The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Long-lived alphaMUPA transgenic mice show reduced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine

Calorie restriction (CR) extends the life span of various species through mechanisms that are as yet unclear. Recently, we have reported that mitochondrion-mediated apoptosis was enhanced in alphaMUPA transgenic mice that spontaneously eat less and live longer compared with their wild-type (WT) control mice. To understand the molecular mechanisms underlying the increased apoptosis, we compared alphaMUPA and WT mice for parameters associated with SOD2 (MnSOD), a mitochondrial antioxidant enzyme that converts superoxide radicals into H(2)O(2) and is also known to inhibit apoptosis. The SOD2-related parameters included the levels of SOD2 mRNA, immunoreactivity and enzymatic activity in the liver, lipid oxidation and aconitase activity in isolated liver mitochondria, and the sensitivity of the mice to paraquat, an agent that elicits oxidative stress. In addition, we compared the mice for the levels of SOD2 mRNA after treatment with bacterial lipopolysaccharides (LPS), and for the DNA binding activity of NFkappaB as a marker for the inflammatory state. We extended SOD2 determination to the colon, where we also examined the formation of pre-neoplastic aberrant crypt foci (ACF) following treatment with dimethylhydrazine (DMH), a colonic organotypic carcinogen. Overall, alphaMUPA mice showed reduced basal levels of SOD2 gene expression and activity concomitantly with reduced lipid oxidation, increased aconitase activity and enhanced paraquat sensitivity, while maintaining the capacity to produce high levels of SOD2 in response to the inflammatory stimulus. alphaMUPA mice also showed increased resistance to DMH-induced pre-neoplasia. Collectively, these data are consistent with a model, in which an optimal fine-tuning of SOD2 throughout a long-term regimen of reduced eating could contribute to longevity, at least in the alphaMUPA mice.     

阴道镜下高频电灼术联合α-2a干扰素凝膏治疗尖锐湿疣疗效分析

目的探讨阴道镜下高频电灼术联合重组人干扰素α-2a治疗尖锐湿疣（CA）的效果。方法将165例CA分为3组，A组应用阴道镜下高频电灼术联合重组人干扰素α-2a；B组单纯采用阴道镜下高频电灼治疗；C组应用NS-FII型多功能光谱治疗仪联合肌注重组人干扰素α-2a。结果治疗后3-6个月A、B、C组复发率分别为0％、4．4％、65．4％：半年后人乳头瘤病毒（HPV）转阴率分别为93．5％、85．4％、43．8％，A组明显优于B组，B组明显优于C组，3组比较差异有统计学意义（P〈0．01）。结论阴道镜下高频电灼术联合重组人干扰素α-2a治疗CA可明显降低CA复发率和提高HPV转阴率。     

人胸腺基质淋巴生成素的研究进展

人胸腺基质淋巴生成素(hTSLP)是一种结构类似IL-7的新型细胞因子.正常情况下其mRNA主要表达于基质细胞、上皮细胞和肥大细胞,病理状态下,TSLPmRNA在一些前炎症细胞因子的作用下表达增加.hTSLP直接作用的效应细胞主要是树突状细胞(DC),通过调节DC的成熟来发挥作用.一方面.hTSLP-DC在外周能够诱导初始T细胞向一类特殊的Th2细胞分化,从而启动炎症反应;另一方面,hTSLP-DC能够促进胸腺中CD4+CD25-T细胞分化成CD4+CD25+调节性T细胞,从而在免疫耐受的发生和调控中发挥作用.     

Cyclooxygenase-2 expression in colorectal cancer liver metastases

Cyclooxygenase-2 (COX-2) is up-regulated in 85-90% of primary human colorectal cancers and is a putative target for the chemopreventative activity of non-steroidal anti-inflammatory drugs. However, COX-2 expression by human colorectal cancer liver metastases has been poorly characterized. We studied a consecutive series of 38 patients who underwent liver resection for metastatic disease, for whom long-term (up to 57 months), prospective follow-up data were available. Semi-quantitative immunohistochemistry for COX-2 was performed on 54 metastases from 35 patients, for whom adequate histological material was available. Diffuse cytoplasmic staining for COX-2 protein was detected in cancer cells in 100% of metastases (COX-2 score 1, n=25; score 2, n=29). There was no relationship between metastasis size or differentiation grade and the level of COX-2 protein expression. There was no difference in colorectal cancer-free or overall survival between patients with high (score 2) and low (score 1) COX-2 scores (Kaplan–Meier survival analysis and log rank test, both P=0.97). Multivariate Cox regression analysis identified age, incomplete resection and presence of extra-hepatic disease as independent predictors of disease-free and overall survival following surgery. COX-2 protein was also localized to a subset of stromal fibroblasts and mononuclear cells within metastases as well as hepatocytes from resection specimens. COX-2 protein was expressed by cancer cells in all human colorectal cancer liver metastases which were studied. Investigation of the effect of selective COX-2 inhibition on metastasis growth and metastasis cancer cell proliferation/apoptosis in vivo are warranted.     

Changed Bcl:Bax ratio in endometrium of patients with unexplained infertility

Apoptosis has been shown to be an important regulator of endometrial function during the menstrual cycle and implantation. Recently, some possible implantation defects were identified in patients with unexplained infertility. In this study, we investigated the role of spontaneous apoptosis, which is regulated by death regulatory genes, such as Bcl-2, Bax, p53, and isoenzymes of nitric oxide synthases; eNOS and iNOS during the implantation window in women with unexplained infertility. Endometrial samples were evaluated from fertile (n=15) and unexplained-infertile women (n=15) during post-ovulatory 7th or 8th day of their menstrual cycles. Apoptotic cells were detected using the dUTP nick-end labelling assay and Bcl-2, Bax, p53, iNOS and eNOS were assessed immunohistochemically. Reduced apoptotic cells, weak immunoreactivity of p53 and strong immunoreactivity of Bcl-2 were observed in the unexplained-infertile group compared with the fertile group (p<0.001). Bax intensity was similar in both groups. While weak iNOS immunoreactivity was detected in both groups, moderately increased eNOS immunoreactivity was observed in infertile cases. Spontaneous apoptosis is reduced in the endometrium of unexplained-infertile women, and is associated with the changed Bcl-2:Bax ratio. This finding may be a contributing factor to defective implantation causing infertility in this group of patients.     

In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers

To identify the advantages of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated polysulfone (PSf) hollow fibers for hemodialyzer and hemofilter minimodules with hollow fibers were made and blood compatibility was evaluated in vitro and ex vivo. Three types of hollow fibers, i.e., pure PSf (no additives), PSf alloyed with poly(1-vinyl-2-pyrrolidone) (PVPy), and PSf coated with the MPC copolymer, were processed in wet conditions. Commercially available hollow fibers (APS) were used as a control sample. The PSf hollow fibers have a condensed structure. A porous structure was observed when the PVPy was alloyed before wet processing, and no effect of the innercoated MPC copolymer on the porous structure was observed. One-tenth-sized minimodules of the conventional hemodialyzer were fabricated with 200 fibers each. The solute permeability of the hollow fibers was evaluated using 10% bovine serum in a buffer solution containing cytochrome C, which is a model protein of ₂-microglobulin. After circulation for 2.5h, the solute permeability of APS and PVPy-alloyed PSf hollow fibers decreased to 50% compared with their initial values. In contrast, the value for the hollow fibers innercoated with the MPC copolymer maintained its initial level. The inner surface of the dialysis membranes was observed with a transmission electron microscope and a layer of adsorbed protein on the PSf, APS, and PVPy-alloyed PSf hollow fibers was observed, but not on the MPC copolymer-coated fibers. Blood cell adhesion was then evaluated by circulation of whole rabbit blood without any anticoagulant ex vivo. Many adherent cells were observed on the PVPy-alloyed PSf hollow fibers; however, blood cells did not adhere or aggregate on the MPC copolymer-coated hollow fibers. From these results, we concluded that the in-situ coating of MPC copolymer on PSf hollow fibers is effective in preventing blood coagulation and maintaining the solute permeability of the fibers.     

The role of COX-2 in angiogenesis and rheumatoid arthritis

Recent evidence suggests that cyclooxygenase (COX)-2 is a mediator of angiogenesis, and COX-2 activity is known to be upregulated in the rheumatoid arthritis (RA) synovium. We examined whether mediation of angiogenesis by COX-2 was occuring in cells of the RA synovium and in microvascular endothelial cells (ECs) that are similar to those found in the RA synovium. We demonstrate that rofecoxib, a selective COX-2 inhibitor, acts directly on human dermal microvascular ECs (HMVECs) to inhibit their chemotactic and tube forming ability. Likewise, pretreatment of HMVECs with rofecoxib significantly inhibited their ability to form tubes induced by conditioned media (CM) of activated RA synovial fibroblasts. When RA synovial fibroblasts were pretreated with rofecoxib for 16 h and then stimulated with interleukin (IL)-1beta, their CM induced significantly less HMVEC tube formation when compared with CM from vehicle-treated RA synovial fibroblasts. ELISAs performed on activated RA fibroblast CM for known proangiogenic factors demonstrated a significant reduction in bFGF, in addition to the expected decrease in PGE(2). Our studies suggest that COX-2-induced angiogenic activity is an active mechanism within diseased synovium and may provide an additional rationale for the use of COX-2 inhibitors in RA.     

High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.     

人颈动脉粥样硬化斑块环氧化物酶-2及Ⅰ型前列腺素合成酶的表达

目的探讨环氧化物酶-2(cyclooxygenase type 2,COX-2)及Ⅰ型前列腺素合成酶(membrane associated prostaglandin E-1,mPGES-1)在人颈动脉粥样硬化斑块中的表达变化及作用机制。方法收集24例人颈动脉粥样硬化斑块标本和10例肠系膜动脉标本做对照组,应用免疫组织化学及逆转录PCR方法测定COX-2及mPGES-1mRNA表达水平,Western印记方法检测COX-2及mPGES-1的蛋白表达水平。比较不同程度动脉粥样硬化组织间COX-2、mPGES-1 mRNA表达水平及蛋白表达水平。结果颈动脉粥样硬化斑块组的免疫组织化学染色检测COX-2和mPGES-1呈阳性表达,斑块组COX-2 mRNA和mPGES-1 mRNA表达与对照组相比上调,差异有统计学意义(P<0.05);COX-2及mPGES-1 mRNA上调水平相关(P<0.05);颈动脉粥样硬化斑块的COX-2蛋白表达上调水平与对照组相比差异有统计学意义(P<0.05);颈动脉粥样硬化斑块COX-2、mPGES-1 mRNA及蛋白表达水平与病理损害程度有关,差异有统计学意义(P<0.05)。结论COX-2及mPGES-1基因表达水平上调可能是进展性动脉粥样硬化损害的关键因素。