Expression of the interleukin-2 receptor (Tac antigen/CD25) in hematologic neoplasms

The expression of interleukin-2 (IL-2) receptor (CD25) has been investigated in 165 cases of hematologic neoplasia by alkaline phosphatase-antialkaline phosphatase (APAAP) labeling of cell smears using two monoclonal anti-IL-2 receptor antibodies (anti-Tac and DAKO-IL2-R). IL-2 receptor was found in 2 of 16 (13%) T-cell malignancies (1 T-ALL, 1 Sézary syndrome). In contrast, among B-cell disorders, IL-2 receptor was expressed in the majority of hairy cell leukemia cases (14 of 16, i.e., 88%) and a smaller proportion of cases of common acute lymphoblastic leukemia (7 of 44, i.e., 16%), B-cell chronic lymphocytic leukemia (8 of 20, i.e., 40%), B-cell prolymphocytic leukemia (3 of 5), and B-cell lymphoma (3 of 8). In addition, IL-2 receptor was present on the neoplastic cells in some cases of acute myeloid leukemia (6 of 44, i.e., 14%) and acute undifferentiated leukemia (4 of 7). IL-2 receptor (Tac antigen) expression is thus found across the spectrum of hematologic neoplasms and is not restricted to T-cell disorders. The explanation for the expression of the IL-2 receptor by such a variety of different hematologic malignancies remains unclear.     

Ligand-dependent selection of the receptor gene: segregation of IL-2 binding activity and anti-Tac reactivity by a single amino acid alteration in the Tac antigen (p55)

The Tac antigen (p55, CD25) is a 55 kDa glycoprotein that binds interleukin 2 at low affinity (Kd congruent to 10-50 nM). Expression of the Tac antigen is induced in the activated human T cells to constitute the functional, high-affinity IL-2 receptors (IL-2Rs) (Kd congruent to 10 pM) in conjunction with p70-75. A monoclonal antibody, anti-Tac, recognizes this molecule and inhibits the binding of IL-2 to both high- and low-affinity IL-2Rs. This observation indicates that IL-2 and anti-Tac binding sites are located close to each other within the Tac molecule. In this report, by utilizing a novel approach, we selected cDNAs encoding the Tac antigen variants whose reactivity with anti-Tac is greatly reduced, while retaining their IL-2 binding activity. Each of the mutant cDNAs contained a point (G----A) mutation resulting in an amino acid substitution at the particular amino-terminal portion of the Tac molecule (Asp-4). These results demonstrate that N-terminal amino acid Asp-4 is involved in the epitope recognized by anti-Tac, and that IL-2 binding site and anti-Tac binding site are structurally separable from each other in the Tac molecule.     

Cloning and characterization of a cDNA specific for bovine retina

A retina-specific cDNA clone (pCR18) was selected from a bovine retinal cDNA library and characterized. The clone pCR18 consisted of 905 base pairs and hybridized to the mRNA of about 12S from the bovine retina, but not that from the brain or liver. The nucleotide sequence revealed a long open reading frame which encodes a 147 amino acid polypeptide of about 15,700 Da. No significant sequence homology with the predicted protein was found in the protein sequence library of about 3500. Messenger RNA which hybridized to pCR18 translated a polypeptide of about 19,000 Da in a reticulocyte translation system. Southern blot analysis indicated that the bovine genome contains a single copy of this gene. Furthermore, RNA dot analysis showed that the poly(A)+ RNA from the human retinoblastoma cell lines (Y79 and WERI) hybridized to pCR18, whose intensity was comparable to that of the bovine retina. In situ hybridization revealed that pCR18 was expressed mostly in some ganglion cells of the rat retina. The results suggest that cDNA clone (pCR18) encodes a protein specific for the retina and mRNA for pCR18 is mostly localized in the retinal ganglion cells and also expressed in the human retinoblastoma cells, although its function remains to be elucidated.     

大鼠脑中催产素受体cDNA的克隆

目的: 克隆大鼠脑中催产素受体(OTR)cDNA编码区全长。方法: 从分娩后SD大鼠脑组织中提取总RNA, 通过RT-PCR扩增出大鼠OTRcDNA, 与pMD18-T载体连接, 构成重组质粒, 进行测序并与子宫中OTRcDNA序列进行比较。结果:获得OTRcDNA编码区全长, 并且与Rozen等报道的子宫OTRcDNA序列完全一致。结论: 克隆了脑中催产素受体(OTR)cDNA, 并且证明中枢神经系统内OTR与外周的OTR一样。     

人IL—2受体α链cDNA体外转录产物的表达

重组质粒pJSB13由载体pGEM3多克隆位点中插入人IL-2受体α链cDNA12-937bp片段构成。本文报导了利用载体上的SP6启动子,在体外合成了与插入cDNA片段互补的正链RNA,并用帽子结构类似物在此RNA 5′端进行了加帽,此加帽RNA能在兔网织红细胞体外翻译体系和爪蟾卵母细胞中有效翻译,且翻译产物能分泌到卵母细胞培养液中。     

犬IL-18 cDNA的克隆及其免疫学特性

目的：获得犬白介素-18(cIL-18)基因并探讨其作为基因疫苗佐剂的免疫效果。方法：从犬的外周血中分离白细胞，经ConA刺激培养5～12h。提取犬白细胞总RNA作为模板，通过RT-PCR技术扩增cIL-18 cDNA。将其克隆到载体pMD18-T中测序，并与已发表的序列进行比较。将cIL-18 cDNA克隆入pIRES1 neo中，构建cIL-18真核表达载体。以200txg：200txg的比例，与狂犬病病毒糖蛋白表达载体plGneo混合免疫犬，并与糖蛋白单独免疫犬的免疫应答进行比较。结果：扩增获得单一长约0．6kh核酸带，测序证明长度为582bp，编码193个氨基酸，与从犬肺巨噬细胞中扩增的cIL-18基因的序列完全一致。以构建的表达载体pIIL18与pIGneo混合免疫与用pIGneo单独免疫相比较，前者抗狂犬病病毒特异性抗体的水平显著低于后者；但混合免疫组犬外周血淋巴细胞对特异性和非特异性抗原刺激的反应性显著高于糖蛋白单独免疫组。结论：cIL-18全长为582bp，其真核表达载体具有增强细胞免疫应答的能力，同时可显著抑制体液免疫应答。本研究为cIL-18作为基因疫苗佐剂的研究奠定了基础。     

Expression of Tac antigen by non-Hodgkin's lymphomas

Anti-Tac is a monoclonal antibody that appears to recognize the interleukin-2 receptor. With the use of a frozen-section immunoperoxidase technic, a large series of non-Hodgkin's lymphomas were investigated for the presence of Tac-antigen on neoplastic cells. Approximately one-fourth of cases expressed the Tac antigen, including 27% of B-lineage lymphomas, 6% of the T-lineage lymphomas, and three of four cases of Ki-1-expressing lymphoma. The B-lineage lymphomas with the highest incidence of Tac antigen expression were the large cell lymphomas, both diffuse and follicular, where about one-half of cases expressed the Tac antigen. All major categories of lymphoma expressed Tac except plasma-cytoma/myeloma, small noncleaved cell (Burkitt's and non-Burkitt's), and lymphoblastic malignancies.     

Expression of p55 (Tac) interleukin-2 receptor (IL-2R), but not p75 IL-2R, in cultured H-RS cells and H-RS cells in tissues.

The authors studied the secretion of interleukin-2 (IL-2), the expression of interleukin-2 receptors (IL-2R; p55/Tac and p75), and the response to exogenous IL-2 by cultured Hodgkin's Reed-Sternberg cells (cell lines HDLM-1, HDLM-1d, and KM-H2) and T cells (H9, HuT78, HuT102, MOLT-4, and MT-2). All of these cells did not produce IL-2 or produced it in undetectable amounts, and their growth was not affected by the addition of anti-IL-2 or anti-IL-2R antibodies. This indicates that H-RS cells in long-term culture, as well as T cells, can grow independently of IL-2. The three H-RS cell lines, as well as two of the T-cell lines (HuT102 and MT-2), expressed Tac, whereas the other three T-cell lines were Tac negative. Expression of p75 was noted in the two Tac-positive T-cell lines, but not in cultured H-RS cells. The expression of Tac and p75 in HuT102 and MT-2 cells correlated well with their capacity to proliferate on treatment with exogenous IL-2. On IL-2 treatment, nucleic-acid uptake in Tac/p75-positive T cells increased approximately four- to sixfold, whereas the Tac/p75-negative T cells did not show increased proliferation. Unlike the T cells, the Tac-positive H-RS cells did not respond to IL-2. The lack of a proliferative response to IL-2 appears to be related to the absence of p75 in H-RS cells. A similar pattern (Tac positivity and p75 negativity) was noted in H-RS cells in lymph nodes involved by Hodgkin's disease. Thus the exogenous IL-2 released by surrounding T lymphocytes may not cause the proliferative activity of H-RS cells because of the lack of high-affinity IL-2 receptors in the latter cells. In contrast to H-RS cells in culture, H-RS cells in tissues were stained by a specific anti-IL-2 monoclonal antibody. This indicates that the expression of IL-2 or an IL-2-like substance by H-RS cells in tissues may be responsible, in part, for the great increase in the number of reactive T lymphocytes in tissues involved by Hodgkin's disease.     

A competitive enzyme-linked immunoassay (ELISA) for the measurement of soluble human interleukin-2 receptors (IL-2R, Tac protein)

A solid-phase, competition enzyme-linked immunosorbent assay (ELISA) was established for the quantitative measurement of soluble (human) interleukin-2 receptors (IL-2R). The ladder of reagents from the solid phase up consisted of: (1) recombinant DNA-derived, purified IL-2R, (2) sample-containing soluble IL-2R and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody, 7G7/B6, directed against the IL-2R, (3) alkaline phosphatase-conjugated rabbit anti-FITC, and (4) substrate. This ELISA was compared with a 'sandwich' ELISA for soluble IL-2R. The competitive ELISA was less sensitive than the 'sandwich' assay, being capable of measuring 5000 versus 31 U/ml, respectively. While both anti-Tac and 7G7/B6 in the IL-2R-containing sample inhibited the 'sandwich' assay, only 7G7/B6 inhibited the competition assay. Anti-mouse immunoglobulin enhanced the 'sandwich' assay and inhibited the competitive assay; both effects could be overcome by the addition of normal mouse immunoglobulin in the sample buffer. Studies of a patient's serum receiving anti-Tac as therapy for the adult T cell leukemia demonstrated that rises in the level of IL-2R occurring with anti-Tac therapy, as measured with the competition assay, were masked in the 'sandwich' assay. This competition ELISA will be useful for measuring soluble IL-2R levels in patients receiving anti-Tac as therapy for various immunologic disorders.     

编码人分化抗原5C5全长cDNA的克隆及功能初探

目的 编码５Ｃ５分化抗原全长ｃＤＮＡ的克隆及功能探索。方法 构建人活化Ｂ细胞株３Ｄ５细胞的λｇｔ１１ ｃＤＮＡ文库，以核苷酸杂交法从ｃＤＮＡ文库中筛选阳性克隆、作核苷酸序列分析，编码蛋白质氨基酸序列的亲疏水分析，Ｎｏｒｔｈｅｒｎ ｂｌｏｔ测５Ｃ５ ｍＲＮＡ转录本长度，用ＲＴ－ＰＣＲ检测５Ｃ５ ｍＲＮＡ在不同细胞株的表达，观察单抗５Ｃ５－Ｇ１对３Ｄ５细胞增殖的影响。结果 从人活化Ｂ细胞株３Ｄ５的λｇ     

Cloning of a Partial cDNA for Rat Interleukin-12 (IL-12) and Analysis of IL-12 Expression In Vivo

Experimental models of autoimmunity in the rat may feature selective activation of either the Th1 or Th2 subset of helper T cells. Interleukin-12 (IL-12) is a key cytokine in the development of Th1 responses. In order to study IL-12 in the rat we used polymerase chain reaction (PCR) primers based on murine IL-12 to amplify a partial cDNA from rat tissue. The product was cloned and sequenced: it shows 94% nucleotide identity with the murine gene and 94% identity of predicted amino acid sequence. Primers based on the rat IL-12 sequence were used to analyse IL-12 expression in vivo using semi-quantitative PCR. We studied RNA from lymphoid tissues of two rat strains which differ in their response to mercuric chloride (HgCl₂): Brown Norway (BN) rats develop autoimmunity with a predominant Th2 response; Lewis rats are resistant. Interleukin-12 expression was higher in Lewis than BN, and higher in spleen than lymph node. After HgCl₂, IL-12 expression increased in BN towards the time when the autoimmune response autoregulates. Variation in baseline levels of IL-12 expression may account for the Th2 predisposition of BN rats compared to Lewis rats; IL-12 may play a role in the autoregulation of the Th2 response induced by HgCl₂     

中国人IL—18结构蛋白cDNA的克隆和序列分析

目的克隆人白介素 - 18结合蛋白 (h IL- 18BP)的 c DNA,以研究其结构与功能的关系。方法从中国汉族人的胎盘组织中提取总 RNA,以 RT- PCR方法扩增出全长 5 85个 bp的 IL- 18BP c DNA,将其与表达载体 pc DNA3连接 ,转化大肠杆菌 DH5 α,建立了 h IL- 18BP的 c DNA克隆。结果序列分析表明 ,与国外文献报道的人 IL- 18BP的 c DNA序列完全一致。结论 h IL- 18BP已成功地得到克隆。     

脂质体介导IL-2、TNF-α联合基因转导体内抗肝细胞癌的实验研究

目的 :探讨脂质体介导IL 2cDNA、TNF αcDNA联合基因转导体内抗肿瘤效果。方法 :建立裸鼠肝癌动物模型 ,在荷瘤部位将脂质体包裹的IL 2cDNA和TNF αcDNA直接注入瘤体中 ,观察肿瘤大小变化 ,并检测此 2种基因在肿瘤组织中的表达情况。结果 :IL 2基因和TNF α基因联合治疗组肿瘤生长明显受到抑制 ,抗肿瘤效果显著优于单纯治疗组和对照组。结论 :IL 2和TNF α基因联合治疗组对肿瘤生长抑制效果明显优于单纯治疗组与对照组 ,荷瘤鼠生存期明显延长 (P <0 0 5 )。