一个新的人B细胞活化抗原—5C5

用活化人B细胞株3D5细胞免疫小鼠和作为筛选的靶细胞,我们建立了产生单克隆抗体5C5的杂交瘤细胞株。此单抗识别的抗原5C5在25μg/ml anti-μ刺激的B细胞,于第10小时开始表达,亦即于G_1期开始表达。5C5细胞百分率随培养时间而增多。在PWM诱导下.外周血单一核细胞中5C5~ 细胞随培养时间而增加,至第3～4天达最高峰,然后减少,至第7天降至本底水平。5C5~ 细胞在不能为BCDF诱导分化至免疫球蛋白分泌细胞(ISC)的B细胞株3D5,Raji和Daudi阳性,但在能为BCDF诱导分化至ISC的CESS和SKW6细胞却不表达。这均表明5C5抗原表达于B细胞活化的早期和中期,但在B细胞终末分化阶段消失。在休止期B细胞、休止期T细胞、PHA激活的T细胞、单核细胞和中性粒细胞,以及在所检测的T细胞株和髓细胞株,5C5抗原均为阴性。~(125)I标记后用单抗5C5免疫沉淀提取的抗原,在还原与非还原条件下电泳,均只有分子量为52000的一条带,表明5C5是一个单链细胞表面蛋白。鉴于5C5抗原的分子量与文献中已报道的B细胞活化抗原分子量不同,以及5C5在细胞株表达的特点,它可能是一个新的人B细胞活化抗原。     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

PBX1基因剪切体表达与SLE的相关研究

了解PBX1基因各种剪切体的表达在SLE患者和正常人中是否存在差异 ,探讨PBX1的表达与SLE发病的相关性。通过PCR扩增及毛细管芯片电泳 ,确证剪切体h、k、l存在于人体 ;通过实时荧光定量PCR技术 ,对剪切体h、k、l分别进行SLE患者组和正常组的mRNA表达定量比较。结果发现这 3种剪切体在患者组中的表达较正常人明显降低 ,正常人的表达是SLE的 9～ 12倍。重度患者的k、l剪切体与轻中度的病人相比表达明显降低 ,并发狼疮性肾炎的病人k剪切体的表达较无肾累及的病人显著降低。说明PBX1基因剪切体h、k、l在SLE患者中mRNA表达水平下降 ,并与SLE活动度及肾累及有关。提示机体通过PBX1的表达量的调节可能参与SLE的发病     

Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons

Neural stem cells are present both in the developing nervous system and in the adult nervous system of all mammals, including humans. Little is known, however, about the extent to which stem cells in adults can give rise to new neurons. We used immunocytochemistry, electron microscopy, fluorescence microscopy (FM imaging) and electrophysiology to demonstrate that progeny of adult rat neural stem cells, when co-cultured with primary neurons and astrocytes from neonatal hippocampus, develop into electrically active neurons and integrate into neuronal networks with functional synaptic transmission. We also found that functional neurogenesis from adult stem cells is possible in co-culture with astrocytes from neonatal and adult hippocampus. These studies show that neural stem cells derived from adult tissues, like those derived from embryonic tissues, retain the potential to differentiate into functional neurons with essential properties of mature CNS neurons.     

Effect of illumination of nitrate and phosphate removal by coimmobilized Chlorella pyrenoidosa and activated sludge

Chlorella pyrenoidosa and activated sludge were co-immobilized with simplifying modified PVA-sulfate method. Effects of light intensity and light:dark ratio on the growth of co-immobilized algae cells and removal of nitrate and phosphate were studied. The results indicate that the growth rate of co-immobilized algae cells reduces when light intensity is decreased from 4000 Lux to 1000 Lux, while optimal light:dark ratio for algal growth is 16:8. The influence of illumination on nitrate removal is so weak that the removal percentage can reach 90 approximately 100% within 12 approximately 24 h during the experimental periods in spite of changing illumination conditions. On the other hand, phosphate removal efficiency reduces when light intensity or light:dark ratio is decreased. The highest phosphate removal percentage is 99.6% under the circumstances of 4000 Lux and full-time illumination in our experiment, while the average phosphate removal is about 78%. The change of pH value in water samples is also observed. When water sample is treated by the co-immobilized system, pH value increases in light and decreases in dark. Microorganisms' physiological action is considered as the main mechanism that leads to the change of pH value.     

原位PCR技术检测石蜡包埋脑组织中人巨细胞病毒DNA

应用原位聚合酶链反应（ＩＳＰＣＲ）技术检测了２５例尸检畸形胎儿石蜡包埋脑组织中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ，并与普通ＰＣＲ及原位杂交（ＩＳＨ）进行了比较。ＩＳＰＣＲ、ＰＣＲ及ＩＳＨ检测阳性率分别为４４％，３６％及２０％。与ＩＳＨ相比较，ＩＳＰＣＲ不仅检出阳性率高，而且信号强度增强。研究结果提示，ＩＳ－ＰＣＲ是诊断ＨＣＭＶ感染的快速、敏感、特异的实用方法。     

PCR-RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea

Summary.  We have evaluated PCR–RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with PCR–RFLP based genotyping, which was developed for this study. Among the 80 case isolates, which had been previously typed by routine neutralization test, only 42 cases (52.5%) were matched with typing by PCR–RFLP. The result clearly demonstrated that the enterovirus serotype does not coincide with the genotype. Therefore, the classification of enteroviruses by genotyping with PCR–RFLP, although rapid and simple, may be complicated by regional or seasonal differences. However, the PCR–RFLP method developed in this study is applicable to the epidemiological study of enteroviruses when regional or seasonal differences exist, and is useful in identifying the source of an infection. Received August 6, 2001; accepted April 15, 2002 Published online July 10, 2002     

The ability of heat-killed Mycobacterium vaccae to stimulate a cytotoxic T-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein

Exogenous antigens are generally presented by Class II major histocompatibility (MHC) molecules. When administered with an adjuvant, however, they are capable of inducing a CD8+ T-cell response where antigen recognition is associated with Class I MHC. Accordingly, immunization with soluble ovalbumin (OVA) alone does not activate CD8+ cytotoxic T cells (CTL) but when given in complete Freund's adjuvant (CFA), or in formulations of a number of novel adjuvants, an OVA-specific CD8+ CTL response can be detected. We show in this report that immunization with soluble OVA mixed with heat-killed Mycobacterium vaccae, but not with other common pathogenic and saprophytic mycobacteria, can activate OVA-specific CD8+ CTL. An OVA-specific CTL response is detected when mice are immunized by either the intraperitoneal or intranasal route and their spleen cells are re-stimulated in vitro. Adjuvant activity of heat-killed M. vaccae is present in M. vaccae culture filtrate, in soluble protein components of whole M. vaccae and in the 65 kDa heat-shock protein (hsp) of M. vaccae. Mycobacterium vaccae has previously been shown to have no adverse side-effects in humans. The current results suggest that M. vaccae may be useful as an adjuvant for vaccines and other immunotherapies where CD8+ CTL responses to exogenous proteins are crucial.