections. Stabilization of IL-2-PE40 at 37 degrees C for the continuous administration by pumps was achieved by adding NAD, the substrate for the enzyme portion of the chimeric toxin.