The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9

We recently demonstrated that stromal cell-derived factor-1(SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4expressed by human primary lymphocytes and macrophages, andHeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signalingmolecule. Here, we demonstrate that SDF-1 strongly acceleratesthe shedding of syndecan-4 ectodomains and to a lesser extentthat of syndecan-1 from HeLa cells. The fact that this accelerationwas not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence.Pre-treating the cells with heparitinases I, III, or with theprotein kinase C (PKC) inhibitor, bisindolylmaleimide, significantlyinhibited this accelerated shedding, which suggests the involvementof both cell-surface heparan sulfate and PKC transduction pathway.In contrast, Map Kinase or NF-B pathway inhibitors had no effect.Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9)mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9silencing by RNA interference strongly decreases the syndecan-1and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1also accelerates in a CXCR4-independent manner, the sheddingof syndecan-1 and -4 from human primary macrophages, which issignificantly inhibited by anti-MMP-9 antibodies. This stronglyindicates the role of MMP-9 in these events occurring in botha tumoral cell line and in human primary macrophages. BecauseMMP-9 plays a crucial role in extracellular matrix degradationduring cancer cell metastasis and invasion, and shed ectodomainsof syndecans may likely be involved in tumor cell proliferation,these data further indicate the multiplicity of the roles playedby SDF-1 on tumor cell biology.     

碎米蕨属的系统学研究——基于4种叶绿体DNA序列片段

碎米蕨属(Cheilosoria Trevis.)隶属碎米蕨类, 由于形态上的趋同进化, 使得该类群的系统分类一直存在争议。该研究对该碎米蕨属植物的4种叶绿体DNA序列片段(rbcL/matK/rps4/rps4-trnS)进行PCR扩增和序列分析, 再结合其它相关类群, 用贝叶斯法和最大似然法构建系统树并探讨其系统发育关系。结果表明, 碎米蕨属不是一个单系类群, 旧世界分布的碎米蕨属植物(薄叶碎米蕨除外)均聚在亚洲Cheilanthes群内, 与粉背蕨属(Aleuritopteris Fée)等类群形成不同亚支。该属孢子形态具有明显异质性。薄叶碎米蕨(Cheilosoria tenuifolia (Burm. f.) Trevis.)与亚洲其它碎米蕨属植物的系统位置相距甚远, 且与隐囊蕨(Notholaena hirsuta (Poir.) Desv.)聚为完全支持的分支, 两者可能均为大洋洲起源, 并属于另一类群。美洲和旧世界分布的碎米蕨属植物关系较远, 二者可能代表了不同的演化路线。     

北京北部农牧交错区C4植物及其形态功能型和生境分析

根据野外调查和文献资料研究了北京北部农牧交错区C4植物的种类组成、形态功能型组成及其与生境的关系.该区共有野生C4植物68种,分布在7科,40个属,其中禾本科43种,莎草科16种,藜科5种.1年生（ANG和ANF）C4物种占C4植物总数的62%,它们在盐碱地、沙地、弃耕干扰地、湿地均超过这些生境C4植物分布的半数,在草地ANG和ANF超过30%;C4植物数量和C4/total在该地区的生境分布中有两个方向的变化：一是从草地到弃耕干扰地,两者均呈增加趋势,二是从草地到沙地和盐碱地呈现前者减少而后者增加的不同变化趋势,体现了农牧交错区植被退化的复杂性.     

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1)

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.     

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.     

RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.     

C3d3通过促进脾脏B细胞高表达CXCR4增强hCGβ DNA免疫体液免疫效应

本文旨在探讨分子佐剂C3d3与hCGβ融合在基因免疫中增强抗hCGβ体液免疫效应的机制。分别用质粒pCMV4-hCGB-C3d3、pCMV4-hCGβ和pCMV4免疫BALB/c小鼠,间接ELISA法检测免疫小鼠外周血IgG/IgA类抗hCGβ抗体水平;ELISPOT分析免疫鼠脾脏组织IgG/IgA类抗体分泌细胞水平(ASC);RT-PCR分析免疫鼠脾脏B细胞趋化因子受体表达,RT-PCR和FCM分析CXCR4表达水平;RT-PCR和ELISA检测脾脏组织CXCL12表达水平。结果显示,pCMV4- hCGβ-C3d3免疫组外周血IgG类抗hCGβ抗体水平明显高于pCMV4-hCGβ免疫组;而IgA类抗hCGβ抗体水平在两组间无明显差异。pCMV4-hCGβ-C3d3免疫组脾脏组织IgG类ASCs水平明显高于pCMV4-hCGβ组;两组间IgA类ASCs水平无明显差异。经pCMV4-hCGB、pCMV4-hCGβ- C3d3免疫鼠脾脏B细胞CXCR4表达明显高于对照组;且pCMV4-hCGβ-C3d3组明显高于pCMV4-hCGβ免疫组。CXCR4~ 细胞与ASCs呈正相关,r=0.966,(P<0.05)。pCMV4-hCGβ-C3d3和pCMV4-hCGβ组脾脏组织CXC L12表达均显著高于对照组。结果表明,分子佐剂C3d3与hCGβ基因融合,在基因免疫小鼠后能够显著升调节脾脏ASCs CXCR4表达,从而可能增强抗hcGβ基因疫苗的体液免疫效应。     

CDK4/6信号通路靶向治疗剂在癌症治疗中的研究进展

多数癌症的发生发展都具有细胞周期高度活化的特性。细胞周期蛋白依赖性激酶4/6 (CDK4/6)不仅在细胞有丝分裂中发挥了巨大作用,而且参与了衰老、凋亡和组蛋白调节等诸多生物学过程,并在多种癌症的发生发展中被异常激活。FDA批准了Palbociclib、Ribociclib和Abemaciclib等3种靶向CDK4/6的抑制剂,在临床上也取得了显著的疗效,有效地延长了内分泌治疗耐药的乳腺癌患者以及其他多种类型癌症患者的生存期。但这些抑制剂的临床应用也面临着获得性耐药等问题。文中综述了CDK4/6参与的生物调控过程,及其抑制剂在癌症治疗中的应用和面临的耐药性挑战。     

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.     

DPC4／Smad4基因RNA干扰靶点设计和慢病毒载体制备

目的：DPC4／Smad4基因RNA干扰靶点的设计和RNA干扰靶点慢病毒载体制备。方法：针对DPC4／Smad4基因序列,并利用网站设计程序,依据RNA干扰序列设计的原则,设计多个RNA干扰靶点序列。根据设计经验和设计软件将其进行评估测定,选择最佳动力学参数靶点进入其后续的实验流程;生工生物合成含干扰序列的DNAoligo,具有严格的检测体系（PAGE纯化体系）,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞,并且对长出的克隆进行酶切鉴定。然后挑选出阳性克隆测序,进行测序比对后,鉴定阳性的克隆即为构建成功的目的基因RNA干扰慢病毒载体。将构建的慢病毒载体以及辅助包装载体质粒共转染到293T细胞。收获含有病毒的细胞培养上清,浓缩后进行滴度测定,并检测其感染性。另外应用荧光实时定量PCR检测在感染的293T细胞中敲减效果。结果：成功构建DPC4／Smad4shRNA的慢病毒载体LVshSmad4,并成功制备DPC4／Smad4shRNA慢病毒,三株病毒感染细胞后均具有有效的敲减效应,其中SHl最为显著。结论：DPC4／Smad4基因RNA干扰靶点的成功设计和RNA干扰靶点慢病毒制备,为以后探讨DPC4／Smad4基因与肿瘤的相关性治疗提供了实验基础。     

DPC4/Smad4基因RNA干扰靶点设计和慢病毒载体制备*

摘要目的：DPC4/Smad4 基因RNA干扰靶点的设计和RNA 干扰靶点慢病毒载体制备。方法：针对DPC4/Smad4基因序列,并利 用网站设计程序,依据RNA干扰序列设计的原则,设计多个RNA干扰靶点序列。根据设计经验和设计软件将其进行评估测定, 选择最佳动力学参数靶点进入其后续的实验流程;生工生物合成含干扰序列的DNA oligo,具有严格的检测体系（PAGE 纯化体 系）,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞,并且对长出 的克隆进行酶切鉴定。然后挑选出阳性克隆测序,进行测序比对后,鉴定阳性的克隆即为构建成功的目的基因RNA 干扰慢病毒 载体。将构建的慢病毒载体以及辅助包装载体质粒共转染到293T 细胞。收获含有病毒的细胞培养上清,浓缩后进行滴度测定,并 检测其感染性。另外应用荧光实时定量PCR 检测在感染的293T细胞中敲减效果。结果：成功构建DPC4/Smad4 shRNA的慢病毒 载体LVshSmad4,并成功制备DPC4/Smad4 shRNA慢病毒,三株病毒感染细胞后均具有有效的敲减效应,其中SH1 最为显著。结 论：DPC4/Smad4 基因RNA干扰靶点的成功设计和RNA 干扰靶点慢病毒制备,为以后探讨DPC4/Smad4 基因与肿瘤的相关性治 疗提供了实验基础。     

Contribution of circulating leukocytes to cytokine production in pancreatic duct obstruction-induced acute pancreatitis in rats

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.     

Characterization of a subclone (D10S) of the D10.G4.1 helper t-cell line which proliferates to attomolar concentrations of interleukin-1 in the absence of mitogens

Most studies have shown that interleukin-1 (IL-1) acts as a helper or co-stimulator in T-lymphocyte activation and proliferation by mitogens or antigens. We describe here a stable subclone (D10S) of the murine D10.G4.1 helper T-cell which proliferates to subfemtomolar (attomolar) concentrations of IL-1 beta or alpha in the absence of mitogens. D10S cells have been maintained in culture for over two years without splenic cell feeder layers nor antigen stimulation. Detection of proliferation can be made by either uptake of tritiated thymidine at 72 h or in 48 h by a colorimetric assay which measures mitochondrial dehydrogenases; the latter assay is rapid and inexpensive. D10S cells are distinct from the parent clone D10.G4., which requires mitogens for IL-1 activity. IL-1-induced proliferation is independent of the elaboration of IL-2, IL-4, or IL-6, although these cells proliferate to these lymphokines at considerably higher concentrations when compared to IL-1. The D10S cells proliferate in direct correlation to the duration of IL-1 presence in the culture. We found no evidence that IL-1 induced more IL-1 in these cells. The subclone is highly specific for IL-1: proliferation was not observed to endotoxin, human or murine interferon-gamma (IFN gamma), tumor necrosis factor (TNF), lymphotoxin, or granulocyte-macrophage colony stimulating factor (GM-CSF). There was no suppressive effect of transforming growth factor (TGF beta). Only at high concentrations (100 ng/ml) did IL-6 induce proliferation. We conclude that this stable, feeder layer-free cell line is highly sensitive to IL-1 which acts as a direct stimulant for these cells; they are also useful for bioassays as well as the study of IL-1 receptors as described in the accompanying paper.     

利用Gal4/ VP16- UAS 和双荧光素酶报告基因 系统检测??- 分泌酶活性

目的：确立基于Gal4／vp16-UAS和双荧光素酶报告基因系统检测γ-分泌酶切割淀粉样前体蛋白活性的方法。方法：将插入上游激活序列（SAS）和萤火虫荧光素酶报告基因的质粒MH100,嵌舍酵母活性转录因子（Gal4）、单纯疱疹病毒蛋白（VP16）和γ-分泌酶切割位点的质粒C99-GVP,以度海肾荧光素酶质粒pRL—CMV,用脂质体转染法转入稳定表达淀粉样前体蛋白C末端的人神经母细胞瘤细胞（SH—SYSY）,用免疫沉淀Western blot分析法检测β-淀粉样蛋白（邶）的生成,利用Gal4／vp16-UAS和双荧光素酶报告基因系统测定荧光素酶报告基因的表达。结果：免疫沉淀Westem blot分析表明A（的生成在γ-分泌酶激活荆神经节苷脂GM1作用下升高并呈剂量依赖性,同时双荧光素酶法检测γ-分泌酶活性也同步升高。在γ-分泌酶抑制荆作用下Aβ的产生呈荆量依赖性的减少,同时γ-分泌酶活性也同步降低。结论：基于Gal4／vp16-UAS和双荧光素酶报告基因系统检测γ-分泌酶活性的方法有效可靠,是一种敏感、定量的检测方法。     

The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1

The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide-generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide-depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.     

Selective identification of HLA-DP4 binding T cell epitopes encoded by the MAGE-A gene family

Because of the high frequency of HLA-DP4 in the Caucasian population, we have selectively delineated HLA-DP4 restricted T cell epitopes in the MAGE-A tumor antigens. We identified 12 good binders to HLA-DP4 and investigated the capacity of the seven best binders to induce in vitro specific CD4+ T cell lines from HLA-DP4 healthy donors. We found that the MAGE-A1 90–104 peptide exhibited a high and constant frequency of CD4+ T cell precursors in all the six tested donors. The MAGE-A1 268–282 peptide was found immunogenic in only two donors but with a high precursor frequency. The MAGE-A12 127–141 peptide was T cell stimulating in six different donors and induced fewer T cell lines. The peptide-specific T cell lines were stimulated by DC loaded with the lysates of cells transfected with MAGE-A1 or MAGE-A12, or loaded with the recombinant protein. We also show that the immunoreactivity of CD4+ T cell epitopes restricted to the same HLA II molecule may vary from one individual to another, as a result of inter-individual variations in the CD4+ T cell repertoire.     

糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。