Chemical conjugation of a novel antibody-interleukin 2 immunoconjugate agains t c-erbB-2 product

Objective To develop a new chemical method to produce a monoclonal antibody (MoAb) 520C9/recombinant human interleukin 2 (rhIL-2) conjugate. Methods MoAb 520C9 reactive with the protooncogene c-erbB-2 product P185 was chemically conjugated with rhIL-2 by using a simple two-step method.First, the rhIL-2 was activated by Sulfosuccinimidyl 4-［N-maleimidomethyl］ cyclohexane-1-carboxylate, a heterobifunctional linker, and N-succinimidyl s-acetylthioacetate was introduced onto 520C9.Then SATA on the 520C9 was reacted with the maleimide group on the activated rhIL-2 to generate 520C9-rhIL-2 immunoconjugate. Results The immunoconjugate retained the antigen binding activity compared to the respective native antibody as determined by an indirect live cell binding assay.The immunoconjugate also possessed IL-2 activity as measured by the standard CTLL-2 cells proliferation assay and the stimulation of human peripheral blood mononuclear cells (PBMCs) into lymphokine-activated killer cells. Conclusion Our method of conjugation of rhIL-2 to 520C9 preserves the binding activity of the antibody and the cytokine function of IL-2.This simple and efficient method of conjugation should be applicable to other types of MoAbs and recombinant cytokines.     

白细胞介素-4对哮喘患者血清IgE的影响

目的：探讨白细胞介素－４（ＩＬ－４）在过敏性支气管哮喘发病机制中对ＩｇＥ的作用。方法：试验采用双盲、交叉、随机、安慰剂对照的设计方法,给予８例过敏性哮患者吸入２０μｇ人重组ＩＬ－４或载体溶液,并分别于吸入前、吸入后２,２４,４８ｈ测定患者血清ＩｇＥ水平。结果：吸入载体溶液与吸入ＩＬ－４前的血清总ＩｇＥ基础值无显著差异（ｐ〉０．０５）,吸入后各时点血清总ＩｇＥ亦无明显变化（ｐ〉０．０５）。结论：吸入     

烧伤患者血清sIL-2R水平的观察及临床意义

为探讨烧伤患者伤后免疫功能紊乱的可能机制以及烧伤患者病情监测、免疫状况评估的观察指标 ,动态观察了 35例大面积烧伤患者伤后一周、二周、三周及四周血清可溶性白介素 - 2受体 (s IL - 2 R)水平 ,并据烧伤严重程度分中、重、特重度烧伤组进行比较观察。结果发现 :(1)烧伤患者 s IL- 2 R水平明显高于正常对照组 (P<0 .0 1) ,至伤后四周仍无恢复 ;(2 )特重度烧伤患者 s IL- 2 R水平明显高于重度烧伤患者 ,重度烧伤患者 s IL- 2 R水平明显高于中度烧伤患者 (P<0 .0 1)。结论 :升高的 s IL- 2 R是影响烧伤患者免疫功能的众多原因中的重要原因之一 ,伤后 s IL- 2 R水平的观察对烧伤患者病情的监测及免疫状况的评估有一定的临床意义。     

IL-1对肾小球系膜细胞增殖和周期素依赖激酶2表达的影响

目的 探讨白细胞介素－１（ＩＬ－１）对肾小球系膜细胞增殖及周期素依赖激酶２（ＣＤＫ２）表达的影响。方法 利用体外培养的大鼠肾小球系膜细胞,分别采用噻唑蓝掺入法、流式细胞仪检测、免疫细胞化学等观察ＩＬ－１作用前后系膜细胞增殖民政部以及ＣＤ听表达变化。结果 ＩＬ－１可以明显促进系膜细胞增殖,同时伴有ＣＤ听表达增高。结论ＩＬ－１对系膜细胞有明显的促增殖春机制可能与影响ＣＤＫ２的表达有关。     

重组人白细胞介素-1β对人红系、粒系造血祖细胞集落形成的影响

为探讨重组人白细胞介素-1β(rhIL-1β)对人红系、粒系造血祖细胞的作用,采用96孔板微量培养法在体外进行培养。在培养中加入不同浓度的rhIL-β1(0～5ug/mL)后,观察BFU-E、CFU-E及CFU-GM的集落形成。结果加入不同浓度的IL-1β后,BFU-E、CFU-E的集落形成均受到抑制,且呈剂量依赖性,在IL-1β剂量为0.1和0.5μg/mL时,抑制作用更明显(JP<0.005,0.05),而IL-1β对CFU-GM的集落形成无明显影响。结果表明,IL-1β对红系造血祖细胞(BFU-E和CFU-E)影响的研究有助于解释某些慢性疾病性贫血(如类风湿性关节炎伴贫血等)的发生机制,提示IL-1β单克隆抗体有可能用于治疗慢性疾病性贫血。     

Interleukin-1β-induced, nitric oxide-dependent and -independent inhibition of vascular smooth muscle contraction

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1β and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1β-induced relaxation, we examined the effects of interleukin-1β on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1β (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 μM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N^G-monomethyl-

Full-size image

-arginine (

Full-size image

-NMMA, 100 μM), prevented the inhibitory effect of interleukin-1β. After treatment with interleukin-1β for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither

Full-size image

-NMMA (100 μM) nor aminoguanidine (100 μM) reversed the inhibition, whereas methylene blue (10 μM) partially reversed the inhibition. After treatment with interleukin-1β for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 μM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1β treatment. In contrast, the 24-h interleukin-1β treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of

Full-size image

-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1β. The 24 h interleukin-1β treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K⁺ channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1β treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1β inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1β, the NO/cyclic GMP system is activated. After a 24-h interleukin-1β treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.     

Chemokine production by human vascular smooth muscle cells: modulation by IL-13

1. The production of chemokines by vascular smooth muscle cells (SMC) is implicated in the pathogenesis of atherosclerosis, although the factors regulating chemokine production by these cells are incompletely characterized.
2. We describe the differential stimulation of interleukin-(IL)-8, monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T-cell expressed and secreted (RANTES) synthesis following treatment of human vascular SMC with IL-1α or tumour necrosis factor α (TNFα). Under basal conditions, cultured SMC release very low amounts of IL-8, MCP-1 and RANTES as assessed by specific ELISA. Concentration-response studies with IL-1α or TNFα revealed that each stimulus induced a similar amount of MCP-1. In contrast approximately three fold more IL-8 was induced by IL-1α than by TNFα whereas significant RANTES production was induced only by TNFα. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL-1α or TNFα stimulation.
3. The T-cell derived cytokines IL-10 and IL-13 were also found to have differential effects on chemokine production by SMC. IL-13, but not IL-10, significantly enhanced IL-8 and MCP-1 release in response to IL-1α or TNFα. This increase in chemokine release appeared to be accounted for by increased mRNA expression.
4. These findings provide support for the concept that smooth muscle cells can have an active role in a local immune response via the production of chemokines which can be selectively modulated by T-cell derived cytokines.

     

Interferon gamma and interleukin 10 levels in preimplantation embryo culture media

Purpose The aim of our study is to elucidate whether human oocyteslembryos secrete IFN and/or IL-10 and whether the fertilization process depends on the balance between these cytokines.Methods A total of 142 embryo culture media from 24 patients were collected and the cytokine levels were tested with ELISA.Results IFN and IL-10 were detectable in 40.1% and 29.6% of culture media respectively. The difference of IFN and IL-10 levels in media from fertilized oocytes between day 1 and day 2 are significant (0.46 vs. 1.47 and 34.2 vs. 12.7, respectively). However there was no significant difference between the IFN levels of the media from fertilized and nonfertilized oocytes 0.46 vs. 0.85 at day 1 and 1.47 vs. 1.49 at day 2, as well as IL-10 levels 34.2 vs. 30.9 at day 1 and 12.7 vs. 9.58 at day 2 respectively.Conclusions Human preimplantation embryos secrete the cytokines IFN and IL-10. No effect of these cytokines on fertilization process could be shown.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, April 3–7, 1995, Vienna, Austria.     

Tumor-associated lymphocytes (TAL) are competent to produce higher levels of cytokines in neoplastic pleural and peritoneal effusions than those found in sera and are able to release into culture higher levels of IL-2 and IL-6 than those released by PBMC

This work was designed to study the proliferative response of tumor-associated lymphocytes (TAL) from neoplastic effusions against autologous tumor cells and the immunophenotype pattern of TAL from neoplastic effusions and that of PBMC of the same patients. We also compared the serum levels of the cytokines interleukin (IL) 1, 2 and 6, tumor necrosis factor- (TNF) and soluble IL-2 receptor (sIL-2R) with those present in neoplastic effusions of the same patients. Moreover, we examined the ability of TAL and peripheral blood mononuclear cells (PBMC) to produce and release the cytokines and sIL-2R and to express membrane CD25 following their stimulation with phytohemagglutinin (PHA) in vitro. Finally, we compared the cytokines/sIL-2R production and membrane CD25 expression by PHA-stimulated PBMC of the patients with neoplastic effusions with a series of 90 cancer patients without neoplastic effusions and 20 normal healthy subjects. Thirteen neoplastic pleural and eight peritoneal effusions were collected from 11 patients with primary lung cancer, 7 with primary epithelial ovarian cancer, 1 with breast cancer, 1 with pleural mesothelioma, and 1 with pancreatic cancer. The proliferative response of TAL from neoplastic effusions against autologous tumor cells was lower than the response to PHA, IL-2, and anti-CD3, but significant. The percentage distribution of CD3⁺ and CD8⁺ lymphocyte subpopulations was higher in peritoneal than in pleural effusions, while the CD16⁺ subset was higher in pleural than in peritoneal effusions. The percentage distribution of CD16⁺ was significantly lower in pleural effusions than in PBMC of patients with pleural effusions. The CD39 antigen was higher on TAL from peritoneal effusions than on PBMC of the same patients. The levels of IL-1 and sIL-2R in peritoneal effusions did not differ from those measured in the sera of the same patients, while the levels of IL-2, IL-6, and TNF were higher in the peritoneal effusions. The levels of IL-2, IL-6, TNF, and sIL-2R, but not IL-1, in pleural effusions were significantly higher than those found in the sera of the same patients. The amounts of IL-2 and IL-6 produced by TAL were generally higher than those released by PBMC. The secretion of cytokines IL-1, IL-2, and sIL2R by PHA-stimulated PBMC was lower, but IL-1 and IL-6 secretion was higher in cancer patients with neoplastic effusions than in either cancer patients without neoplastic effusions or normal subjects. The CD25 expression on PHA-stimulated PBMC derived from cancer patients with neoplastic effusions was in the same range as that of cancer patients without neoplastic effusions and normal subjects. These findings suggest that TAL may be able to produce cytokines and may be amenable to immune manipulation.Abbreviations FITC Fluorescein-isothiocyanate - IL Interleukin - mAb Monoclonal antibody - MHC Major histocompatibility complex - NK Natural killer - PBMC Peripheral blood mononuclear cells - PHA Phytohemagglutinin - TAL Tumor-associated lymphocytes - TIL Tumor-infiltrating lymphocytes - TNF Tumor necrosis factor- - sIL-2R Soluble interleukin-2 receptor     

白细胞介素-1受体拮抗剂对博莱霉素致大鼠肺纤维化模型的影响

目的：探讨白细胞介素１受体拮抗剂（ｉｎｔｅｒｌｅｕｋｉｎ１ｒｅｃｅｐｔｏｒａｎｔａｇｏｎｉｓｔ,ＩＬ１ｒａ）治疗博莱霉素（ｂｌｅｏｍｙｃｉｎ,ＢＬＭ）致大鼠肺纤维化模型的作用机制。方法：利用ＭＴＴ法测定经ＩＬ１ｒａ作用前、后１周时该模型大鼠肺泡巨噬细胞产生肿瘤坏死因子α（ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒα,ＴＮＦα）,及其培养上清促肺成纤维细胞增殖能力的变化;利用Ｎｏｒｔｈｅｒｎ杂交测定肺泡巨噬细胞ＴＮＦα、血小板衍化生长因子（ｐｌａｔｅｌｅｔｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ,ＰＤＧＦ）ｍＲＮＡ的表达。结果：ＢＬＭ致肺纤维化模型１周时,肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性与正常对照组相比均明显增强,而ＩＬ１ｒａ（１０ｍｇ·Ｌ－１）对肺泡巨噬细胞ＴＮＦα的产生及该组细胞上清的促成纤维细胞增殖活性有明显的抑制作用。ＢＬＭ致肺纤维化模型１周时肺泡巨噬细胞ＴＮＦα、ＰＤＧＦｍＲＮＡ的表达较正常对照组增高,而ＩＬ１ｒａ对其表达无明显影响。结论：ＩＬ１ｒａ通过对肺泡巨噬细胞的ＴＮＦα产生及其对成纤维细胞增殖活性的抑制,可能对该模型的肺纤维化起到抑制作用