Heptad motifs within the distal subdomain of the coiled-coil rod region of M protein from rheumatic fever and nephritis associated serotypes of group A streptococci are distinct from each other: Nucleotide sequence of the M57 gene and relation of the deduced amino acid sequence to other M proteins

Streptococcal M protein, a dimeric alpha helical coiled-coil molecule, is an antigenically variable virulence factor on the surface of the bacteria. Our recent conformational analysis of the complete sequence of the M6 protein led us to propose a basic model for the M protein consisting of an extended central coiled-coil rod domain flanked by a variable N-terminal and a conserved C-terminal end domains. The central coiled-coil rod domain of M protein, which constitutes the major part of the M molecule, is made up of repeating heptads of the generalized sequence a-b-c-d-e-f-g, wherein a and d are predominantly apolar residues. Based on the differences in the heptad pattern of apolar residues and internal sequence homology, the central coiled-coil rod domain of M protein could be further divided into three subdomains I, II, and III. The streptococcal sequelae rheumatic fever (RF) and acute glomerulonephritis (AGN) have been known to be associated with distinct serotypes. Consistent with this, we observed that the AGN associated M49 protein exhibits a heptad motif that is distinct from the RF associated M5 and M6 proteins. Asn and Leu predominated in the a and d positions, respectively, in subdomain I of the M5 and M6 proteins, whereas apolar residues predominated in both these positions in the M49 protein. To establish whether the heptad motif of M49 is unique to this protein, or is a general characteristic of nephritis-associated serotypes, the amino acid sequence of M57, another nephritis-associated serotype, has now been examined. The gene encoding M57 was amplified by PCR, cloned into pUC19 vector, and sequenced. The C-terminal half of M57 is highly homologous to other M proteins (conserved region). In contrast, its N-terminal half (variable region) revealed no significant homology with any of the M proteins. Heptad periodicity analysis of the M57 sequence revealed that the basic design principles, consisting of distinct domains observed in the M6 protein, are also conserved in the M57 molecule. However, the heptad motif within the coiled-coil subdomain I of M57 was distinct from M5 and M6 but similar to M49. Similar analyses of the heptad characteristics within the reported sequences of M1, M12, and M24 proteins further confirmed the conservation of the overall architectural design of sequentially distinct M proteins. Furthermore, the heptad motif within subdomain I of the AGN-associated serotypes M1 and M12 was similar to M49 and M57, whereas that of the RF associated M24 was similar to the M5 and M6 proteins. These results clearly demonstrate a correlation between the heptad motifs within the distal coiled-coil subdomain of the M proteins from different streptococcal serotypes and their epidemiological association with the sequelae AGN and RF.     

Carbohydrate specificity of IgM autoantibodies to CD45 in Systemic Lupus Erythematosus

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinantE. coli fusion proteins encoded by exons 3–7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.Abbreviations SLE systemic lupus erythematosus - SBA soybean agglutinin - RCAI Ricinus communis agglutinin - SNL Sambucus nigra lectin - MBP maltose binding protein - mAb monoclonal antibody - WGA wheat germ agglutinin     

大鼠肾脏和其它组织中磷酸酪氨酸蛋白的研究

本文采用P-tyr-BSA为免疫原免疫家无得抗血清。将纯化的IgG与HRP偶联,建立了P-tyr-Pr的ELISA法,并测定了正常大鼠肾脏等组织中P-tyr-Pr含量,其分布规律如下:上清中P-tyr-Pr含量高者,其颗粒部分则低,反之亦然;其中肾脏上清中含量远比其它组织(脾、肺、肝等)高。在此基础上,又研究了膜性肾炎大鼠肾脏P-tyr-Pr含量,发现其上清中的含量远远高于正常大鼠肾脏中的含量。     

Urinary proteomics revealed prostaglandin H(2)D-isomerase, not Zn-α2-glycoprotein, as a biomarker for active lupus nephritis

Although renal histopathology is the gold standard for the diagnosis and prognosis of lupus nephritis (LN), the invasiveness of renal biopsy warrants the discovery of novel non-invasive diagnostic and prognostic biomarkers. In the present study, urine samples from 10 LN patients (5 active and 5 inactive) were analyzed by two-dimensional gel electrophoresis (2-DE) to screen for potential biomarkers of active LN. Quantitative analysis and statistics revealed 16 protein spots whose levels significantly differed between groups. These proteins were successfully identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS). Among these potential candidates, differential levels of urinary Zn-α2-glycoprotein (ZA2G) and prostaglandin H(2)D-isomerase (PGDS) were further validated by enzyme-linked immunosorbent assay (ELISA) in another independent group of 78 subjects, including 30 active LN, 26 inactive LN, 14 non-LN glomerular diseases, and 8 healthy normal individuals. Whereas ZA2G levels were elevated in urine of patients with active LN and non-LN glomerular diseases, PGDS was elevated only in the urine of the active LN group. Urinary PGDS, not ZA2G, may serve as a biomarker for active LN and upon validation in larger studies, may become the non-invasive test to evaluate the disease activity in future management of LN.     

系统性红斑狼疮合并甲状腺功能减退症50例临床分析

目的:分析系统性红斑狼疮合并甲状腺功能减退症50例临床症状和治疗效果.方法:选取2010年6月至2012年6月间于我院接受诊断和治疗的系统性红斑狼疮合并甲状腺功能减退症患者50例作为观察组,同时选取同期入院的单纯系统性红斑狼疮患者50例作为对照组,分析两组临床症状的差异.观察组患者分别给予系统性红斑狼疮合并甲状腺功能减退症联合治疗和甲状腺功能减退症单独治疗,观察比较治疗前后甲状腺功能差异.结果:观察组患者关节病变发病率为64.00％明显高于对照组的22.00％(P＜0.05):观察组患者雷诺现象的发病率为36.00％明显高于对照组的6％(P＜0.05).联合治疗患者治疗后促甲状腺素水平明显低于单独治疗(P＜0.05),联合治疗患者治疗后游离甲状腺素和游离甲状腺三碘原氨酸水平均明显高于单独治疗(p＜0.05).结论:系统性红斑狼疮合并甲状腺功能减退症患者的关节病变和雷诺现象发病率明显较高,联合治疗后甲状腺功能减退症恢复较佳.     

狼疮性肾炎患者血清sTM、KIM-1、sCD134水平的表达及临床意义

摘要 目的：研究狼疮性肾炎（LN）患者血清可溶性血栓调节蛋白（sTM）、肾损伤分子-1（KIM-1）及可溶性CD134（sCD134）水平的表达及临床意义。方法：选择2016年12月至2018年12月我院收治的LN患者100例,根据系统性红斑狼疮疾病活动度指数(SLEDAI)将患者分为活动期组(SLEDAI≥10分)56例,非活动期组(SLEDAI<10分)44例。另取同期于我院接受体检的健康志愿者50例记为对照组。比较各组受试者的各项肾功能指标、血清sTM、KIM-1及sCD134水平,分析血清sTM、KIM-1及sCD134水平与肾功能指标的相关性。应用受试者工作特征（ROC）曲线分析血清sTM、KIM-1及sCD134水平在LN诊断中的能效。结果：活动期组血尿素氮（BUN）、血肌酐（Scr）以及红细胞沉降率（ESR）水平均高于非活动期组、对照组,且非活动期组BUN、Scr以及ESR水平均高于对照组（P＜0.05）。活动期组血清sTM、KIM-1及sCD134水平均高于非活动期组、对照组,且非活动期组血清sTM、KIM-1及sCD134水平均高于对照组（P＜0.05）。经Pearson相关性分析显示,LN患者血清sTM、KIM-1、sCD134水平与患者BUN、Scr、ESR水平呈正相关（P＜0.05）。ROC曲线分析显示,sTM最佳临界值为24.46 ng/mL,曲线下面积为0.823;KIM-1最佳临界值为8.27μg/L,曲线下面积为0.823;sCD134最佳临界值为15.25 ng/mL,曲线下面积为0.823。结论：LN患者血清sTM、KIM-1及sCD134水平与患者疾病活动程度密切相关,对LN具有很好的诊断效能,临床可能通过联合检测血清sTM、KIM-1及sCD134水平,为LN的诊断以及疾病活动程度提供评估参考。     

IgA1 isolated from Henoch‐Schönlein purpura children promotes proliferation of human mesangial cells in vitro

Previous studies show that the proliferation of human mesangial cells (HMCs) played a significant part in the pathogenesis of Henoch‐Schönlein purpura nephritis (HSPN). The aim of this study was to explore the proliferation of HMCs induced by IgA1 isolated from the sera of HSP patients. HMCs were cultured in three different types of media, including IgA1 from patients with HSP (HSP IgA1 group), healthy children (healthy IgA1 group) and medium (control group). The proliferation of HMCs incubated with IgA1 was determined by cell counting kit‐8 assay and bromodeoxyuridine incorporation. The expression of ERK1/2 and phosphatidylinositol 3 kinase/protein kinase B/mammalian targets of the rapamycin (PI3K/AKt/mTOR) signals and transferrin receptor (TfR/CD71) was detected with the methods of immunoblotting. The results indicated that the proliferation of HMCs significantly increased in the HSP IgA1 group compared with that in the control group or the healthy IgA1 group (P < 0.001). Moreover, we found that IgA1 isolated from HSP patients activated ERK and PI3K/AKt/mTOR signals, and markedly increased TfR/CD71 expression in HMCs. These effects induced by IgA1 isolated from patients with HSP were inhibited by human TfR polyclonal antibody (hTfR pAb) and soluble human transferrin receptor (sTfR), indicating that IgA1‐induced HMC proliferation and ERK1/2 and PI3K/AKt/mTOR activation were dependent on TfR/CD71 engagement. Altogether, these data suggested that TfR/CD71 overexpression and ERK1/2 and PI3K/AKt/mTOR activation were engaged in HMC proliferation induced by IgA1 from HSP patients, which might be related to the mesangial injury of HSPN.     

过敏性紫癜性肾炎肾组织中KIM-1的表达与临床意义

目的:探讨过敏性紫癜性肾炎肾组织中肾损伤分子1(kidney injury molecule 1,KIM-1)的表达与临床意义。方法:选择2015年4月到2018年1月在我院诊治的过敏性紫癜性肾炎患者150例作为研究对象,采用免疫组化法检测患者肾组织中KIM-1表达,采用半定量评分系统进行肾脏病理损害评分,并对二者进行相关性分析。结果:肾炎组织与肾旁组织的KIM-1相对表达量分别为(9.28±1.38)和(2.74±1.30),肾炎组织中KIM-1的表达显著高于肾旁组织(P=0.000);肾炎组织的毛细血管外肾小球活动、系膜增殖、内皮增殖、肾间质炎症、肾小球慢性化、肾小管间质慢性化指数评分均显著高于肾旁组织(P0.05);肾组织KIM-1表达量与肾小球慢性化指数、肾间质炎症指数、肾小管间质慢性化指数均呈显著正相关性(P0.05)。结论:过敏性紫癜性肾炎组织中KIM-1呈高表达,可能作为评估肾脏病理病变程度的参考指标。     

霉酚酸酯联合糖皮质激素治疗小儿紫癜性肾炎的疗效观察

目的:探究霉酚酸酯联合糖皮质激素治疗小儿紫癜性肾炎的疗效及安全性。方法:选择2012年3月~2015年9月70例于我院就诊的紫癜性肾炎儿童,按照不同的治疗方法将其分为观察组(35例)和对照组(35例)。两组患者入院后均给予常规治疗,在此基础上观察组患者给予泼尼松联合霉酚酸酯治疗,对照组给予泼尼松联合环磷酰胺治疗。比较两组的总有效率、临床症状消失时间、生化指标、免疫功能及不良反应发生情况。结果:观察组的临床总有效率为94.29%(33/35),对照组的总有效率为85.17%(30/35),两组比较差异无统计学意义(P=0.232)。治疗后,观察组蛋白尿、血尿等临床症状消失时间与对照组比较差异无显著意义(P0.05);两组患儿的24 h蛋白定量、CD19~+水平均较治疗前显著降低,且观察组CD19~+水平显著低于对照组(P0.05),两组的血清白蛋白、总蛋白含量、CD3~+、CD3~+CD4~+水平在均较治疗前明显上升,且观察组的CD3~+、CD3~+CD4~+水平显著高于对照组(P0.05)。观察组治疗期间不良反应发生率显著低于对照组(P=0.012)。结论:霉酚酸酯联合糖皮质激素治疗小儿紫癜性肾炎的临床效果较泼尼松联合环磷酰胺治疗更好,安全性更高。