M型磷脂酶A2受体及其抗体和Nephrin在不同类型膜性肾病患者的表达

目的:检测不同类型膜性肾病(membranous nephropathy,MN)患者肾组织PLA2R、Nephrin及血清anti-PLA2R的表达量,判断它们之间是否存在相关性及其意义。方法:收集MN患者37例为实验组,包括特发性膜性肾病(idiopathic membranous nephropathy,IMN)患者23例、HBV相关性MN(HBV-MN)患者8例、V型狼疮性肾炎(stage V lupus nephritis,LN-V)患者6例,选取10例肾肿瘤行一侧肾切术患者为对照组。应用免疫组织化学法检测各组肾组织中PLA2R及Nephrin的表达,通过专业图像处理软件对其表达进行半定量分析,并应用ELISA检测各组血清anti-PLA2R的表达量。结果:1)各实验组及对照组均可见PLA2R及Nephrin沿肾小球毛细血管袢沉积。IMN组PLA2R较其余各组表达量明显升高,差异有统计学意义,而剩余三组比较无统计学差异。各组Nephrin相对表达量,对照组>LN-V>HBV-MN>IMN,差异有统计学意义;2)IMN组肾组织Nephrin相对表达量同肾组织PLA2R及血清anti-PLA2R浓度间均负相关,肾组织PLA2R相对表达量同血清anti-PLA2R浓度呈正相关;3)IMN组中血清anti-PLA2R阳性者其肾组织PLA2R均表达升高。结论:IMN患者肾组织中PLA2R的表达较继发性膜性肾病(secondary membranous nephropathy,SMN)及正常对照组高,IMN组中血清anti-PL A2R阳性者其肾组织PL A2R均表达升高,临床上可联合二者共同鉴别MN是否原发,结合肾组织中Nephrin的检测,可增强其敏感性。PLA2R同足细胞裂孔隔膜蛋白Nephrin于IMN患者肾组织表达量呈负相关,为IMN的发病机制的研究提供进一步支持。     

特发性膜性肾病患者血清PLA2R抗体的检测技术及意义

特发性膜性肾病(idiopathic membranous nephropathy, IMN),即原发性膜性肾病,易发生在40岁以上男性群体中。IMN诊断的主要依据为临床表现及肾活检病理改变,后者是一种有创检测,对患者有一定影响。自肾小球足细胞表面M型磷脂酶A2受体(phospholipase A2 receptor, PLA2R)发现以来,相关领域对IMN的发病机制有了新的认识。随着对PLA2R抗体研究的不断深入,发现其不仅可以作为IMN的诊断指标,而且有助于判断疾病活动情况及疗效监测。该文就抗PLA2R抗体检测技术在IMN中的研究进展作一简要综述。     

血清中抗磷脂酶A2 受体特异性抗体的两种检测方法比较

目的:比较两种不同方法检测特发性膜性肾病(Idiopathic membranous nephropathy,IMN) 患者血清磷脂酶A2受体(M-Phospholipase A2 Receptor,PLA2R)抗体检出率,评价两种方法检测抗体对疾病的诊断价值。方法:病例为2014 年12月至2015 年10 月中国医科大学附属盛京医院收治的IMN 及其他明确病理诊断的入院患者,分为IMN 组和非IMN 组,整理临床资料;采用间接免疫荧光法(Indirect immunofluorescence assay,IFA) 和酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)对患者血清抗PLA2R 抗体进行检测。结果:IFA 方法与ELISA 方法检测抗PLA2R 抗体的敏感度分别为71.3%、68.5%,特异度均为100%,两者ROCAUC 分别为0.860、0.839,两种方法的诊断效能无统计学差异(P>0.05),且两种方法有较好的一致性(资=0.876),一致率达93.8%;抗体阳性的患者,易出现血清血白蛋白水平的降低(P<0.05);抗体滴度越高患者的低蛋白血症越严重,发生大量蛋白尿比例越高(P<0.05)。结论:两种方法检测血清中PLA2R 抗体均有较高的敏感度和特异度,该抗体可作为IMN 患者良好的诊断指标。     

抗激活小鼠T细胞抗原单克隆抗体（2H3）识别的靶抗原分析

2H3为一株抗激活小鼠T细胞表面抗原的IgG_1型单克隆抗体。间接ELISA实验表明:它能与ConA 激活的小鼠脾细胞及IL—2依赖的CTLL—2细胞发生特异性结合。靶细胞免疫荧光染色法表明:上述两种靶细胞可与荧光标记的抗IL—2R单抗结合并可被事先与2H3作用所阻断,但该两种靶细胞与荧光标记抗Ia抗体的结合不能被2H3所阻断。对2H3所结合的靶细胞膜蛋白提取物进行的Western-Blotting实验表明:2H3识别的靶抗原分子量约为50～60KD。结果提示:2H3识别的靶抗原与小鼠细胞膜表面的IL—2R(P_(55)蛋白)是一致的,其免疫生物学活性正在进行分析中。     

特发性膜性肾病患者血清抗磷脂酶A2受体抗体与尿中IgG4检测的临床意义

目的：探讨特发性膜性肾病（IMN）患者血清抗磷脂酶A2受体（PLA2R）抗体及尿IgG4检测的临床意义。方法：将90例膜性肾病（MN）患者按照病理类型分为特发性膜性肾病（IMN）组（52例）和继发性膜性肾病（SMN）组（38例）;另选同期体检健康者35例作为对照组。检测各组血清抗PLA2R抗体表达和尿IgG4水平;分析治疗后不同转归的IMN患者之间的血清抗PLA2R抗体阳性率及尿IgG4水平差异。结果：IMN组的血清抗PLA2R抗体阳性率及尿IgG4水平均明显高于SMN组和对照组（P＜0.05）;SMN组的尿IgG4水平明显高于对照组（P＜0.05）,而血清抗PLA2R抗体阳性率与对照组差异无显著统计学意义（P＞0.05）;治疗后,IMN缓解组的血清抗PLA2R抗体阳性率明显低于未缓解组（P＜0.05）,治疗后尿IgG4水平较本组治疗前及未缓解组治疗后均降低（P＜0.05）,而治疗后未缓解组的尿IgG4水平较治疗前不降反升（P＜0.05）;IMN复发患者的血清抗PLA2R抗体阳性率及尿IgG4水平均明显高于无复发者（P＜0.05）。结论：IMN患者血清抗PLA2R抗体阳性率和尿IgG4水平明显升高,其变化与IMN的病情及远期结局均有关,二者联合检测有助于IMN的诊断、病情活动及预后评估。     

M2e通用流感疫苗的研究进展

M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.     

M2e通用流感疫苗的研究进展

M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.     

M2e通用流感疫苗的研究进展

M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.     

M2e通用流感疫苗的研究进展

M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.     

M2e通用流感疫苗的研究进展

M2基质蛋白是A型流感病毒膜蛋白,在A型流感病毒的生命周期中,M2具有重要的生物学功能,已成为抗病毒药物研究的靶蛋白.其胞外区M2e(M2 eetodomain,M2e)为24个氨基酸残基,该片段在多病毒株中具有极高的保守性.针对M2e产生IgG型抗体能够防止流感病毒引发的死亡,减少动物模型中流感的发病率.了解有关M2e疫苗的研究进展,以及关于M2e作为A型流感疫苗靶抗原的关键问题很重要.     

Membranous nephropathy: A ten-year journey of discoveries

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Over the last decade important research discoveries have revealed that most “idiopathic” cases are caused by autoantibodies to podocyte antigens including phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain containing 7A (THSD7A). In this review, we will discuss the histopathology of primary MN, recent revelations regarding pathogenesis, and ancillary tests.     

Immunology of membranous nephropathy: from animal models to humans

Membranous nephropathy (MN), the leading cause of nephrotic syndrome in adults, is characterized by the deposition of subepithelial immune deposits that consist mainly of immunoglobulin (Ig)G and complement. Most of the cases are primary or idiopathic (iMN), while only approximately 25% of the cases are secondary to some known disease such as systemic lupus erythematosus, hepatitis B, drugs and malignancies. Most of our knowledge on the pathogenesis of iMN has relied upon old experimental models (i.e. Heymann nephritis) that have shown that immune deposits are formed in situ by the reaction of autoantibodies against the respective podocyte antigen. Recent findings indicate that podocyte proteins also act as an autoantigen in human iMN. The M‐type phospholipase A2 receptor (PLA2R) has been identified as the main target antigen, as it can be found in approximately 70% of iMN patients but only rarely in other glomerulonephritides. Podocytes damage in the experimental model of Heymann nephritis is complement‐mediated. In humans, the presence of complement within the subepithelial deposits is well established, but IgG4, which does not activate complement by classical or alternative pathways, represents the predominant subclass of IgG anti‐PLA2R. Some evidence suggests that IgG4 anti‐PLA2R autoantibodies can bind mannan‐binding lectin (MBL) and activate the lectin complement pathway. A genetic background for iMN has been demonstrated by genome‐wide association studies that have shown highly significant associations of the PLA2R1 and the human leucocyte antigen (HLA)‐DQA1 loci with iMN. In addition to their diagnostic value, anti‐PLA2R antibodies may be useful to monitor disease activity and predict response to treatment.     

Course monitoring of membranous nephropathy: Both autoantibodies and podocytes require multidimensional attention

A variety of podocyte antigens have been identified in human membranous nephropathy (MN), which is divided into various antigen-dominated subtypes, confirming the concept that MN is the common pattern of glomerular injury in multiple autoimmune responses. The detection of autoantibodies, which has been widely used in the clinical practice of MN, may lead to personalized precision medicine. However, given the potential risks of immunosuppressive therapy, more autoantibodies and biomarkers need to be identified to predict the prognosis and therapeutic response of MN more accurately. In this review, we attempted to summarize the autoantigens/autoantibodies and autoimmune mechanisms that can predict disease states based on the current understanding of MN pathogenesis, especially the podocyte injury manifestations. In conclusion, both the autoimmune response and podocyte injury require multidimensional attention in the disease course of MN.     

A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A2.

Notexin and nigexine are monomeric phospholipases A2(PLA2s) from the venoms of Notechis scutatus scutatus and Naja nigricollis, respectively. Polyclonal antibodies raised in mice against these antigenic proteins displayed non-reciprocal cross-reactivity; anti-notexin antibodies recognized notexin but not nigexine, whereas anti-nigexine antibodies recognized both antigens. Polyclonal antibodies raised by successive immunization with nigexine and notexin contained cross-reacting antibodies with affinities for both antigens that differed from those of antibodies present in anti-nigexine antiserum. A monoclonal antibody has been obtained from a mouse immunized with both PLA2s. This monoclonal antibody, called MN1, recognized notexin and nigexine with comparable high affinity (Kd = 10(-9) M). It also recognized most purified PLA2s from elapid snake venoms and all PLA2-containing venoms from cobras and sea-snakes. This offers the first demonstration that most PLA2s from cobras and sea-snakes share a fine structure which is not restricted to the common catalytic site.     

Differential diagnosis of membranous nephropathy with autoantibodies to phospholipase A2 receptor 1

Membranous nephropathy (MN) accounts for most cases of the nephrotic syndrome in adults. Recently, studies on the underlying pathomechanisms led to the identification of the podocyte M-type receptor for secretory phospholipase A2 (PLA₂R1) as a target antigen of circulating autoantibodies.Autoantibodies to PLA₂R1 may not only play a role in the development of primary MN, but also serve as a marker for diagnosis, disease activity and therapy monitoring. Antibody detection is crucial to discriminate between patients with primary MN and those with a secondary form of the disease, as both forms require different diagnostic approaches and treatment strategies. Standardized test systems based on recombinant PLA₂R1 allow for the sensitive and specific analysis of anti-PLA₂R1 autoantibodies. Further research into pathogenic mechanisms and other disease markers can pave the way for improved patient care.     

THSD7A-associated membranous nephropathy in a patient with neurofibromatosis type 1

Target antigens in idiopathic membranous nephropathy (MN) include the phospholipase A2 receptor (PLA₂R), and in some cases, the thrombospondin type 1 domain-containing 7A (THSD7A). A notable phenomenon is the high rate of cancer (reported to be as high as 20%) in patients with THSD7A-associated MN. Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by NF1 gene mutation, and clinically characterized by multiple cutaneous neurofibromas and café-au-lait spots. In this article, we report a patient with NF1 who developed THSD7A-associated MN when the NF1 skin lesions deteriorated. The patient, a 62-year-old male, was referred to us for nephrotic syndrome for 6 months. Physical examination revealed multiple cutaneous nodules throughout the entire body, and the patient noted recent increase in the numbers of these skin lesions. Cutaneous nodules excisional biopsy suggested NF1 and Sanger sequencing using genomic DNA extracted from peripheral blood revealed a previously reported heterozygous frameshift NF1 mutation (c.1541_1542delAG, p. Gln514fs). Renal biopsy revealed MN and immunohistochemistry (IHC) showed enhanced staining of THSD7A as well as PLA₂R along the glomerular basement membrane whereas the serum level of THSD7A and PLA₂R were both within normal range. The neurofibroma tissues were positive for THSD7A but not for PLA₂R on IHC. The patient did not respond to 6-month treatment with glucocorticosteroid and cyclophosphamide. In this exceptional case, strong positive staining of THSD7A in both skin and renal biopsy samples, together with the temporal association between nephrotic syndrome and skin lesions and lack of treatment response, suggested the possibility that MN could be the result of immune response to THSD7A in NF1. This report may improve understanding of the mechanistic link between MN and cancer.     

Sex-related differences in the initiation of allergic rhinitis in mice

BACKGROUND: Several clinical and epidemiologic studies have investigated sex differences in the prevalence of allergic rhinitis. At present, however, no reports have demonstrated such differences in experimental models with local, but not parenteral, sensitization with antigens that may reflect natural exposure to allergens. We have recently developed murine models of allergic rhinitis after repeated intranasal sensitization with antigens in the absence of adjuvants. In this study, we investigated the role of sex in the initiation of the disease in vivo. METHODS: Male and female CBA/J and BALB/c mice were sensitized intranasally with phospholipase A2 (PLA2) and Schistosoma mansoni egg antigen (SEA), respectively, in the absence of adjuvants. After the repeated sensitization, serum Ab titers against the sensitizing antigen and nasal eosinophilia were determined. In addition, the involvement of androgen in IgE synthesis was investigated in castrated CBA/J male mice with or without testosterone administration. RESULTS: Females produced significantly higher levels of PLA2-specific IgE than males in CBA/J mice sensitized with PLA2. On the other hand, both titers of PLA2-specific IgG1 and nasal eosinophilia did not significantly differ between the two groups. Castrated male mice produced significantly higher amounts of PLA2-specific IgE than sham-treated male mice. In addition, PLA2-specific IgE production decreased in castrated mice treated with testosterone. Sexual differences in the production of Ag-specific IgE were not seen in BALB/c mice after the sensitization with SEA. CONCLUSION: These results suggest that sex is responsible for the production of Ag-specific IgE, but not IgG1 or nasal eosinophilia, and that androgen appears to be involved in the in vivo production of specific IgE in male mice.     

Binding of monoclonal antibodies to glomerular endothelium, slit membranes, and epithelium after in vivo injection. Localization of antigens and bound IgGs by immunoelectron microscopy.

The antigens recognized by seven monoclonal antibodies (MAbs) raised against rat glomerular proteins were localized, and the sites of binding of the MAbs after in vivo injection were determined by immunoelectron microscopy. The antigens were localized in situ by immunoperoxidase and immunogold labeling to different domains and microdomains of the glomerular endothelium and epithelium. 23A recognized an antigen expressed exclusively on the luminal (apical) domain of the endothelium. 5A (anti-podocalyxin) and 26C (anti-DPPIV) recognized antigens expressed on the apical domains of both the endothelium and podocytes. 13A, 14A, 20B (anti-gp330), and 27A recognized antigens restricted to podocytes in the glomerulus. The 13A antigen was present on their basal surface and the 27A and 14A antigens were expressed on both their apical and basal domains. The 14A antigen also was associated with the filtration slit membranes. All these MAbS bound to their antigens after injection in vivo. Those that recognize endothelial antigens were rapidly cleared from the circulation and rapidly disappeared from glomeruli, whereas those that recognize epithelial antigens persisted in the circulation and were detectable in glomeruli for hours or days. The sites of binding of the MAbs differed: 23A and 5A IgG (antipodocalyxin) bound exclusively to the luminal domain of the endothelium, whereas 26C IgG (anti-DPPIV) bound to both the luminal endothelial membrane and the apical and basal domains of podocytes. The MAbs that recognize podocyte antigens bound to different domains of the podocyte plasmalemma: 13A and 27A IgGs to the basal domain, 14A to the slit membranes, and 20B to coated pits on the entire plasma membrane. 27A IgG led to the formation of small subepithelial immune deposits that remained up to 10 days. It is concluded that 1) glomerular membrane proteins vary considerably in their distribution among plasmalemmal domains and microdomains of endothelial and epithelial cells; 2) virtually all structures in the glomerulus and all domains and micro-domains of the endothelium and podocyte are accessible to circulating antibodies; and 3) the fate of immune complexes formed by binding to glomerular components varies with the location of the antigen within the glomerulus, with those that bind to the basal domain and slit membranes of the podocyte persisting longer than the others.     

Expression of Fc epsilon receptors on activated human T lymphocytes

Our results clearly demonstrate that the low-affinity receptor for IgE (Fc epsilon R) is an activation antigen transiently expressed on a subpopulation of human T lymphocytes. It can be selectively induced by stimulation with certain antigens or lectins, but it is not found on resting T cells. The increased numbers of activated Fc epsilon R+ T cells observed after stimulation of peripheral blood mononuclear cells (PBMC) from bee venom allergic patients with the specific allergen phospholipase A2 (PLA2) suggest that Fc epsilon R+ T cells might very well be involved in the regulation of the human IgE response against the respective antigen. These results were obtained by the use of two monoclonal antibodies, M-L25 and M-L47, which were raised against the human low-affinity Fc epsilon R in our laboratory. After stimulation of PBMC with phytohemagglutinin a peak of 7.6 +/- 6% Fc epsilon R+ T cells was observed on day 3, with pokeweed mitogen of 0.8 +/- 0.8% on days 2 and 3, and with concanavalin A of 0.6 +/- 0.7% Fc epsilon R+ T cells on day 2. Stimulation of PBMC with tetanus toxoid (TT) induced Fc epsilon R on maximally 0.6 +/- 0.8% of the total T cells (day 4), stimulation with purified protein derivative from tuberculin (PPD) on 0.2 +/- 0.6% of the T cells (day 2). In contrast to these antigens, stimulation of PBMC from bee venom allergic patients with PLA2 induced as a peak 2.5 +/- 2.5% of the total T cells to express Fc epsilon R (day 5), although the stimulated T cell population was much smaller than with TT or PPD, as was shown by their stimulation indices. The allergen-stimulated Fc epsilon R+ T cells were exclusively T4+. The Fc epsilon R-expression index was determined, which for a specific antigen or lectin correlates the percentage of Fc epsilon R+ T cells to the stimulated T cell population, respectively.     

Type specificity and use of human sera for rapid typing of herpes simplex virus isolates by an indirect peroxidase-labeled antibody technique: a comparison with three other methods

Summary Human sera from patients showing seroconversion following a primary herpes simplex virus type 1 (HSV-1) or HSV type 2 (HSV-2) infection, as well as 19S and 7S fractions obtained from the same sera, have been used for typing 191 HSV isolates by an indirect peroxidase-labeled antibody (PLA) method. Typing has also been performed on all of the isolates using a microneutralization (MN) and the indirect hemagglutination (IHA) inhibition test. Furthermore, 30 isolates have been typed by a plaque method. Results obtained with the PLA method was in complete agreement with those of the other two procedures. The PLA method is rapid and simple, offers easy interpretation and a permanent record of results.With 3 Figures