自身免疫性肝病患者自身抗体免疫学特点分析

我们对自身免疫性肝病患者血清中出现循环的自身抗体进行分析,探讨自身抗体在鉴别自身免疫性肝病及病毒性肝炎中免疫学之间的关系。一、资料与方法1.研究对象:收集2000年10月至2005年6月来我院门诊及入院就诊的肝功能生化指标。ALT≥40 U/L,且反复异常,HBsAg阴性的3500例患者。参照国际自身免疫性肝炎组修订的评分标准和美国肝病学会原发性胆汁性肝硬化(PBC)指导建议,结合相关临床资料及部分患者肝组     

Specific epstein-barr virus serological response in patients with nasopiiaryngeal carcinoma detected by immunoblotting

The specific humoral immune response against Epstein-Barr virus (EBV) antigens in patients with nasopharyngeal carcinoma (NPC) was compared to that of patients with infectious mononucleosis (IM) and other EBV-seropositive subjects using immunoblotting technique. Almost all sera from E,BV serologically associated NPC reacted reproducibly with a major group of polypeptides (four to six) of early antigen (EA) complex with molecular weights ranging from 50 kD to 58 kD, and with some additional polypeptides. Sera from IM-patients reproducibly recognized only one polypeptide of 50 kD belonging to the major group of polypeptides of EA-complex. Sera from patients with other types of head and neck cancer and relatively high levels of IgG antibody against viral capsid antigen (VCA) and EA did not react reproducibly with any of the EBV-associated proteins.     

Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases

In 1988 and in 1989 consensus workshops were organized in order to define the interlaboratory concordance in detecting autoantibody specificities in selected sera from patients with rheumatoid disorders and to determine the possible causes of discrepancies. In total 20 sera were tested for the presence of antibodies against nRNP, Sm, Ro (SS-A), La (SS-B), Scl-70, centromeric antigens, ribosomal RNP and Jo-1. The methods used for detection by the 28 European laboratories who participated included immunofluorescence, counter-immunoelectrophoresis, immunodiffusion, immunoblotting and ELISA.

The results showed that only a combination of two or more techniques was able to detect all specificities with an adequate efficiency. Recommendations to improve the efficiency of autoantibody detection and to standardize laboratory protocols are given.     

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with ¹²⁵I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.     

乙肝病毒e抗原反式激活蛋白的亚细胞定位研究

目的 验证HBeAgTP基因在细胞内的表达及表达蛋白的亚细胞定位。方法 构建HBeAgTP基因的酵母表达诱饵质粒pGBKT7-HBeAgTP及绿色荧光蛋白表达质粒DEGFP-C1-HBeAgTP，pEGFP-C1．HBeAgTP转染HepG2细胞，24h后荧光显微镜下观察蛋白表达的亚细胞定位。pGBKT7-HBeAgTP转化AH109酵母细胞，提取转化了质粒的酵母蛋白质，进行Western免疫印迹分析。结果 成功构建出HBeAgTP基因。HBeAgTP基因的酵母表达诱饵质粒pGBKT7-HBeAgTP及绿色荧光蛋白表达质粒pEGFP-C1．HBeAgTP，Western免疫印迹法印证HBeAgTP可表达蛋白，其表达的蛋白亚细胞定位于细胞质。结论 HBeAgTP基因可表达蛋白，其表达蛋白亚细胞定位于细胞质。     

Synaptophysin: A reliable marker for medulloblastomas

Summary Synaptophysin is an acidic, integral membrane glycoprotein (M_r 38000) of presynaptic vesicles in various neurons and neuroendocrine cells, and in tumours derived from such cells. By indirect immunofluorescence microscopy of cryostat sections, using the monoclonal antibody SY 38 to synaptophysin, a consistent positive immunoreactivity was observed in all medulloblastomas (n= 6) and neuroblastomas (n=3) as well as a ganglioneuroma and a glioneuronal hamartoma. The presence of synaptophysin in medulloblastomas was confirmed biochemically by immunoblotting experiments. For purpose of comparison, the expression of intermediate-sized filament (IF) proteins was also examined. While neurofilament proteins were consistently expressed in the neuroblastomas (3/3), the ganglioneuroma and the glioneuronal hamartoma, IF distribution in medulloblastomas was variable. A neurofilament-positive type of tumour (1/6) could be distinguished from vimentin-expressing neoplasms (4/6) by immunocytochemistry. These data indicate that synaptophysin is a reliable marker for medulloblastomas as well as other differentiated and undifferentiated neuronal tumours and in this respect is superior to the more heterogeneous expression patterns of IF proteins in these tumours.     

RA33/36抗原的纯化及相应抗体的检测

目的纯化RA33/36抗原,观察其检测效率。方法从小鼠Ehrlich腹水癌细胞中提取RA33/36粗抗原,使用肝素-琼脂糖凝胶CL-6B对RA33/36粗抗原进行亲和层析,SDS-聚丙烯酰胺凝胶(SDS-PAGE)和免疫印迹法(IBT)检测富含RA33/36的组分,比较粗抗原和纯化抗原的检测效果。结果SDS-PAGE和IBT显示RA33/36抗原主要存在于缓冲液C的洗脱峰中,纯化抗原较粗抗原杂蛋白条带明显减少,且更加清晰,在检测RA33/36抗体时得到了相同的阳性和阴性结果。结论纯化的RA33/36抗原较粗抗原提高了抗体检测效率,可广泛应用于临床。     

Platelet autoantigens: Identification and characterization using immunoblotting

Summary Antiplatelet autoantibodies are important in the etiology of idiopathic (or immune) thrombocytopenic purpura (ITP). Studies using immunoblotting techniques have been helpful in identifying the antigenic target proteins for the antibodies. Antibodies against the glycoprotein (GP) IIIa portion of the GPIIb/IIIa complex were the first to be demonstrated by this approach. Similar GPIIIa autoantigens have also been found to be the most frequent targets of ITP antibodies. Not all anti-GPIIIa antibodies are directed against the same epitope on GPIIIa. A subset of anti-GPIIIa antibodies found in patients with an acquired qualitative platelet dysfunction actually interfere with fibrinogen binding to normal platelets. Antibodies directed against targets on GPV have been found in patients with acute ITP of childhood. In patients with ITP associated with lupus erythematosus, antibodies which bind to intracellular proteins of apparent molecular weights of 66 and 108 kDa have been detected. Thus, ITP antibodies can have a variety of target antigens. Study of larger series of patients will determine whether identification of platelet autoantigens correlates with clinical course of ITP.Presented at the International Workshop on ITP, August 26 and 27, 1988, Lucerne, Switzerland