CR2-CD59和CD59-CR2靶向补体抑制物的构建、表达与鉴定

CD59是补体的细胞膜抑制剂,它通过与C8和C9结合,可达到阻止MAC(membrane attack complex)组装的目的.而且CD59不影响补体调理素、C3a或C5a的产生.目前研究结果表明可溶性CD59(soluble forms of CD59,sCD59)对补体相关疾病的治疗作用甚微[1,2].直接将靶向补体抑制物定位至补体相关疾病位点的策略可能提高对补体的抑制效率,同时可以避免由于系统性和长期性补体抑制带来的副作用.已有研究表明,抗体-CD59靶向融合蛋白比非靶向补体抑制物能更有效保护目标细胞[3].因此,本研究拟采用可溶性CR2作为将补体抑制物(CD59)定位至补体相关疾病位置的载体,从而构建一种更有效的靶向补体抑制物,使其只对补体激活位点产生抑制,而不影响机体免疫系统正常的补体调节,为研制炎症相关性疾病新的治疗药物奠定基础.     

CD137/CD137L在抗病毒免疫中作用的研究进展

病毒持续感染常导致抗原特异性T细胞功能损害,病毒则可逃避免疫监视以致不能被清除.CD137/CD137L是除CD28/B7以外另一对具有多种活性的T细胞共刺激分子,在抗病毒免疫中有重要调节功能,尤其对增强和维持CD8+T细胞应答起关键作用.近年研究表明,CD137/CD137L在抗病毒免疫治疗中具有潜在的临床应用价值.     

LAIR-1/LAIR-2(CD305/CD306)推定配体分布的研究

目的：研究LAIR-1（CD305）及LAIR-2（CD306）推定配体在多种细胞系上的分布，为鉴定LAIR-1／LAIR-2推定配体提供了实验依据。方法：建立稳定表达LAIR-1-Fc和LAIR-2-Fc融合蛋白的细胞系。以纯化的融合蛋白进行活细胞免疫荧光染色，用流式细胞仪检测LAIR-1及LAIR-2推定配体在多种细胞系膜表面的表达，并比较LAIR-1和LAIR-2推定配体的分布及可能结合的位点。结果：LAIR-1推定配体及LAIR-2推定配体均在人羊膜来源的上皮细胞系WISH上高表达，在人黑色素瘤细胞系C32、人胚肾上皮细胞系293T及人脐静脉内皮细胞系ECV304上有一定程度的表达。LAIR-1-Fc和LAIR-2-Fc融合蛋白与推定配体的结合，可分别被可同时识别LAIR-2和LAIR-1的单克隆抗体（mAb）FMU-LAIR-2．2和FMU-LAIR-2．1阻断。结论：LAIR-1推定配体及LAIR-2推定配体的分布基本一致，主要表达于WISH、C32、293T和ECV304细胞系。LAIR-1和LAIR-2可能拥有共同的配体。     

CD23表达与B细胞增殖分化的研究进展

本文探讨了生理和病理情况下B细胞表面CD23的表达，分析了其表达与B淋巴细胞增殖分化的关系，总结了影响B细胞表达CD23的各种细胞因子的作用特点及其可能机制，同时归纳了（可溶性CD23sCD23）的生物学作用及其介导的信号传递途径的新研究进展。本文还探讨了EBV感染的B细胞表面CD23的表达与B细胞体外长期存活的分子生物学机制。     

经CR2激活MAPKs与抑制HIV-gp160处理的CD4+细胞增殖

目的建立CR2稳定表达的HOS-CR2、HOS-CD4CR2细胞系,研究细胞中MAPKs的活化和细胞增殖变化,阐明经CR2的信号传导、CR2的表达对HIV-gp160等所致的CD4+细胞的影响.方法用哺乳动物细胞稳定转染法建立稳定表达CR2的HOS-CR2、HOS-CD4CR2细胞系,细胞经磁珠阳性纯化后,用FACS和Western blot对CR2的表达进行鉴定.用PMA、10% NHS、HIV-gp160等处理细胞后,经Western blot检测细胞的MAPKs的活化水平;用Cell Titer 96 Aqueous One Solution Reagent检测和分析处理后细胞的增殖差异.结果 FACS检测和统计学方法分析结果表明CR2表达的阳性率高达96%以上;Western blot结果显示所建HOS-CR2、HOS-CD4CR2细胞系的CR2的表达水平与Raji细胞相近;细胞经PMA、预先激活补体的10% NHS处理细胞,发现在处理10 min时ERK、JNK、P38的活化达高峰;PD98059、Wortmanin和抗-CR2能阻断ERK、JNK、P38的活化.HIV-gp160、预先激活补体的10% NHS能相应激活HOS-CR2, HOS-CD4 和HOS-CD4CR2细胞ERK、JNK、P38,这些激活均可被相应的抗体所阻断;细胞增殖实验结果显示HIV-gp160能抑制细胞增殖(P＜0.01),CR2的表达能加重HIV-gp160所致的细胞增殖抑制(P＜0.01).结论磁珠阳性纯化法可浓集稳定表达靶基因的细胞,提高阳性率和表达水平.刺激因素可经由CR2激活MAPKs,CR2的表达促进HIV-gp160所诱导的细胞死亡,与HIV感染所致的CD4+细胞缺损密切相关,CD4+细胞缺失机制的阐明将有助于AIDS致病机制和防治策略的研究.     

头孢地嗪增加老年细菌性肺炎患者外周血CD4/CD8和Th1/Th2细胞比例

目的探讨头孢地嗪对老年细菌性肺炎患者外周血T淋巴细胞CD4/CD8和Th1/Th2细胞比例的影响。方法选取老年细菌性肺炎患者63例,以随机数字表分为两组;对照组31例采用头孢三嗪静脉滴注疗法,观察组32例采用头孢地嗪静脉滴注疗法治疗。分别于治疗前后抽取患者空腹静脉血,以流式细胞仪测定CD4/CD8和Th1/Th2的细胞计数比;以ELISA试剂盒测定Th1细胞分泌因子白细胞介素2(IL-2)、γ干扰素(IFN-γ)与Th2细胞分泌因子IL-4、IL-10的血清浓度。结果治疗前,两组患者的各项指标均无明显差异。治疗后,观察组患者CD4+细胞、Th1细胞比例增加,Th2细胞比例降低,CD4/CD8和Th1/Th2比值均显著高于对照组;同时,观察组患者血清IL-2、IFN-γ浓度显著高于对照组,IL-4、IL-10浓度显著低于对照组。结论头孢地嗪能够提高老年细菌性肺炎患者细胞免疫功能,纠正外周血T淋巴细胞CD4/CD8和Th1/Th2比例失调。     

CD3/CD8设门在FCM检测Th1/Th2亚型中的应用

目的　探讨流式细胞术准确、灵敏检测Th1/Th2细胞的新方法。方法　采用荧光mAbCD3和CD8设门 ,准确找到CD4 + T细胞群 ,然后进行Th1/Th2细胞分析 ,并与单用CD4设门进行比较。结果　以CD3/CD8设门较单用CD4设门 ,对靶细胞的定位较为准确 ,测量所获图形美观、清楚并有规律。结论　以CD3/CD8设门 ,可提高检测Th1/Th2细胞的准确性 ,同时也可应用于Tc1/Tc2细胞的检测分析 ,是检测基础和临床免疫样品较理想的方法     

CD21-independent Infection of Epstein-Barr Virus in Human Signet Ring Gastric Carcinoma Cell Line

Epstein Barr virus ( EBV), a ubiquitous human her pesvirus, is the etiologic agent of infectious mononucleo sis, and is closely associated with several human malig nancies including Burkitt′s lymphoma (BL), undifferenti ated nasopharyngeal carcinoma (NPC) and opportunisticlymphoma in immunocompromised hosts. In recent years,there have been increasing evidences of association of EBVwith additional malignancies, such as gastric carcinoma.EBV has been found in tumor cells of m…     

食管鳞状细胞癌组织中miR-21与ERK1/2表达及临床意义的研究

目的：研究miR-21和ERK1/2在食管鳞状细胞癌(esophageal squamous carcinoma,ESCC)组织中的表达并分析二者的临床意义。方法：原位杂交技术和免疫组化方法分别检测80例食管鳞状细胞癌及癌旁正常组织中miR-21和ERK1/2的表达,Spearman相关统计方法分析miR-21和ERK1/2表达的相关性,并分析二者与ESCC患者临床病理资料的关系。结果：miR-21和ERK1/2在ESCC组织中的阳性表达率和表达量都显著高于癌旁正常组织,尤其在有淋巴结转移的ESCC组织中显著增高,但在ESCC不同性别、年龄、民族、病理类型、肿瘤侵及范围和肿瘤大小间的差异无统计学意义。进一步分析发现miR-21与磷酸化的ERK1/2(p-ERK1/2)在ESCC及癌旁正常组织的表达呈正相关,相关系数为0.570。结论：miR-21和p-ERK1/2与ESCC发生发展密切相关,二者可能协同通过促进肿瘤细胞淋巴结转移增加ESCC的恶性程度。     

乙型肝炎患者红细胞CR1密度相关基因多态性与CR1活性的相关性

为研究乙型肝炎患者红细胞ＣＲ１活性与ＣＲ１密度相关基因多态型别的关系,采用ＰＣＲ加ＨｉｎｄⅢ酶切技术测定红细胞ＣＲ１密度相关基因多态型别（ＨＨ、ＨＬ、ＬＬ型）,采用红细胞ＣＲ１（ＥＣＲ１）免疫粘附活性（ＲＣＩＡ）和粘附ＩＣ量免疫酶联（ＥＬＩＳＡ）法测定红细胞ＣＲ１活性。乙型肝炎患者ＨＨ型比率是７３９％（３４／４６）,正常人ＨＨ型比率是８０％（６４／８０）,ＨＨ型乙型肝炎患者ＥＣＲ１ＲＣＩＡ（３７８±０２９ｎｇＡＨＧ／１０７ＲＢＣ）明显高于ＨＬ型乙型肝炎患者（１８８±０２６ｎｇＡＨＧ／１０７ＲＢＣ）和ＨＨ型正常人（２６０±０３０ｎｇＡＨＧ／１０７ＲＢＣ）。提示乙型肝炎患者红细胞免疫功能低下是获得性的。     

Origin and properties of soluble CD21 (CR2) in human blood

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L-cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR-LON-HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130 kD found by SDS–PAGE analysis. This may be partly accounted for by the non-globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.     

Distribution of the chromatin protein DEK distinguishes active and inactive CD21/CR2 gene in pre- and mature B lymphocytes

DEK is an abundant and ubiquitous chromatin protein that has only recently attracted attention. DEK preferentially binds to cruciform and superhelical DNA and induces positive supercoils into closed circular DNA. It is quite likely therefore that DEK performs an important architectural function in chromatin. However, it is not known how DEK is distributed in chromatin. As the first study of its kind, we investigate the distribution of DEK at the CD21/complement receptor 2 gene regulatory regions in two B lymphocyte lines, namely Ramos, which expresses the CD21 gene, and Nalm-6, which does not. We use a chromatin immunoprecipitation approach and show that DEK appears to be distributed over various regions of the expressed and silent genes, but occurs in 2- to 3-fold higher amounts at a promoter-proximal site of the expressed gene. Moreover, induction of CD21 expression in Nalm-6 cells leads to accumulation of DEK at this site. We propose that the accumulation of DEK is functionally linked to gene expression.     

Expression of Epstein-Barr virus nuclear antigen-2 (EBNA2) induces CD21/CR2 on B and T cell lines and shedding of soluble CD21

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) expressed as a fusion protein with the hormone-binding domain of the estrogen receptor was used to study expression of CD21 and other surface markers in different cell lines. Special emphasis was placed on cell lines with a normally low expression of CD21, especially on T cell lines. After induction of EBNA2, a substantial increase in CD21 mRNA was observed, as well as increased production of membrane CD21. This was found not only in cell lines of B cell origin, but also in the T cell line Jurkat. The amount of CD21 was quantitated by means of a fluorescence immunoassay, and found to correlate with the presence of EBNA2 protein. A decrease in EBNA2 abundance was associated with complete loss of cell-associated CD21. As we could also detect large amounts of soluble CD21 (sCD21) in the supernatant of the transfected cell lines, which exceeded the total amount contained in the respective cell lysates, this indicates considerable shedding of the newly synthesized receptor molecules induced by EBNA2, comparable to the situation described for CD23. It further provides an explanation of the recent findings of increased sCD21 levels in sera of patients with EBV-associated disease, and suggests a possible additional function of EBNA2 in vivo.     

The CR2/CD19 complex on human B cells contains the src-family kinase Lyn

The complement receptor 2 (CR2 or CD21) can be found in non-covalentassociation with the Blymphocyte specific CD19 complex at thesurface of mature human B cells. Upon ligation of the B cellantigen receptor complex (BCR), members of the CR2-CD19 complexmay associate with membrane immunoglobulin (mlg). Moreover,CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein—Barrvirus, are known to have profound effects on B cell activation.We here show that CD19 is tightly linked to the non-receptorsrc kinase Lyn and that the CD19 glycoprotein itself servesas a substrate for a yet undefined serine/threonine kinase presentwithin the complex. In the process of antigen recognition, mlgand the CR2-CD19 complex may bind different sites of a complement-opsonizedantigenic particle. We hypothesize that in this process, approximationto the BCR allows CD19-associated Lyn kinase to phosphorylatepotential substrates within the antigen—receptor complex,thereby effecting its coupling to the intracellular compartment.     

Complement receptor type two (CR2,CR21)

Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed.     

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor,CR2) that binds iC3b and CD23

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short concensus repeats (SCR) 1–2, SCR 4–5 and SCR 9–11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore^TM technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1–2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.     

Participation of CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in membranoproliferative glomerulonephritis type I.

Intraglomerular expression of complement receptors (CR) was investigated chronologically in 22 repeatedly biopsied patients with membranoproliferative glomerulonephritis (MPGN) type I by indirect immunoperoxidase staining using MoAbs. Patients were divided into two groups based on whether intraglomerular C3c deposition was decreased at the second biopsy (2nd Bx) (group A, n = 12), or not (group B, n = 10). At the first biopsy (1st Bx), the severity of glomerular injury and the degree of glomerular C3c deposition were compatible between the two groups. Four patterns of CR1 (CD35) expression on podocytes were recognized: normal; generally decreased; focally/segmentally lost; and completely lost. The numbers of CR3 (CD11b/CD18)- and CR4 (CD11c/CD18)-positive cells per glomerular cross-section were counted. At the 1st Bx, no significant difference was found in the number of CR3+ or CR4+ cells between the two groups. At the 2nd Bx, the numbers of both the CR3+ and CR4+ cells were significantly decreased only in group A (P < 0.01). The numbers of CR3+ and CR4+ cells were significantly higher in cases with moderate or marked C3c deposits than in those with no or mild C3c deposits. The intensity of CR1 expression in group B was less than that in group A at both the 1st and 2nd Bx (1st, P < 0.05; 2nd, P < 0.01), and chronological improvement of CR1 expression was observed only in group A. The severity of glomerular injury was increased only in group B (P < 0.01), and was associated with persistent massive proteinuria and hypocomplementaemia. Our results suggest that, in cases with an adverse outcome, a more severe defect of CR1 initially exists and the expression of CR1 is not recoverable chronologically. This irreversible decrease or loss of CR1 may partly contribute to the continuous C3c deposition and intraglomerular infiltration of CR3+ and CR4+ cells.     

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein-Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as-yet-unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon-11-encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21-pool. CD21 shedding is induced by stimulation with PMA plus Ca(2+) ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.