Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter

DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (d(m)C) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.     

逆转录病毒介导的IL－2和TNF－α融合基因在卵巢癌细胞中的表达及其对肿瘤细胞生长的影响

ＩＬ－４和ＴＮＦ－α是引人注目的抗肿瘤活性因子，两者在抗肿瘤及免疫调节方面具有协同作用。我们利用逆转录病毒载体Ｘｄ１构建了插入ＩＬ－２和ＴＮＦ－α融合基因的重组逆转录病毒载体ＸｄＦ，融合基因包括编码ＩＬ－２前导肽和ＩＬ－２和ＴＮＦ－α成熟肽的序列。重组载体用Ｌｉｐｏｆｅｃｔｉｎ方法引入病毒包装细胞ＰＡ３１７，挑选Ｇ４１８抗性克隆，用ＮＩＨ３Ｔ３细胞测定病毒滴度，获得一滴度为１×１０￣４ＣＦＵ／ｍｌ的高滴度克隆。超速离心浓缩这一重组病毒上清，转导人卵巢癌细胞系ＳＫＯＶ３，经Ｇ４１８筛选获得抗性克隆。ＰＣＲ可从融合基因转导的细胞ＤＮＡ中扩增出１．１ｋｂ的融合基因片断，证明目的基因完整的整合在细胞的基因组ＤＮＡ中，测其上清中的ＩＬ－２和ＴＮＦ－α活性结果显示：ＩＬ－２的活性为８－１５Ｕ／１０￣６ｃｅｌｌｓ／２４ｈ，ＴＮＦ－α的活性为１５０－４００Ｕ／１０￣６ｃｅｌｌｓ／２４ｈ。这一结果表明逆转录病毒介导的融合基因可以在卵巢癌细胞中获得有效表达，表达的融合基因产物在体内和体外部对肿瘤细胞的生长具有抑制作用。     

胰岛素样生长因子Ⅱ在肝细胞癌的免疫组化研究

以兔抗胰岛素样生长因子Ⅱ(IGF-2)的抗体,ABC法,对50例肝癌石蜡切片染色,结果:(1)正常肝有极微弱阳性,表明可分泌少量IGF-2;(2)癌旁肝27例,全部阳性,“++”者19例(70%),表明有大量IGF-2产生和存在;(3)50例肝癌,48例(96%)阳性,IGF-2呈胞浆弥散型分布,其中26例(52%)呈较强阳性(++),8例核内也有IGF-2,表明癌细胞能自己合成IGF-2为其恶性增殖创造条件,且不因分化程度之降低而有所减少;(4)IGF-2呈较强阳性(++)之细胞(包括小多角形肝细胞)既见于癌周肝,又见于慢性肝炎,表明胰岛素样生长因子Ⅱ直接和增殖而非与癌变相关。     

人大肠癌中Ⅳ型胶原酶与基底膜免疫组织化学双重染色研究

目的观察Ⅳ型胶原酶和基底膜在大肠癌生长中的相互关系及Ⅳ型胶原酶的表达与ｐ２１的表达之间的关系。方法应用免疫组化双标记方法，对８６例人大肠癌组织的Ⅳ型胶原酶表达及基底膜的改变作对照研究。结果大肠癌组织中Ⅳ型胶原酶的阳性率为８３．７２％（７３／８６），与对照组癌周正常肠粘膜的阳性率１０％差异有非常显著意义（Ｐ＜０．０１）。同时，双染色显示Ⅳ型胶原酶阳性的癌组织基底膜表现为不连续，出现缺口、片段缺失或完全消失等形态。此外，肠癌中Ⅳ型胶原酶的分布与ｐ２１的表达呈正相关（ｒ＝０．９７４，ｐ＜０．０１）。结论Ⅳ型胶原酶的分泌对肠癌基底膜破坏起重要作用，可以作为判断大肠癌生长特性的辅助指标。     

单侧外周伤害性刺激诱发大鼠脊髓中转录因子CREB的双侧磷酸化(英文)

c AMP反应成分结合蛋白 (c AMP response elem ent-binding protein,CREB)是一种转录因子 ,它在磷酸化之后可调节靶基因的转录。 CREB在 13 3位置的丝氨酸的磷酸化 ,与脊髓中伤害性传入的处理有关 ,本文作者等用特殊抗体对此进行了免疫细胞化学研究。在正常大鼠 ,虽然几乎所有脊髓神经元的核中都可见 CREB的轻度着色 ,但磷酸化的 CREB仅见于双侧腰段脊髓的 ～ 层 (75± 15 vs60± 18)和 层 (9± 3 )。用福尔马林注射引起一侧后脚掌出现炎症时 ,可在双侧腰段脊髓看到磷酸化CREB细胞核的快速 (小于 5 min)和节段性的显著增多 ;它们主要分布在双侧背角表层 ～ 层 (2 5 4± 2 0 vs 2 62± 2 3 )、 层(115± 13 )和双侧 ～ 层 (3 46± 2 0 vs3 2 8± 2 6) ;而在对照和炎症组大鼠的胸段脊髓中均未见磷酸化 CREB的增加。在注射CFA诱发一侧炎症或切断一侧坐骨神经的实验组大鼠 ,也可看到至少延续到第三天的强而双侧性的 CREB的磷酸化。这种由一侧后肢伤害性传入引致腰段脊髓中镜像式双侧 CREB磷酸化的出现 ,与一般看到的损伤传入只在同侧脊髓背角引起某些神经化学改变的结果不同 ,可能是人神经损伤后或在实验动物中出现对侧镜像式疼痛过敏现象的基础     

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

Rapid and Sensitive Assay for Detection of Enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is an obligatory anaerobic, gram-negative bacterium found among the normal intestinal flora of humans. Enterotoxigenic strains of B. fragilis (ETBF) have been associated with diarrheal diseases in humans and animals. The enterotoxin of ETBF induces fluid changes in ligated intestinal segments and cytotoxic response in HT29/C1 cells. By using a pair of monoclonal antibodies (MAbs; MAb C3 and MAb 4H8) specific for the lipopolysaccharide of B. fragilis, an assay based on immunomagnetic separation (IMS) in combination with PCR (IMS-PCR) was developed. After DNA extraction, a 294-bp fragment was amplified. The specificity of the IMS-PCR assay was evaluated by adding previously isolated and confirmed ETBF strains to normal fecal samples. All fecal samples to which ETBF strains were added were positive, showing a 100% specificity. The spiked fecal samples were also used for evaluation of the sensitivity of the assay. The detection limit was found to be ~50 CFU/g of feces. By this method 10 clinical fecal samples (5 from patients with diarrhea and 5 from healthy controls) were examined. The results of PCR were in accordance with the results of the HT29/C1 cell assay for all samples. The minimum time to retrieval of the final result by the IMS-PCR method is 36 h. The proposed IMS-PCR assay is rapid and sensitive for the direct detection of ETBF in stool samples.     

21－羟化酶CYP21B基因Ile~（172）→Asn错义突变

为了解先天性肾上腺皮质增生症患者的２１－羟化酶ＣＹＰ２１Ｂ基因中Ｉｌｅ~（１７２）→Ａｓｎ错义突变的发生率，根据放大受阻突变体系（Ａｍｐｌｉｆｉｃａｔｉｏｎｒｅｆｒａｃｔｏｒｙｍｕｔａｔｉｏｎｓｙｓｔｅｍ，ＡＲＭＳ）的要求，设计了３种引物：５＇ｄ（ＴＴＧＧＧＡＧＡＣＴＡＣＴＣＣＣＴＧＣＴＣＴ）３＇（共同引物）、５＇ｄ（ＡＧＧＴＧＡＧＧＴＡＡＣＡＧＡ）３＇（正常引物）、５＇ｄ（ＡＧＧＴＧＡＧＧＴＡＡＣＡＧＴ）３＇（突变引物），在７例患儿中进行了检测，发现具有本突变者３例。对其中一例进行的家系分析，结果提示：这组引物有快速、简便的优点，不需使用同位素就能对具有Ｉｌｅ~（１７２）→Ａｓｎ变异的高危家庭成员作产前诊断。     

Functional CD4+T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependentepitope of CD45 molecules(JH6.2) and one a natural thymocytotoxicautoantibody (NTA) with an unknown reactive epitope (NTA260),we subdivided splenic CD4⁺ T cells from 2-month-old BALB/c miceinto five phenotypically distinct subsets. CD45RC⁺NTA260^–(SI) cells were phenotypically analogous to CD4⁺ T cells predominatingin newborn mice and produced a significant amount of IL-2, butnot so IL-4, IL-10 or IFN- when stimulated with immobilizedanti-CD3 mAb in vitro. They appeared to consist mainly of naiveT_hP cells. The CD45RC⁺;NTA260⁺ (S II) subset also produced IL-2,but not other cytokines; however, the IL-2 levels produced weremuch higher than seen with the S I subset, thereby suggestingthe predominance of further maturated T_hP cells. The D45RC^–NTA260⁺(S III) subset mainly produced IL-4, IL-10, IFN- and less IL-2,and contained memory cells that helped the secondary antibodyresponse to a recall antigen, and hence contained T_h2 and probablya mixture of T_h0 and T_h1 cells. The CD45RC^–NTA260^–(S IV) subset was a poor responder to the immobilized anti-CD3mAb. The CD45RC^brightNTA260^dull(S V) subset consisted of a smallnumber of cells that were phenotypically analogous to activatedCD4⁺ T cells. While an age-associated decrease in the proportionof S I and less markedly in S II and in turn increase in S IIIsubsets of CD4⁺ T cells occurred in normal BALB/c mice, autoimmunedisease-prone (NZBxNZW)F₁ mice showed a marked age-associateddecrease in the proportion of not only S I, II but also IIIsubsets. As aged (NZBxNZW)F₁ mice carry CD4⁺ T helper cellsfor IgG anti-DNA antibody production, such age-associated polarizationto the S IV subset appears to be critical in the pathogeneslsof autoimmune disease in these mice.