Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

蛋白酪氨酸磷酸酶SHP-1/SHP-2催化活性域的表达和动力学分析

为了分离纯化SHP-1/SHP-2催化活性域蛋白(分别命名为D1C/D2C), 并估测其动力学常数, 将已经构建好的D1C/D2C重组质粒转化Escherichia coli BL21菌株, 经IPTG诱导表达、菌体裂解缓冲液悬浮和超声波破碎后, 通过HPLC分离纯化D1C/D2C蛋白, 所得产物进行SDS-PAGE电泳检测。然后, 以pY作为去磷酸化反应的底物, 利用孔雀绿显色法, 通过双倒数作图法对纯化的D1C/D2C蛋白进行动力学分析。结果表明, 本试验已成功地表达了D1C和D2C蛋白, 主要以可溶性蛋白的形式表达; 利用HPLC技术可有效地对D1C/D2C蛋白进行分离纯化; D1C的相对分子质量为34.6 kD, 米氏常数Km=2.04 mmol/L, 催化常数Kcat=44.98 s, 特异性常数Kcat/Km=22.05 L/(mmol·s); D2C的相对分子质量为35.3 kD, 米氏常数Km=2.47 mmol/L, 催化常数Kcat=27.45 s, 特异性常数Kcat/Km=11.11 L/(mmol·s); D1C的磷酸酶活性较强于D2C。     

Gelsolin蛋白在类风湿性关节炎和系统性红斑狼疮的临床诊断及疾病活动度评价中的意义

目的:分析gelsolin蛋白对类风湿性关节炎(rheumatoid arthritis,RA)和系统性红斑狼疮(systemic lupus erythematosus,SLE)的临床诊断及疾病活动度评价的意义。方法:采集RA 30名和SLE 47名及健康人群50名的临床资料及血清标本,定量Western Blot法检测血清gelsolin水平。分析gelsolin蛋白与RA和SLE患者临床表现及疾病活动度的相关性。结果:RA、SLE和正常对照组之间性别、年龄、血红蛋白、血小板、血红细胞、血白细胞之间没有显著差异;RA患者出现CRP、转氨酶、RF、CCP异常的阳性率明显高于SLE患者(P0.05);而SLE患者出现白蛋白、尿蛋白、尿红细胞、尿素氮、ANA、肌酐异常增高的几率高于RA患者(P0.05)。gelsolin蛋白在SLE和RA血清中的含量均显著低于正常人(P0.05),且RA患者含量更低(P0.05)。gelsolin蛋白滴度与RA的疾病活动度无明显相关性(r=0.089,P=0.652),而与SLE的疾病活动度呈显著负相关(r=0.646,P0.05)。gelsolin蛋白正常组RA患者的转氨酶升高、CRP、RF、CCP阳性率均显著高于SLE患者(P0.05)。gelsolin蛋白降低组SLE患者的白蛋白、尿蛋白、尿红细胞、尿素氮、ANA、肌酐阳性率显著高于RA患者(P0.05)。结论:gelsolin蛋白滴度检测可作为RA和SLE临床辅助诊断手段,其滴度变化可作为SLE疾病活动度进展的预判指标。     

NLRP3炎症小体与自身免疫性疾病的相关性研究进展

炎症小体是存在于细胞内由激活自身免疫应答的多种蛋白质组成的复合体,可诱导半胱天冬蛋白酶(caspase)-1自我剪切,caspase-1能够调控白细胞介素(IL)-1β、IL-18的产生,并进而刺激炎症小体的形成和分泌,调控机体的自身免疫应答反应。NLRP3炎症小体属于NOD样受体家族,是一种胞内模式识别受体,主要存在于巨噬细胞和树突状细胞,发挥激活机体免疫炎症的关键作用。病原相关分子模式及损伤相关分子模式与NLRPs结合,启动固有免疫应答,从而导致自身免疫性疾病的发生和发展。本文通过分析归纳近年来炎症小体与自身免疫性疾病的相关性的研究进展,以期为以炎症小体为作用靶点,防治自身免疫性疾病的研究提供指导。     

腺苷脱氨酶及同工酶对自身免疫病诊断和病情监测的作用

摘要 目的：检测腺苷脱氨酶(ADA)及其同工酶在自身免疫病患者血清中的变化,探讨其诊断和病情监测作用。方法：收集70例系统性红斑狼疮（SLE）、114例类风湿性关节炎(RA)、55例强直性脊柱炎(AS)、及其年龄性别对应的健康血清标本。测定血清总ADA（tADA）、ADA1及ADA2活性。ROC曲线分析其诊断价值。结果：与健康对照相比,ADA活性在SLE患者血清中显著升高[tADA：15（12,20）vs 8（7,10）U/L;ADA1：3.5（2,5）vs 3（2,3）U/L;ADA2：11（8,15）vs 6（5,7）U/L;P<0.01）]。与健康对照相比,RA患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性水平升高[8（5.25,10）vs 7（5,9）U/L,P<0.05）];与健康对照相比,AS患者血清中tADA和ADA1活性无显著变化(P>0.05),ADA2活性升高[7（5,9）vs 6（5,7）U/L,P<0.05）]。ROC分析显示tADA及ADA2对SLE具有较好诊断价值（tADA：88.6%特异性、77.1%敏感性;ADA2：92.9%特异性、68.6%敏感性）。ADA活性对RA及AS患者无诊断价值。spearman相关性分析显示,tADA活性与SLE患者疾病活动度有一定正相关性（r=0.303,P=0.011)。结论：血清tADA活性检测可作为SLE辅助诊断和病情监测指标。     

Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity

AimImbalance of T-helper-cell (TH) subsets (TH1/TH2/TH17) and regulatory T-cells (Tregs) is suggested to contribute to the pathogenesis of Systemic lupus erythematosus (SLE). Therefore, we evaluated their cytokine secretion profile in SLE patients and their possible association with disease activity.MethodsSixty SLE patients, 24 rheumatoid arthritis (RA) patients and 24 healthy volunteers were included in this study. Demographic, clinical, disease activity and serological data were prospectively assessed. Plasma cytokines levels of TH1 (IL-12, IFN-γ), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-β) were measured by enzyme linked immunosorbent assays (ELISA).ResultsSLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL-12 (p < 0.001) and IL-10 (p < 0.05) but comparable levels of IL-23 and IL-4 and slight reduction (but statistically insignificant) of TGF-β levels compared to controls. IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) with disease activity. The RA group exhibited significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-β (p < 0.5) and lower IFN-γ (p < 0.001) and IL-10 (p < 0.01) than those of healthy subjects.ConclusionOur study showed a distinct profile of cytokine imbalance in SLE patients. Reduction in IFN-γ (TH1) and TGF-β1 (Treg) with the elevation in IL-6 and IL-17 (TH17) could imply skewing of T-cells toward TH17 cells. Breaking TH17/Treg balance in peripheral blood may play an important role in the development of SLE and could be responsible for an increased pro-inflammatory response especially in the active form of the disease.     

Potential role of melatonin in autoimmune diseases

Autoimmune diseases are a broad spectrum of disorders involved in the imbalance of T-cell subsets, in which interplay or interaction of Th1, Th17 and Tregs are most important, resulting in prolonged inflammation and subsequent tissue damage. Pathogenic Th1 and Th17 cells can secrete signature proinflammatory cytokines, including interferon (IFN)-γ and IL-17, however Tregs can suppress effector cells and dampen a wide spectrum of immune responses. Melatonin (MLT) can regulate the humoral and cellular immune responses, as well as cell proliferation and immune mediators. Treatment with MLT directly interferes with T cell differentiation, controls the balance between pathogenic and regulatory T cells and regulates inflammatory cytokine release. MLT can promote the differentiation of type 1 regulatory T cells via extracellular signal regulated kinase 1/2 (Erk1/2) and retinoic acid-related orphan receptor-α (ROR-α) and suppress the differentiation of Th17 cells via the inhibition of ROR-γt and ROR-α expression through NFIL3. Moreover, MLT inhibits NF-κB signaling pathway to reduce TNF-α and IL-1β expression, promotes Nrf2 gene and protein expression to reduce oxidative and inflammatory states and regulates Bax and Bcl-2 to reduce apoptosis; all of which alleviate the development of autoimmune diseases. Thus, MLT can serve as a potential new therapeutic target, creating opportunities for the treatment of autoimmune diseases. This review aims to highlight recent advances in the role of MLT in several autoimmune diseases with particular focus given to novel signaling pathways involved in Th17 and Tregs as well as cell proliferation and apoptosis.     

The PD-1/PD-Ls pathway and autoimmune diseases

The programmed death (PD)-1/PD-1 ligands (PD-Ls) pathway, is a new member of the B7/CD28 family, and consists of the PD-1 receptor and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). Recently, it is reported that PD-1, PD-L1 and PD-L2 also have soluble forms aside from their membrane bound forms. The soluble forms increase the diversity and complexity of PD-1/PD-Ls pathway in both composition and function. The PD-1/PD-Ls pathway is broadly expressed and exerts a wider range of immunoregulatory roles in T-cell activation and tolerance compared with other B7/CD28 family members. Studies show that the PD-1/PD-Ls pathway regulates the induction and maintenance of peripheral tolerance and protects tissues from autoimmune attack in physiological conditions. In addition, it is also involved in various diseases mediated by T cells, such as autoimmunity, tumor immunity, chronic viral infections, and transplantation immunity. In this review, we will summarize the relevance of the soluble forms and the latest researches on the role of PD-1/PD-Ls pathway in autoimmune diseases.     

血清腺苷脱氨酶检测在几种自身免疫性疾病中的临床意义研究

目的:探讨血清腺苷脱氨酶(ADA)检测在几种自身免疫性疾病诊断中的应用价值。方法:收集确诊的系统性红斑狼疮(127例)、类风湿性关节炎(103例)和重症肌无力患者(34例)的血清标本,以同时期体检的正常成人(149例)为对照,采用ADA偶联嘌呤核苷磷酸化酶(PNP)和黄嘌呤氧化酶(XOD)法检测血清中ADA活性。结果:系统性红斑狼疮、类风湿性关节炎、重症肌无力患者和正常体检者血清中的ADA活性均值分别为(12.91±5.82)U/L、(10.91±3.70)U/L、(9.21±3.57)U/L、(8.73±2.79)U/L。系统性红斑狼疮、类风湿性关节炎患者血清中ADA活性均显著高于正常体检者(P0.05),而重症肌无力患者血清中ADA活性和正常体检者相比差别无统计学意义(P0.05)。ROC结果显示将血清ADA活性10U/L同时作为RA和SLE诊断的阳性值,ADA活性诊断SLE的AUC为0.748,敏感性为59.8%,特异性为83.2%。ADA活性诊断RA的敏感性为42.7%,特异性为83.2%。血清ADA活性为10 U/L作为诊断的参考值上限,其诊断SLE的效果优于ESR和hs-CRP,其诊断RA的效果低于ESR和hs-CRP。结论:系统性红斑狼疮、类风湿性关节炎患者血清ADA活性均有升高,在系统性红斑狼疮患者中升高更为明显,可作为系统性红斑狼疮潜在的辅助诊断标志物。     

An extended HLA-DQ-DR haplotype rather than DRB1 alone contributes to RA predisposition

 In the present study, we tested our hypothesis on the role of a DQ-DR haplotype in rheumatoid arthritis (RA) predisposition. Using two groups of patients and controls, one from The Netherlands and one from Switzerland, we found that DQA1*0301-homozygous and DQA1*0301//DQA1*0101/04-heterozygous individuals are highly predisposed to RA in both populations, while DQA1*0101/04-homozygous are not. The DQA1*0301-DRB1*0403/06/07 and DQA1*0301-DRB1*0901 haplotypes are not associated with RA by themselves but strongly increase the risk of developing disease in DQA1*0301- and DQA1*0101/04-heterozygous. DRB1 alleles carrying the motif DERAA in their third hypervariable region, i.e., *0103, *0402, *1102, *1103, *1301, and *1302, provide a long-lasting protection against RA in DQA1*0101/04- but not in DQA1*0301-positive individuals. These data show that considering both DQ and DR gives a better distinction between patients and controls than the shared epitope hypothesis. Received: 5 March 1998 / Revised: 21 April     

Association of the interferon-γ receptor variant (Val14Met) with systemic lupus erythematosus

 Genetic factors seem to play a significant role in susceptibility to systemic lupus erythematosus (SLE). The purpose of this study was to investigate whether the amino acid polymorphism (Val14Met) found within the IFN-γ receptor gene (IFNGR1) plays a prominent role in susceptibility to SLE. We found Val14Met located at the COOH terminal of the signal peptide of the IFN-γ receptor. There was a significant difference in this polymorphism frequency between SLE patients and healthy populations. To clarify whether this amino acid substitution resulted in the alteration of the receptor function, we evaluated the induction of HLA-DR antigen expression on B cells by IFN-γ stimulation. There was also a significant difference in the induction of HLA-DR by IFN-γ stimulation between B cells. Furthermore, an intracellular cytokine assay indicated that the Th1/Th2 balance of Th cells bearing the variant receptor shifted to Th2. The genetic polymorphism found within the IFN-γ receptor gene (Val14Met) may result in a shift to Th2, and this shift may increase susceptibility to SLE. Received: 13 April 1998 / Revised: 30 July 1998     

高分压氧下淋巴细胞SHP-1和CD45的功能状态

目的: 探讨高分压氧下淋巴细胞内酪氨酸磷酸酶SHP-1和CD45功能状态的变化.方法: 分别用能引起功能发生不同变化的高分压氧处理淋巴细胞,检测细胞内酪氨酸磷酸酶SHP-1和CD45的催化活性、蛋白量及蛋白磷酸化水平.结果: 经各压力-时程的高分压氧处理后,SHP-1的活性均降低;而CD45仅在具有抑制细胞功能的氧剂量处理后其活性才降低.两种酶的蛋白表达量及酪氨酸磷酸化水平没有发生显著变化.结论: 高分压氧下SHP-1和CD45活性降低可能是由于酶结构受增多的活性氧破坏引起;SHP-1和CD45可能是所选高分压氧方案引起淋巴细胞功能变化的作用位点.     

Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum

C1q is of interest in systemic lupus erythematosus (SLE) research due to deficiencies in its activity being associated with the disease. Current published protocols for measuring C1q vary greatly in their results and ease of reproducibility. Due to this, average C1q concentrations have been reported between 56 and 276 μg/mL in non-SLE serum. We present an improved method for quantifying C1q concentrations, which employs a sandwich ELISA. This method has improved precision, cost efficiency, up-scaling, reproducibility, and uses significantly lesser volumes of serum sample when compared to RID and other methods for quantifying C1q. We report an average concentration of 113 ± 40 μg/mL for C1q in non-SLE serum. The assay designed here will be useful in the high-throughput measurement of serum C1q in SLE cases.     

LST1/A is a myeloid leukocyte-specific transmembrane adaptor protein recruiting protein tyrosine phosphatases SHP-1 and SHP-2 to the plasma membrane

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.     

Novel imidazopyridine suppresses STAT3 activation by targeting SHP-1

The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthesized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16 on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.     

NT FRZ 基因多态性与SEL 的相关性研究

本研究通过调查中国南方SLE人群和健康对照人群中TNFR2基因两个位点（nt587,nt694）的多态性频率,探讨TNFR2基因多态性是否与中国汉族SLE人群的易感性相关。结果发现128例SLE中,nt587G的等位基因频率为54个（21．1％）;而135例健康人群中nt587G的频率为35个（13．0％）;SLE组明显高于健康对照人群（P〈0．05）,携带nt587G的个体SLE发病危险性大。同时128例SLE中,舶94A的等位基因频率为41个（16．0％）;而健康人群中舶94A的频率为32个（11．9％）;两组比较无显著差异（P=0．149）。提示TNFR2基因nt587的多态性与中国南方SLE人群相关,可能通过影响TNFR2的表达而参与SLE的发病,而nt694（G—A）的多态性与中国南方SLE人群不相关。     

Differential regulation of leukemia inhibitory factor-stimulated neuronal gene expression by protein phosphatases SHP-1 and SHP-2 through mitogen-activated protein kinase-dependent and -independent pathways

The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor alpha (LIFR) and gp130. gp130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP-2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen-activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild-type, SHP-2 reduced LIFR- and gp130-mediated activation of MAPK up to 75%. Conversely, coexpression of the wild-type, but not catalytically inactive, SHP-1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP-2 and SHP-1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor-mediated tyrosine phosphorylation of SHP-2. In contrast, SHP-1 association with gp130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP-1 has a positive regulatory role on LIF-stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP-2 is negative. Furthermore, LIF-stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP-2 also negatively regulates LIF-dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP-1 and SHP-2 are important regulators of LIF-dependent neuronal gene expression via both MAPK-dependent and -independent pathways.     

Protein-protein interaction between caveolin-1 and SHP-2 is dependent on the N-SH2 domain of SHP-2

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected H₂O₂-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes. [BMB Reports 2015; 48(3): 184-189]