Autoimmunity to heat shock protein 60 and antigen-specific production of interleukin-10.

The immunopathologic sequelae of chlamydial infection are correlated with immune responses to the Chlamydia trachomatis heat shock protein 60 (hsp60). One pathogenic mechanism that may explain this association is the induction of autoimmune responses to self hsp60, since these two proteins share a high degree of amino acid sequence identity. To investigate the conditions under which autoimmune responses can be generated against self hsp60, groups of CBA mice were immunized with recombinant mouse hsp60, recombinant chlamydial hsp60, or both proteins. The data show that autoimmune responses characterized by strong T-cell proliferation and high titers of antibody to self hsp60 are induced only by concurrent immunization with mouse and chlamydial hsp60. Immunization with mouse hsp60 alone induced lymphocytes that secreted high levels of interleukin-10 (IL-10) but did not proliferate in response to in vitro stimulation with mouse hsp60; coimmunization with mouse and chlamydial hsp60s induced lymphocytes that proliferated strongly in response to mouse hsp60, secreted 6-fold less IL-10, and exhibited a 12-fold increase in the ratio of gamma interferon/IL-10 production. Switches in cytokine production patterns may mediate the pathogenesis of hsp60-associated diseases such as C. trachomatis immunopathology.     

Cocrystallization of an immunoglobulin light chain dimer with bis(dinitrophenyl) lysine: tandem binding of two ligands, one with and one without accompanying conformational changes in the protein

Previous studies showed that the Mcg dimer of immunoglobulin light chains bound bis(dinitrophenyl)lysine both in trigonal crystals and in solution. On prolonged storage in ammonium sulfate, mixtures of ligand and protein produced small trigonal cocrystals in low frequency. These crystals were nearly isomorphous with those of the unliganded dimer in which the subunits were covalently linked by an interchain disulfide bond. By difference Fourier analyses at 3.5 A resolution and subsequent crystallographic refinement, the cocrystals were found to contain molecules with two ligands aligned in tandem along the interface of the variable (V) domains of the protein. One ligand molecule adopted an almost fully extended conformation, with the epsilon-DNP ring situated near the floor, the alpha-carboxyl group directed toward the solvent at the entry, and the alpha-DNP ring outside the rim of the main cavity. As if architecturally designed, the ligand was located symmetrically between the two domains in an orientation that was compatible with both the unaltered structure of the cavity lining and with the known crystal packing interactions of neighboring protein molecules. The second ligand molecule in the cocrystal lodged in the deep pocket immediately under the floor of the main cavity. The ligand adopted a very compact conformation with the two DNP rings roughly antiparallel to each other. This molecule appeared to be semi-permanently sequestered in the pocket since it could not be dislodged by exhaustive perfusion with ammonium sulfate crystallizing media. Relative to its volume in the native dimer, the pocket was expanded to accommodate the oversized ligand. Within a single protein molecule, therefore, two types of binding of a flexible ligand were observed, one with and one without accompanying conformational changes in the protein. The number of cocrystals which could be produced was markedly increased if the interchain disulfide bond between the Mcg monomers was first reduced and alkylated.     

No association between thrombospondin-4 A387P polymorphism and acute coronary syndrome in Chinese Han population

Objective: The thrombospondin-4 (TSP-4) gene G29926C (A387P) polymorphism was recently reported to be associated with an increased risk of MI (myocardial infarction) in American population. However, several subsequent studies produced controversial findings. The aim of this study was to explore the possible association between TSP-4 A387P polymorphism and ACS (acute coronary syndrome) in Chinese Han population. Methods :A case-control study including 412 patients with ACS and 337 controls free from CAD (coronary artery disease) was conducted. TSP-4 A387P polymorphism was determined by PCR (polymerase chain reaction) and RFLP (restriction fragment length polymorphism) analysis. Results:Slightly decreased frequency of GC genotype was observed in patients with ACS, compared with controls (5.3% vs. 7.1%), but the difference did not reach statistical significance (P = 0.31 ). Similarly, the prevalence of C allele was 2.7% and 3.6% for ACS and control groups, respectively (P = 0.32). None of homozygote was detected for C allele. Further analyses in subjects subgrouped according to sex and age also showed no association of TSP-4 A387P polymorphism with ACS. Furthermore, after adjustment for conventional risk factors by multiple logistic regression analysis, the carrier prevalence of C allele did not differ significantly between ACS and control groups (OR = 0.85; 95% CI:0.45-1.59; P = 0.60). Conclusion:The present study suggested that the TSP-4 A387P variant showed a low prevalence compared with western populations and failed to associate with an altered risk of ACS in Chinese Han population. The findings further supplement experimental data for TSP-4 gene study of coronary disease.     

森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。     

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.     

Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.     

颞下颌关节与颅面指标的计算机相关及回归分析

本文用计算机分析统计了118例(236侧)颞下颌关节的12项骨性指标及10项颅面指标的正常值及相关关系。结果表明颞下颌关节各项指标之间、颞下颌关节与颅面各主要指标之间,均存在良好的相关关系。说明颞下颌关节,特别是关节窝和髁突的形态大小具有明显的规律性。由于颅面诸指标与颞下颌关节主要指标之间高度相关,因而本文用逐步回归方法,建立了由颅面特征指标推算髁突形态的回归方程。本文的结果可为该关节的形态研究及人工关节的设计和应用提供有益的依据。     

妊娠高血压患者血浆中降钙素基因相关肽的测定及其临床意义

本文测定了３０例正常育龄妇女，２８例正常妊娠以及３６例妊娠征患者血浆中降钙素基因相关肽的水平，结果发现，轻度妊高生与正常妊娠相比，血浆中ＣＧＲＰＫ是有下降但不显著（Ｐ〉０．０５）。中重度妊高征与正常妊娠相比，差异有非常显著性，因此推测，妊娠状态下血浆中降钙素基因相关肽的减少可能是妊娠高血压症产生的重要原因之一。