Polymorphisms in the gene encoding thymus-specific serine protease in the extended HLA complex: a potential candidate gene for autoimmune and HLA-associated diseases

Positive selection plays a role, together with negative selection, in the prevention of autoimmunity. Thymus-specific serine protease is highly expressed in the thymus and is believed to be involved in positive selection of T cells. The gene encoding thymus-specific serine protease (PRSS16) maps to the extended HLA complex, which harbours several genes predisposing for autoimmune diseases. Here we report the results of scanning the genetic region containing PRSS16 for polymorphisms. Twenty-two polymorphisms were identified, including one missense polymorphism, one deletion leading to elimination of five amino acids, as well as several SNPs in the promoter region.     

Quantification of mouse glial cell-line derived neurotrophic factor family receptor alpha 2 alternatively spliced isoforms by real time detection PCR using SYBR Green I

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFR-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFR-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFR-2b and GFR-2c). All three isoforms are expressed in all mammalian tissues examined, including human fetal brain. However, the expression levels of these isoforms have yet to be quantified. In this report, we have developed a real time polymerase chain reaction (PCR) detection method using SYBR Green I to detect the expression levels of the three splice variants (GFR-2a, GFR-2b and GFR-2c). Of the three isoforms, GFR-2a was found to be the most abundant receptor expressed in the whole murine brain. The real time PCR detection method using SYBR Green I developed in this report can be used to unambiguously quantitate expression levels of the GFR-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.     

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression,interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.     

Expression of CD44 molecules and CD44 ligands during human thymic fetal development: expression of CD44 isoforms is developmentally regulated

It has recently been recognized that CD44 comprises a largefamily of alternatively spliced forms.In the thymus, CD44 hasbeen postulated to play an important role in immature T cellmigration and maturation. In this paper, we have studied theexpression of CD44 molecules and two CD44 ligands, hyaluronan(HA) and fibronectin (FN), during human thymic fetal development.We found that mAbs against all CD44 isoforms (A3D8 or A1G3)reacted with both thymic epithelial (TE) cells and thymocytesbeginning at the time of initial colonization of the human thymusby hematopoietic stem cells at 8.2 weeks of fetal gestation.However, mAbs specific for splice variants of CD44 containingmembrane-proximal inserts (11.24, 11.10 and 11.9) reacted onlywith terminally differentiated TE cells in and around Hassall'sbodies beginning at 16–19 weeks of fetal gestation. Studiesof differentiated versus undifferentiated TE cells in vitroconfirmed the selective expression of CD44 variant isoformson terminally differentiated TE cells. Expression of HA andFN was determined by fluorescence microscopy using either biotlnylated-HAbinding protein or an anti-FN mAb. We found that whereas FNwas present throughout the human fetal thymus beginning at 8.2weeks, HA was not present until 16 weeks of gestational age.These data demonstrate the differential expression of standardversus variant CD44 isoforms during thymic ontogeny and implicateCD44 interactions with ligands other than HA as important inthe earlier stages of humanthymus development     

TCR-Vβ Usage in the Thymus and Blood of Myasthenia Gravis Patients

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vβ families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vβ usage was found in the thymi of MG patients and controls, except Vβ2, which showed a small increase in MG patients’ thymi. Blood T cells of MG patients used Vβ4, Vβ6, Vβ15, Vβ16 and Vβ24 significantly more than those of the controls. Vβ4 and Vβ6 are the gene families most frequently used by anti-AChR CD4⁺cells in MG patients. Blood T cells from MG patients used Vβ12, Vβ14, Vβ17 and Vβ18 significantly less than controls. MG patients used Vβ4 and Vβ6 significantly more in the blood than in the thymus, while the opposite occurred for Vβ7, Vβ12 and Vβ14. Controls used Vβ17 more and Vβ24 less in the blood than in the thymus. The preferential expansion of Vβ4 and Vβ6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vβ usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vβ usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vβ usage in blood and thymi of MG patients are likely to be due to preferential Vβ usage by the anti-AChR T cells in the blood.     

系统性红斑狼疮患者脱氧核糖核酸酶1基因表达研究

目的　研究脱氧核糖核酸酶 1(deoxyribonuclease ,DNASE1)基因表达及 m RNA剪接形式与系统性红斑狼疮 (systemic lupus erythematosus,SL E)的关联性。　方法　以实时荧光定量聚合酶链反应 (real- time PCR)方法检测 DNASE1的 m RNA表达水平 ,以毛细管电泳技术分析 m RNA编码区替代剪接体 ,结合单核苷酸多态性 (single nucleotide polymorphisms,SNPs)单倍型了解基因结构对表达的影响。　结果　SL E患者 DNASE1基因表达水平显著高于正常对照 (P<0 .0 0 1) ,未发现 SL E疾病活动性指数积分与基因表达水平存在相关性 ,但性别分析显示女性患者 DNASE1基因表达水平高于男性患者 (P<0 .0 1)。 8名正常人与 18例患者的毛细管电泳结果显示 ,患者与正常人替代剪接谱系不同 ,且 380 bp处存在明显条带 ,具有不同单倍型的 SL E患者替代剪接谱系亦有差异。　结论　 SL E患者 DNASE1基因表达异常 ,并存在与正常人不同的 m RNA剪接体。 DNASE1基因与 SL E发病相关