重组人可溶性CD14在昆虫细胞表达系统中的表达

BAC-TO-BAC杆状病毒表达系统是一种快速、高效、便捷的表达系统.将人可溶性CD14(sCD14)基因克隆入pFASTBAC1转移质粒中,重组质粒转化DH10BAC感受态细胞,目的基因通过同源重组插入BacmidDNA中,后者转染sf21昆虫细胞获得重组杆状病毒.利用重组蛋白C-末端的6×His@Tag,经TALON金属螯合色谱将重组病毒感染昆虫细胞获得的无血清培养上清--步纯化得到重组蛋白,计算表明从1L培养基中可纯化到约8mg纯度大于95%的重组sCD14蛋白,免疫印迹结果表明重组蛋白具有与抗6×His单抗和抗CD14单抗结合的抗原性.疑胶迁移实验和细胞活性实验表明重组sCD14蛋白能在体外与LPS结合,并能增强LPS诱导THP-1细胞产生细胞毒性因子.     

Varying importance of soluble and membrane CD14 in endothelial detection of lipopolysaccharide

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-beta) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.     

Lipopolysaccharide needs soluble CD14 to interact with TLR4 in human monocytes depleted of membrane CD14

Toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. TLR4 recognizes lipopolysaccharide (LPS) in monocytes/macrophages with the help of other molecules like CD14 and MD-2, which indicates that the functional LPS receptor forms a large complex. The functional relationship between the components has been the subject of debate, as have the modifications induced by the ligand in the expression of some of these components. Moreover, as for other members of this family of receptors, the possible direct interaction of receptors and their ligands is a matter of discussion. In this paper we address the question of whether the expression of some of the components influences the expression of the rest. Human monocytes in which CD14 has been downregulated through interference in the turnover of the molecule at the Golgi level, show normal membrane TLR4 expression, when compared with control cells. On the other hand, LPS alters membrane TLR4 expression by monocytes devoid of membrane CD14 only in the presence of human serum. The effect of serum is blocked by anti-CD14 monoclonal antibodies, which strongly suggests a functional role for soluble CD14/LPS complexes in the interaction with TLR4. Our data add information on the relationship between the components of the LPS receptor and the characteristics of the interaction of LPS and TLR4 in cells devoid of membrane CD14.     

Differential regulation of soluble and membrane CD40L proteins in T cells

CD40 ligand is an important immunoregulatory protein expressed by T cells. This protein exists as two isoforms, a membrane glycoprotein and a truncated soluble form. Here we demonstrate that membrane and soluble CD40L (sCD40L) are differentially regulated depending upon the activation stimulus. In T cell receptor activated cells, both membrane and sCD40L proteins are expressed and CD28 costimulation further increases their expression. The dissection of TCR generated signals into calcium and PKC-dependent pathways demonstrates that calcium is sufficient to induce membrane CD40L yet insufficient for sCD40L. In contrast, sCD40L is preferentially induced by PKC. Moreover, sCD40L production is blocked by Zn(2+)-dependent metalloproteinase inhibitors while membrane CD40L is concurrently increased. This profile suggests the potential involvement of the ADAM-10 protease which was subsequently shown to cleave membrane CD40L to generate sCD40L. Given the role of sCD40L in numerous disease pathologies and its ability to activate proximal and distal immune responses, the regulated cleavage of CD40L may likely contribute to disease mechanisms.     

Expression of the surface membrane receptors of neutrophils in myeloproliferative diseases

For 147 patients with myeloproliferative diseases, a study was made of the expression of Fc-receptors to immunoglobulins IgC, IgA, and that of FcH-receptor and receptors to C3-components of the complement in peripheral blood neutrophils. The data obtained show the lower level of neutrophils having membrane receptors in patients with acute myeloblastic leukemia and chronic granulocytic leukemia at the stage of blast transformation. A decrease in expression of membrane receptors of neutrophils was shown in patients with chronic granulocytic leukemia and benign subleukemic myelosis. The finding of a higher level of neutrophils having surface receptors revealed in patients with real polycythemia is close to the data obtained in the study of expression of membrane receptors in patients with chronic myeloproliferative diseases and healthy persons.     

Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes

CD14 presents as a glycosylphosphatidylinositol-linked membrane protein on the surface of monocytes/macrophages and as a soluble protein in the serum. Our previous studies have shown that an 80-kilobase pair (kb) genomic DNA fragment containing the human CD14 gene is sufficient to direct CD14 expression in a monocyte-specific manner in transgenic mice. In addition, we discovered that human CD14 is highly expressed in hepatocytes. Here, we report the generation of transgenic mice with either a 24- or 33-kb human CD14 genomic DNA fragment. Data from multiple transgenic lines show that neither the 24- nor the 33-kb transgenic mice express human CD14 in monocytes/macrophages. However, human CD14 is highly expressed in the liver of the 33-kb transgenic mice. These results demonstrate that human CD14 expression is regulated differently in monocytes and hepatocytes. Furthermore, we identified an upstream regulatory element beyond the 24-kb region, but within the 33-kb region of the human CD14 gene, which is critical for CD14 expression in hepatocytes, but not in monocytes/macrophages. Most importantly, the data demonstrate that the liver is one of the major organs for the production of soluble CD14. These transgenic mice provide an excellent system to further explore the functions of soluble CD14.     

可溶性CD14基因克隆及序列分析

针对人可溶性CD14(sCD14）的cDNA序列设计引物,通过反转录PCR技术,从U937细胞中,扩增出编码人可溶性CD14的基因序列,并将其克隆至质粒pGEM－T中,通过PCR、酶切及序列测定鉴定重组载体。结果表明获得了人可溶性CD14基因片段,为下一步进行CD14表达及生物学活性研究奠定了基础。     

The potential biological and clinical significance of the soluble tumor necrosis factor receptors

The role of TNF receptors (TNF-Rs) is not limited to signal transduction but includes extracellular regulatory functions affecting systemic TNF bioavailability. This review summarizes the regulation of TNF-R shedding and its kinetics, the complex interaction between the soluble receptors and their ligand in vitro and in vivo, and the potential diagnostic, prognostic and therapeutic value of the soluble receptors in malignant, inflammatory, infectious and autoimmune disorders.     

Dendritic cells,chemokine receptors and autoimmune inflammatory diseases

Dendritic cells (DC) have been implicated in the induction of autoimmune diseases and have been identified in lesions associated with several autoimmune inflammatory diseases. Since DC are regarded as the professional antigen-presenting cell (APC) of the immune system and the only APC capable of activating na?ve T cells, they are likely to play a significant role in breaking tolerance of self-reactive lymphocytes and in supporting autoimmune responses in these diseases. A number of studies have revealed that small molecular weight chemotactic proteins known as chemokines are present within the autoimmune lesions and may contribute to the recruitment not only of DC populations, but also of immune cells such as T cells, B cells, neutrophils and monocytes into the site, and to the formation of organized lymphoid tissue structures within the target organ. The focus of this review will be a discussion of the role of chemokines in the recruitment of DC in human autoimmune inflammatory disorders, specifically the trafficking of DC into the inflammatory sites and the subsequent migration of differentiated DC from the inflammatory sites into the draining lymph nodes. Once DC are properly positioned within the lymph nodes, circulating antigen specific na?ve T cells can interact with DC and become activated, clonally expanded and stimulated to undergo differentiation into antigen-experienced memory T cells. Subsequent reactivation of memory T cells that enter the autoimmune lesions by DC present in the inflammatory lesion is thought to play a central role in tissue inflammation.     

CD133在胃肠间质瘤的表达及其临床意义

目的 探讨胃肠间质瘤（gastrointestinal stromal tumor,GIST）中的CD133的表达及其与GIST临床病理特征的关系.方法 采用免疫组织化学法,检测122例胃肠间质瘤患者组织中的CD133、CD117、CD34、DOG-1、KI-67的表达情况.结果 CD133、CD117、CD34 、DOG-1、KI-67的阳性表达率分别为74.6%（91/122）、98.4%（120/122）、86.9%（106/122）、95.1%（116/122）、47.5%（58/122）.CD133的表达水平与胃肠间质瘤危险度分组高低、核分裂像数目、肿瘤部位有关（P〈0.05）,而与患者性别、年龄、肿瘤大小无明显相关性（P〉0.05）.CD133的表达水平与DOG-1的表达水平无明显相关性（P〉0.05）,而与CD117、CD34、KI-67的表达水平呈正相关.结论 CD133蛋白的表达可能与GIST的恶性行为与预后有关,与CD117、CD34和DOG-1联合检测对于判断GIST的病理性质可能具有重要价值.     

Occurrence, regulation, and significance of progesterone receptors in human meningioma

The abundant expression of progesterone receptors (PR) in human meningiomas is well established. It is unknown, however, how PR expression is regulated, especially since estrogen receptors (ER) are virtually absent in these tumors. At the mRNA level, ER splice variants occur in meningioma but these appear not to be involved in the apparently autonomous PR expression. In an earlier study, because other ER-inducible proteins were either not expressed at all (pS2) or were expressed at a very low level compared to their expression in breast cancer (Cathepsin-D), the authors have postulated that the autonomous PR expression in meningioma is PR promoter-related rather than ER-related and have studied PR expression in cultured meningioma cells. PR levels appeared to decrease rapidly in vitro in monolayer as well as in three dimensional spheroid cultures. Culture conditions thus are not yet sufficient for the quantitative evaluation of PR expression. To evaluate whether PR deterioration is associated with cell turnover (meningiomas grow much faster in vitro than in vivo), the relationship between expression of the apoptotic proteins Bcl-2 and Bax and PR expression was investigated. Bcl-2 expression was found to be highest in meningioma with low PR levels, and in breast cancer tissue with high PR levels. Bax expression was not related to PR expression in any of the two tissues. Given the potential benefit of antiprogestin treatment and the occurrence in meningiomas of a protein capable of binding to the estrogen-responsive element, the expression of PR in meningioma remains a fascinating phenomenon which requires further investigation.     

alpha1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, alpha1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2h) cell exposure to AAT/LPS significantly enhances LPS-induced NF kappaB (p50 and p65) activation in conjunction with increased TNFalpha, IL-1 beta and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFalpha, IL-1 beta and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.     

Cestode regulation of inflammation and inflammatory diseases

Helminth parasites are masters of immune regulation; a likely prerequisite for long-term survival by circumventing their hosts’ attempt to eradicate them. From a translational perspective, knowledge of immune events as a response to infection with a helminth parasite could be used to reduce the intensity of unwanted inflammatory reactions. Substantial data have accumulated showing that inflammatory reactions that promote a variety of auto-inflammatory diseases are dampened as a consequence of infection with helminth parasites, via either the mobilization of an anti-worm spectrum of immune events or by the direct effect of secretory/excretory bioactive immunomodulatory molecules released from the parasite. However, many issues are outstanding in the definition of the mechanism(s) by which infection with helminth parasites can affect the outcome, positively or negatively, of concomitant disease. We focus on a subgroup of this complex group of metazoan parasites, the cestodes, summarizing studies from rodent models that illustrate if, and by what mechanisms, infection with tapeworms ameliorate or exaggerate disease in their host. The ability of infection with cestodes, or other classes of helminth, to worsen a disease course or confer susceptibility to intracellular pathogens should be carefully considered in the context of ‘helminth therapy’. In addition, poorly characterised cestode extracts can regulate murine and human immunocyte function, yet the impact of these in the context of autoimmune or allergic diseases is poorly understood. Thus, studies with cestodes, as representative helminths, have helped cement the concept that infection with parasitic helminths can inhibit concomitant disease; however, issues relating to long-term effects, potential side-effects, mixed pathogen infections and purification of immunomodulatory molecules from the parasite remain as challenges that need to be addressed in order to achieve the use of helminths as anti-inflammatory agents for human diseases.     

RhoC和Ki67在非小细胞肺癌中的表达及意义

目的探讨RhoC、Ki67在非小细胞肺癌中的表达状况及其与临床病理学参数间的关系。方法利用免疫组织化学(ElivisionTMplus法),检测肺癌组织芯片中151例非小细胞肺癌患者RhoC、Ki67的表达情况。结果肺癌组织的RhoC蛋白阳性表达率为59.60%(90/151),相应癌旁组织RhoC阳性表达率为32.7%(16/49),差别有显著意义(P<0.05)。RhoC在TNMIII/IV期的NSCLC组织中阳性表达率为68.9%(51/74),明显高于在Ⅰ/II期NSCLC组织中阳性表达率50.6%(39/77,P<0.05);RhoC在出现淋巴结转移的表达率为49.2%(32/65),显著高于没有淋巴结转移的阳性表达率67.4%(58/86,P<0.05);非小细胞肺癌中RhoC高表达同Ki-67高表达呈显著正相关关系(χ2=21.634,r=0.377,P<0.01)。结论RhoC的高表达与非小细胞肺癌的分期,淋巴结转移及增殖有关。     

Toll receptors,CD14, and macrophage activation and deactivation by LPS

This review will focus on the molecular mechanisms of macrophage activation and desensitization by bacterial lipopolysaccharide (LPS). The most recent advances in the understanding of the function of the LPS receptor complex and its role in the development of the septic shock syndrome and endotoxin tolerance will be discussed.     

水牛CD14 cDNA的克隆、表达及鉴定

目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定.方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入E coli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定.结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带.结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础.     

MCM7与RACK1在肺癌中的表达及其临床意义

目的探讨MCM7与RACK1在各类型肺癌中的表达及其相关性。方法收集肺腺癌150例、鳞癌150例、大细胞癌20例和小细胞癌50例及其癌旁正常肺组织石蜡标本,采用免疫组织化学S-P法检测各类型肺癌组织和癌旁正常肺组织中MCM7与RACK1的表达,并分析二者与肺癌临床病理因素的关系。结果 MCM7与RACK1在癌组织中的表达明显高于癌旁正常肺组织;χ~2检验结果显示肺癌中MCM7与RACK1的表达均与肺癌的病理分级、淋巴结转移、组织学分类和临床TNM分期相关;Pearson相关性分析结果显示,肺癌中MCM7与RACK1的表达呈正相关;Kaplan-Meier法分析显示MCM7与RACK1高表达患者生存时间缩短。结论肺癌中MCM7与RACK1的表达呈正相关,二者表达在肺癌中起促进作用,可作为检测肺癌细胞的增殖状态、新的判断预后的重要因子,以其为靶向进行临床治疗的重要指标。     

High concentration of soluble HLA-DR in the synovial fluid: generation and significance in "rheumatoid-like" inflammatory joint diseases

In the search for its role in inflammatory joint diseases, soluble HLA-DR (sHLA-DR) was quantitated in 72 synovial fluids (SF) by a newly established immunoenzyme assay. Unlike other soluble receptors which accumulated only moderately (sCD25, sCD4) or negligibly (sHLA class I, sCD8) in the SF, SF sHLA-DR levels exceeded serum levels by up to 3 orders of magnitude and varied disease dependently from "control" values (traumatic synovitis and osteoarthritis: 9.9 +/- 6.1 ng/ml). Clear-cut different SF sHLA-DR values in HLA-DR-associated "rheumatoid-like" (136.5 +/- 130.0 ng/ml) vs HLA-B27-associated "spondylarthropathy-like" arthritic forms (28.4 +/- 29.1 ng/ml) were most significant comparing oligoarticular juvenile chronic arthritis type I (147.6 +/- 112.6 ng/ml) and type II (3.3 +/- 1.1 ng/ml), thus offering a new classification marker. Also ex vivo, large amounts of sHLA-DR were released spontaneously by SF mononuclear cells and found to be related to the T-cell activation state. SF sHLA-DR may be shed in large complexes or micelles, as it eluted mainly at >450 kDa on gel filtration. Western blotting revealed that the majority of SF sHLA-DR consisted of full-length alpha- and beta-chains. Minor fractions of smaller sized antigens seemed to be generated by proteolytic cleavage rather than by alternative splicing, since only minute amounts of HLA-DRB mRNA lacking the transmembrane exon could be amplified by RT-PCR. Distinct forms of high-dose sHLA-DR, able to provoke rather than to suppress T-cell responses, are discussed as contributing to some HLA-DR disease association.