A new multivalent B cell activation model--anti-IgD bound to Fc gamma RI: properties and comparison with CD40L-mediated activation

CHO cells permanently transfected with mouse Fc gamma RI alpha chain were prepared and used as a model to polyclonally activate murine B cells. The transfected CHO cells were treated with mitomycin C and placed into culture with varying quantities of anti-IgD. Using this model, murine splenic B cells (from BALB/c or C57Bl/6) were activated by mouse IgG2a-anti-IgD (10.4.22 or AF3.33) in a manner that is analogous to the activation of B cells seen with highly polyvalent anti- IgD (H delta(a)/1) prepared by chemical cross-linking to dextran. Efficient B cell activation was seen with nanogram quantities of anti- IgD. In the presence of IL-4 and IL-5, IgG1 production levels were equivalent to or better than seen when stimulation was with H delta(a)/1-dextran; however, IgE induction was not seen in either situation. The Ig production capacity was compared to that seen when B cells were activated with CD40L, using either CD40L-transfected CHO or a soluble CD40L construct. In the presence of IL-4 and IL-5, once a critical threshold of B cells was present, IgE and to a lesser extent IgG1 production was inversely proportional to B cell number when CD40L was the activating agent. In contrast, with Fc gamma RI-anti-IgD, IgM and IgG1 production was directly proportional to B cell number, while IgE production was never seen. Finally, when B cells were co-activated with immobilized anti-IgD and CD40L simultaneously, the IgE production from B cells induced by CD40L was strongly inhibited, while IgG1 and IgM production were not affected. Since B cell co-activation via sIg and CD40L would be a common scenario in secondary follicles, this inhibition of IgE production may be one of the reasons why serum IgE levels are much below IgG in normal immune situations.      

Trinucleotide expansion mutations in the cartilage oligomeric matrix protein (COMP) gene

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are two human autosomal dominant skeletal dysplasias characterized by variable short stature, joint laxity and early-onset degenerative joint disease. Both disorders can result from mut-ations in the gene for cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein. About one-third of PSACH cases result from heterozygosity for deletion of one codon within a very short triplet repeat, (GAC)5, which encodes five consecutive aspartic acid residues within the calmodulin-like region of the COMP protein. We have identified two expansion mut-ations in this repeat: an MED patient carrying a (GAC)6allele and a PSACH patient carrying a (GAC)7allele. These are among the shortest disease-causing triplet repeat expansion mutations described thus far, and are the first identified in a GAC repeat. A unique feature of this sequence is that expansion as well as shortening of the repeat can cause the same disease. In cartilage, both patients have rough endoplasmic reticulum inclusions in chondrocytes. The inclusions are also present in tendon tissue and can be reproduced in cultured tendon cells, suggesting that the pathophysiology of disease is similar in both cartilage and tendon.      

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

The neurofibromatosis 2 ( NF2 ) gene product, merlin, is a tumor suppressor protein mutated in schwanno-mas and several other tumors. Merlin, which shares significant homology with the actin-associated proteins ezrin, radixin and moesin (ERM proteins), inhibits cell growth when overexpressed in cell lines. The similarities between merlin and ERM proteins suggest that merlin's growth-regulatory capabilities may be due to alterations in cytoskeletal function. We examined this possibility in rat schwannoma cell lines overexpressing wild-type merlin isoforms and mutant merlin proteins. We found that overexpression of wild-type merlin resulted in transient alterations in F-actin organization, cell spreading and cell attachment. Merlin overexpression also impaired cell motility as measured in an in vitro motility assay. These effects were only observed in cells overexpressing a merlin isoform capable of inhibiting cell growth and not with mutant merlin molecules (NF2 patient mutations) or a merlin splice variant (isoform II) lacking growth-inhibitory activity. These data indicate that merlin may function to maintain normal cytoskeletal organization, and suggest that merlin's influence on cell growth depends on specific cytoskeletal rearrangements.      

由重组腺相关病毒1／2载体携带的LacZ报告基因在脐血间质干细胞中的表达

目的:观察由重组腺相关病毒1/2载体携带的LacZ报告基因在体外培养的脐血间质干细胞中的表达情况。方法:实验于2005-10/2006-02在上海交通大学附属新华医院科研中心完成。①以孕龄近60d的杂种妊娠犬的脐带血作为实验用脐血间质干细胞的标本来源。重组腺相关病毒1/2载体-lacZ基因(北京本原正阳基因技术有限公司)。②无菌条件下采集妊娠犬脐带血,置于预装有肝素的离心管中,与Hanks液1∶1混合均匀,叠加于相对密度为1.077的淋巴细胞分离液上,梯度离心分离后进行培养和扩增。③取第4代脐血间质干细胞,经胰酶消化后吹打成单细胞悬液,以5×104/孔接种于24孔板内,随机数字表法分为转染组20孔、空白对照组4孔。转染组将重组腺相关病毒1/2载体-lacZ报告基因(1×1012v.g.mL-1)用不含血清的IMDM培养液作系列滴度稀释,分为5个滴度,即感染复数分别为每个细胞1×102,1×103,1×104,1×105,1×106v.g,各感染复数均设4孔;空白对照组未加入病毒,只加入相同体积的不含血清的IMDM培养液。④转染72h后采用X-gal化学染色法进行检测,成功转染上LacZ基因的脐血间质干细胞其胞浆内会因合成半乳糖苷酶而呈阳性蓝染。每组样本随机选取5个视野,相差显微镜下计数半乳糖苷酶阳性细胞数,取均值即为LacZ基因细胞转染率。结果:①脐血间质干细胞生长情况及LacZ报告基因表达的检测:转染组病毒基因转染后未再见到明显的细胞增殖,转染72h后大部分细胞表达LacZ基因并合成半乳糖苷酶,X-gal染色呈蓝色,长梭形;4～6周后细胞形态趋向老化,长梭形逐渐变为宽扁形;8周后细胞均被蓝染,颜色明显较转染72h时深,细胞形态由长梭形变为不规则,明显老化。空白对照组细胞反应均为阴性。②LacZ报告基因细胞转染率测定结果:转染72h后,感染复数为每个细胞1×102v.g时,仅有少数细胞蓝染呈阳性;感染复数为每个细胞1×103,1×104,1×105,1×106v.g时,转染率分别为(43±5)%,(82±4)%,(95±4)%,(97±3)%。结论:体外培养的脐血间质干细胞能高效转染重组腺相关病毒1/2载体-lacZ报告基因,在一定感染复数范围内,细胞转染率随着感染复数的增加而升高。提示脐血间质干细胞是重组腺相关病毒1/2载体-lacZ报告基因转染的适宜靶细胞。     

胃十二指肠转移性黑色素瘤1例

对1例皮肤恶性黑色素瘤胃十二指肠转移患者进行报道,并分析其内镜特点、治疗方式及预后.由此认为恶性黑色素瘤的胃肠道转移常被低估,有必要对其进行常规内镜检查并采取相应的治疗措施,提高患者的生存率.     

高效液相色谱法测定人血清及尿中奥拉西坦的浓度

以阿昔洛韦(acyclovir)为内标,NH2柱为分析柱;乙腈:水(80:20,v/v)为流动相,检测波长为210nm,建立了测定人血清及尿中奥拉西坦(oxiracetam)浓度的HPLC方法。奥拉西坦和内标保留时间分别为6.3min和8.1min;二峰分离良好。本法平均回收率:血清99.7±5.9%;尿99.0±5.6%,日内及日间相对标准偏差(RSD)为5.0～10.9%,血清及尿标准曲线相关良好,最低检测浓度血清为1μg·mL^-1,尿为20μg·mL^-1。本方法操作简便,迅速,灵敏,可靠。可以满足临床药代动力学测试的要求。     

Alopecia in association with severe seborrhoeic dermatitis following combination antiretroviral therapy for acute retroviral syndrome

We present a case where alopecia occurred with severe seborrhoeic dermatitis associated with the commencement of combination antiretroviral therapy for acute retroviral syndrome. We postulate that the eruption could represent a novel manifestation in association with immunological response to antiretroviral therapy.