Membrane-bound/soluble IL-2 receptor (IL-2R) and levels of IL-1α, IL-2, and IL-6 in the serum and in the PBMC culture supernatants from 17 patients with hematological malignancies

The present study investigated the peripheral blood mononuclear cells (PBMC) blastic responses to PHA, PHA plus recombinant IL-2 (rIL-2) and rIL-2 alone; the expression of membrane-bound IL-2R on PHA-stimulated PBMC; and the levels of IL-1α, IL-2, IL-6, and sIL-2R in serum and in culture supernatants from PHA-stimulated PBMC in 17 patients with hematological malignancies (mean age 58.5 yr, range 22–82): 6 with non-Hodgkin’s lymphoma (NHL), 4 with Hodgkin’s lymphoma (HL), 5 with Hairy cell leukemia, 1 with chronic myelogenous leukemia, and 1 with chronic lymphocytic leukemia. The patients with HL and NHL with active disease (AD) were separated from those in clinical remission. The patients with AD were studied at diagnosis (obviously before therapy) and the patients in clinical remission were out of therapy since at least 6 mo. The lymphocyte blastogenic response to PHA was significantly lower in patients with HL and NHL with AD than in the control group. The response to rIL-2 alone was in the same range in the control group and in HL and NHL AD patients. By adding rIL-2 to PHA there was an increase of the blastogenic response of the same patients. The percentage of CD25 expressed on PHA-stimulated lymphocytes from patients with HL and NHL AD and from normal subjects is in the same range. Serum levels of IL-2, IL-6, and sIL-2R were significantly higher in HL and NHL AD patients than in controls as well as in all other hematological malignancies. Supernatants derived from PHA-stimulated PBMC were assessed for the presence of cytokines and sIL-2R by ELISA. The levels of IL-2, IL-6, and sIL-2R were significantly lower in HL and NHL AD patients than in controls as well as in all other hematological malignancies.     

哮喘患儿血白细胞介素-10、13检测的临床意义

目的探讨哮喘患儿外周血白细胞介素-10(IL-10)、13的变化及其在哮喘发病机制中的作用。方法用ELISA双抗体夹心法测定20例哮喘患儿及18例正常儿童血浆IL-10、13的含量。结果哮喘组血浆IL-13水平明显高于正常对照组,IL-10水平明显低于正常对照组(p均<0.05)。结论IL-10、13等细胞因子参与儿童哮喘发病的病理生理过程,可为判断病情提供较好的实验室参数。     

GTPγS restores nucleophosmin (NPM) localization to nucleoli of GTP-depleted HeLa cells

Previous studies showed that localization of nucleophosmin/B23 (NPM) to nucleoli requires adequate cellular GTP levels (Finchet al., J Biol Chem 268, 5823–5827, 1993). In order to study whether hydrolysis of GTP plays a role in NPM localization, we introduced a nonhydrolyzable GTP analog into HeLa cells. Cells were first depleted of GTP with the IMP dehydrogenase inhibitor, mycophenolic acid (MA), to induce translocation of NPM from the nucleoli to the nucleoplasm. Non-hydrolyzable GTP analogs were then introduced into cells by electroporation. We found that introduction of the non-hydrolyzable analog, GTPS, was effective in restoring NPM localization to nucleoli. Cells incubated in medium containing G-nucleotides without electroporation showed no effect. To reduce the possibility that cells use guanine from degraded nucleotide to supplement GTP pools via salvage pathways, experiments were also performed in the presence of (6-mercaptopurine) 6MP, a competitive inhibitor of the salvage enzyme, HGPRT (hypoxanthine guanine phosphoribosyl transferase), in addition to MA. Under these conditions, introduction of GTPS still effectively restored the localization of NPM into nucleoli. This study demonstrates that electroporation can be used effectively to introduce nucleotides into cultured cells without excessive loss of viability. Our results also indicate that the GTP dependent localization of NPM to the nucleoli may not require GTP hydrolysis.     

Peroxisome proliferator-activated receptor-alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis

The interleukin-12 (IL-12) family of cytokines which includes IL-12, IL-23, and IL-27 play critical roles in T cell differentiation and are important modulators of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Previously, we demonstrated that peroxisome proliferator-activated receptor (PPAR) -α agonists suppress the development of EAE. The present studies demonstrated that the PPAR-α agonist fenofibrate inhibited the secretion of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 by lipopolysaccharide-stimulated microglia. The cytokines interferon-γ and tumor necrosis factor-α also stimulated IL-12 p40 and IL-27 p28 expression by microglia, which was suppressed by fenofibrate. Furthermore, fenofibrate inhibited microglial expression of CD14 which plays a critical role in TLR signaling, suggesting a mechanism by which this PPAR-α agonist regulates the production of these pro-inflammatory molecules. In addition, fenofibrate suppressed the secretion of IL-12p40, IL-23, and IL-27p28 by lipopolysaccharide-stimulated astrocytes. Importantly, fenofibrate suppression of EAE was associated with decreased expression of IL-12 family cytokine mRNAs as well as mRNAs encoding TLR4, CD14, and MyD88 known to play critical roles in MyD88-dependent TLR signaling. These novel observations suggest that PPAR-α agonists including fenofibrate may modulate the development of EAE, at least in part, by suppressing the production of IL-12 family cytokines and MyD88-dependent signaling.     

Production of interleukin-1 (IL-1) and a specific IL-1 inhibitor during human monocyte-macrophage differentiation: Influence of GM-CSF

Mononuclear phagocytes release several factors involved in host defense and inflammation. Of these, interleukin-1 (IL-1) has multiple biological activities which are controlled at different levels including modulation of gene expression, protein synthesis or secretion, and interaction with inhibitors. We have investigated the production of IL-1 alpha and beta as well as the production of a specific IL-1 inhibitor (IL-1 INH) during the in vitro maturation of human monocyte-macrophages. Highly purified monocytes isolated by counterflow centrifugal elutriation were cultured up to six weeks, producing high levels of IL-1 alpha and beta during the first week of culture. Shortly after the first week bioactivity of IL-1 decreased, preceding a decrease of IL-1 immunoreactivity. In contrast, IL-1 inhibitory activity reached a peak during the third week and remained detectable up to six weeks. Granulocyte-monocyte-colony-stimulating factor GM-CSF increased the production of IL-1 INH by approximately 20%, but did not affect IL-1 production. The IL-1 INH, apparent molecular weight approximately 23 kD, blocks the binding of [125I]IL-1 alpha to its receptor. The balance between the production of IL-1 and its antagonist may be important for the regulation of the immune response and chronic inflammation during pathological processes.     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract     

白介素10和白介素17基因启动子区多态性位点与儿童哮喘的相关性研究

目的：探讨IL-10基因启动子区-627A/C和IL-17基因启动子-152A/G位点多态性与儿童哮喘发生的相关性。方法：采用聚合酶链反应-限制性片段长度多态性分析（PCR-PFLP）方法检测186名哮喘儿童、198名健康儿童各个多态性位点的基因型,采用SPSS13.0进行统计学分析。结果：IL-17基因-152A/G位点的基因型及等位基因频率分布在哮喘组与正常对照组均存在显著性差异（p〈0.05）,哮喘组-152A/G位点等位基因A频率显著高于正常对照组（x2=6.077,p=0.014,OR=1.430,95%CI=1.076-1.902）。结论：IL-17基因-152A/G位点可能与儿童哮喘的发病存在关系,其中A等位基因可能是易感基因,携带A的个体可能更易患有哮喘。     

白介素18结合蛋白研究进展

白介素18结合蛋白（IL-18BP）是新近发现的一种糖蛋白,属免疫球蛋白超家族成员,目前已发现有6种IL-18BP的同工蛋白,IL-18BP可以在体内外有效抑制IL-18的作用。因而被认为是IL-18的天然拮抗剂,另外发现几种痘病毒编码的蛋白质与IL-18BP有高度同源性,其病毒产物可减弱IL-18诱导的Th1反应,利用IL-18BP拮抗IL-18的作用进行基因治疗将为某些自身免疫性疾病的治疗开辟一条新的途径。     

A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18

IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 by forming a 1:1 high affinity (Kd=400 pM) complex, exhibiting a very low dissociation rate. We have developed a sandwich ELISA for IL-18BPa and determined its limit of detection (62 pg/ml). Interference by IL-18 and related cytokines, as well as cross reactivity with other IL-18BP isoforms (b, c, and d) were determined. Using this ELISA, we measured serum IL-18BPa in large cohorts of healthy individuals and in septic patients. Serum IL-18BPa in healthy individuals was 2.15+/-0.15 ng/ml (range 0.5-7 ng/ml). In sepsis, the level rose to 21.9+/-1.44 ng/ml (range 4-132 ng/ml). Total IL-18 was measured in the same sera by an electrochemiluminescence assay and free IL-18 was calculated based on the mass action law. Total IL-18 was low in healthy individuals (64+/-17 pg/ml) and most of it ( approximately 85%) was in its free form. Total IL-18 and IL-18BPa were both elevated in sepsis patients upon admission (1.5+/-0.4 ng/ml and 28.6+/-4.5 ng/ml, respectively). At these levels, most of the IL-18 is bound to IL-18BPa, however the remaining free IL-18 is still higher than in healthy individuals. We conclude that IL-18BPa considerably inhibits circulating IL-18 in sepsis. Yet, exogenous administration of IL-18BPa may further reduce circulating IL-18 activity.     

Direct evidence of a heterotrimeric complex of human interleukin-4 with its receptors.

The mode of binding of interleukin-4 (IL-4) to its two known receptors, specific receptor IL-4R and a shared receptor gamma c, was investigated using gel filtration and gel electrophoresis. A ternary complex between IL-4 and the soluble domains of the two receptors was shown to exist in solution. The association constant between gamma c and the stable complex of IL-4/sIL-4R is in the millimolar range, making the ternary complex a feasible target for crystallization studies.     

白介素10和白介素17基因启动子区多态性位点与儿童哮喘 的相关性研究

目的:探讨IL-10基因启动子区-627A/C和IL-17基因启动子-152A/G位点多态性与儿童哮喘发生的相关性。方法:采用聚合酶链反应-限制性片段长度多态性分析(PCR-PFLP)方法检测186名哮喘儿童、198名健康儿童各个多态性位点的基因型,采用SPSS13.0进行统计学分析。结果:IL-17基因-152A/G位点的基因型及等位基因频率分布在哮喘组与正常对照组均存在显著性差异(p<0.05),哮喘组-152A/G位点等位基因A频率显著高于正常对照组(x2=6.077,p=0.014,OR=1.430,95%CI=1.076-1.902)。结论:IL-17基因-152A/G位点可能与儿童哮喘的发病存在关系,其中A等位基因可能是易感基因,携带A的个体可能更易患有哮喘。     

白细胞介素12在细胞免疫及抗肿瘤中的作用

IL-12是由巨噬细胞和B细胞产生的一种二聚体细胞因子,是具有多种生物活性的免疫效应细胞生长刺激因子。细胞因子网络在维持免疫功能,促进机体抗感染方面起重要作用。特别是能促进T细胞和NK细胞的增殖与杀瘤作用,诱导IFN-γ等多种细胞因子的产生,调节Th1细胞发育,因而具有良好的抗癌作用。     

Co-expression of IL-18 binding protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced arthritis

We constructed a recombinant adenoviral vector containing a murine interleukin （IL）-18 binding protein （mlL-18BP） and murine IL-4 （mIL-4） fusion gene （AdmIL-18BP/mIL.4） and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in modulating the T-helperl and T-helper2 （Th1/Th2） balance in mice with collagen-induced arthritis （CIA）. Mice with CIA were intra-articularly injected with 107 pfu/6 μl ofeitherAdmIL.18BP/mIL-4, or a controladenovirus, or with the control vehicle （phosphate-buffered saline）. After intra-articular gene therapy with AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-α （TNF-α）, T-interferon （IFN-γ）, IL-4, IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-T-expressing and IL-4-expressing CD4^＋ T cells from mice splenocytes were monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after intraarticular injection of AdmIL-18BP/mIL-4 showed significantly increased serum concentrations of IL-4 and IL-10 （P〈0.01 at all time points） but greatly decreased serum concentrations ofIFN-γ, TNF-α and IL-β （P〈0.01 at all time points ） compared to both the con trol adenovirus and phospha tebuffered saline control groups. The percentage of LFN-γ- producing CD4^＋ T cells was significantly decreased in response to local AdmIL-18BP/mIL-4 treatment. The percentage of IL-4-producing CD4^＋ T cells increased significantly at 1 week after local injection of AdmIL-18BP/ mIL-4 then returned to normal by week 4. These data indicated the significant modifying effects on the Th1/Th2 imbalance in murine CIA produced by local overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4 is a promising potential therapy for rheumatoid arthritis.     

Immunolocalization of IL-17A, IL-17B, and their receptors in chondrocytes during fracture healing.

Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in the epiphyseal growth plate.     

Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins. (c) 1992 John Wiley & Sons, Inc.