Reactivation of Epstein-Barr virus in Sj?gren''s syndrome

Summary Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by severe dryness of the eyes and mouth, resulting from lymphocytic infiltration of the lacrimal and salivary glands. SS may exist as a primary condition (primary SS, 1° SS) or as a secondary condition (2° SS) in association with rheumatoid arthritis, systemic lupus erythematosus, or progressive systemic sclerosis. In some 1° SS patients, there may be involvement of the extraglandular organs, including skin, kidney, liver, lung and nervous system. Furthermore, these patients may develop a lymphoproliferative syndrome that includes lymphadenopathy and increased risk of lymphoma. In the pathogenesis of SS, a role for Epstein-Barr virus (EBV) has been suggested because: (a) EBV is present in salivary gland epithelial cells of normal individuals and exaggerated immune responses against EBV could play a role in the destruction of salivary glands in SS; (b) SS salivary gland biopsies contain increased levels of EBV DNA in comparison to normal salivary glands, indicating viral reactivation and inability of lymphoid infiltrates to control EBV replication in SS patients; and (c) salivary gland epithelial cells in SS patients express high levels of HLA-DR antigens and may present EBV-associated antigens to immune T cells in SS patients. Therefore, SS may represent a situation in which genetically predisposed individuals (i. e., HLA-DR3-DQA4-DQB2) have a persistent but ineffectual T cell immune response against EBV at its site of latency. Among 14 non-Hodgkin's lymphomas that developed in SS patients, EBV DNA was detected in increased amounts in the tumor tissue of one patient. Characterization of this tumor DNA revealed: (a) polyclonal immunoglobulin gene rearrangements; (b) EBV DNA with an unusual restriction fragment length polymorphism pattern involving the Bam M fragment; and (c) EBV terminal repeat sequences suggestive of viral replication, similar to those reported in EBV lymphomas occurring in other immunocompromised individuals. Early recognition of this clinical problem may allow beneficial use of antiviral agents.     

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.     

Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N_1-525), an amino-terminal construct (N_1-179), and a carboxy-terminal construct (N_414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N_414-496 was much more antigenic than GST-N_1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues ⁴⁴⁰VPQVRKETRASSR⁴⁵² (site 1), ⁴⁷⁹PEADTDPL⁴⁸⁶ (site 2), and ⁵²⁰DKDLL⁵²⁴ (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.     

Ultrastructural localization of phosphoglycerate kinase in adult<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis>

Phosphoglycerate kinase (PGK) is an enzyme that produces one ATP molecule in the glycolytic pathway. Clonorchis sinensis is largely dependent on glycolysis for energy production. We performed immunoelectron microscopy on adult C. sinensis by using mouse immune serum raised against recombinant C. sinensis PGK. A high density of gold particles was found in the microvilli of the intestinal epithelium and in lamellae of the sperm duct. PGK was common in the somatic cells of intra-uterine eggs and in excreted products. It was localized with moderate intensity in muscular fibers of the subtegumental muscle layer, and in the myoepithelia of the intestine and excretory bladder. We suggest that PGK plays an essential role in C. sinensis energy production for movement via muscle contraction.     

Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method

Porous PLGA/PVA scaffolds were fabricated by blending poly(lactic-co-glycolic acid) (PLGA) with polyvinyl alcohol (PVA) to improve the hydrophilicity and cell compatibility of the scaffolds for tissue engineering applications. PLGA/PVA blend scaffolds with different PVA compositions up to 20wt% were fabricated by a melt-molding particulate-leaching method (non-solvent method). The prepared scaffolds were investigated by scanning electron microscopy (SEM), mercury intrusion porosimetry, the measurements of water contact angles and bi-axial tensile strengths, etc. for their surface and bulk characterizations. The scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200-300 microm; porosity, about 90%). The PLGA/PVA blend scaffolds with PVA compositions more than 5% were easily wetted in cell culture medium without any prewetting treatments, which is highly desirable for tissue engineering applications. In vitro cell compatibility of the control hydrophobic PLGA and hydrophilized PLGA/PVA (5wt%) blend scaffolds was compared by the culture of human chondrocytes in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the PLGA/PVA blend scaffold had better cell adhesion and growth than the control PLGA scaffold. For in vivo evaluation of tissue compatibility, the scaffolds were implanted into the skull defects of rabbits. The results were evaluated by histology examinations. The PLGA/PVA (5wt%) blend scaffold showed better bone ingrowth into the scaffold and new bone formation inside the scaffold than the PLGA scaffold. It seems that 5% addition of PVA to PLGA to fabricate PLGA/PVA blend scaffolds is enough for improving the hydrophilicity and cell compatibility of the scaffolds.     

脑内和肌内接种小鼠胶质母细胞瘤株后神经末梢的观察

本实验应用Nonidez及Glees二种镀银法,对诱发的小鼠胶质母细胞瘤株(G 422)进行了观察。脑内及肌内接种胶质母细胞瘤后,在肿瘤边缘可见大小不等的神经束,伴随或不伴随血管伸入瘤内。有的在血管周围间隙形成血管周围神经丛。这些神经纤维与肿瘤周围宿主的脑组织、皮下、毛囊和肌肉间隙的神经纤维相联系。因此我们推测肿瘤内的神经是由肿瘤周围宿主的器官组织的神经延伸来的。肿瘤边缘的神经纤维的数量多于核心区,走行于肿瘤的间质或实质,沿途不断分支,终末分布到肿瘤细胞的表面。我们观察到球形、游离分叉状、梭形、环形、树枝状、杵状及丛刷状等类型的神经末梢。以上观察表明,恶性肿瘤——小鼠胶质母细胞瘤是受神经支配的。     

抗核糖体抗体阳性判断标准的探讨

目的 :分析针对分子量为 38kD和 或 15 16 5kD多肽抗原的抗核糖体抗体 (anti ribosomalantibodies)与SLE的关系 ,探讨抗核糖体抗体的阳性判断标准及其在SLE中的检出情况。方法 :收集病人血清 2 6 2例 ,包括SLE115例、RA5 7例、硬皮病 2 0例、MCTD39例及其他免疫性疾病 31例 ,用免疫印迹法 (Western blot)检测其抗ENA抗体谱。结果 :分别以同时出现 38kD和 16 5 15kD蛋白条带以及出现 38kD蛋白条带为判断抗核糖体抗体阳性的标准 ,则该抗体对诊断SLE的敏感性和特异性结果分别为 2 8 7% (33 115 )、96 6 % (14 2 14 7)及 17 4 % (2 0 115 )、97 3% (14 3 14 7) ,两者比较敏感性有显著性差异 (P <0 0 5 ) ,特异性无显著性差异 (P >0 0 5 ) ;仅 38kD分子阳性的 14例样本中 ,SLE占大多数 ,显著多于其它疾病 (71 4 %比 2 8 6 % ,P <0 0 5 ) ;38kD分子阳性同时伴抗Sm抗体阳性者多于抗Sm抗体阴性者 ,但无显著性差异 (6 4 3%比 35 7% ,P >0 0 5 )。结论 :抗核糖体抗体 38kD分子与SLE密切相关 ,而与抗Sm抗体相关性不强。以 38kD为主要抗原多肽判断抗核糖体抗体 ,不仅可以提高该抗体的阳性检出率 ,同时也不会降低其对SLE诊断的特异性 ,该判断方法值得推广应用。     

ctB和ureⅠ双基因融合表达及其免疫特性分析

目的：构建霍乱毒素B亚单位（CtB）和幽门螺杆菌尿素膜通道蛋白（UreⅠ）融合的原核表达质粒pET32a（＋）ctB/ure Ⅰ,并初步研究融合蛋白Ct B/Ure Ⅰ的表达特性和免疫特性.方法：PCR从pUC18 ctB中克隆ctB基因,定向在pET32a（＋）/ureⅠ的ureⅠ基因5＇端插入ctB基因,构建ctB和ure Ⅰ双基因原核表达质粒pET32a（＋）ctB/ure Ⅰ,转该质粒于E.coli BL-21（DE3）,经酶切和序列分析鉴定工程菌.IPTG诱导表达,HP-His亲和层析纯化,SDS-PAGE和Gel-Pro Analizer4分析,重组蛋白免疫BALB/c小鼠.用Western blot和ELISA分析重组蛋白的免疫特性.结果：工程菌含完整的ctB和ure Ⅰ基因,与相对应基因的序列同源性分别为100%.在22℃,1 mmol/L IPTG诱导4 h后,重组蛋白的表达占菌体总蛋白12%,亲和层析纯化后蛋白纯度为94.3%.Western blot表明重组蛋白分别能与相应的抗体反应,该蛋白免疫小鼠后能产生相应的IgG抗体.结论：成功构建了能表达CtB/Ure Ⅰ蛋白的大肠杆菌表达菌株.对融合蛋白表达和纯化后,初步证明了该重组蛋白有CtB和Ure Ⅰ的双特异反应原性和免疫原性,为研究新型幽门螺杆菌疫苗奠定了坚实的基础.