1例伴发恶性胸腺瘤的副肿瘤性天疱疮临床及免疫学研究

目的报告我国首例伴发恶性胸腺瘤的副肿瘤性天疱疮(PNP)临床及免疫学特征。方法对患者进行系统体检、医学影像学、组织病理学和免疫学检查,使用重组蛋白免疫印迹法对由胸腺瘤分泌抗体所针对的自身抗原进行了研究。结果患者确诊为PNP,伴发恶性胸腺瘤,患者血清中含有抗重组人包斑蛋白(envoplakin)和周斑蛋白(periplakin)L亚区GST融合蛋白的抗体。结论PNP具有独特的临床表现、组织病理以及免疫学特点,患者血清中的自身抗体针对多种抗原,包斑蛋白和周斑蛋白的L亚区很可能是PNP患者血清抗体结合的主要位点。     

副肿瘤性天疱疮患者免疫荧光与免疫印迹检测研究

目的 研究副肿瘤性天疱疮患者的免疫学特点。方法 采用免疫荧光和免疫印迹方法检测4例副肿瘤性天疱疮患者肿瘤切除前、后的血清。结果 以鼠膀胱为底物行间接免疫荧光示IgG和C3棘细胞间沉积，肿瘤切除后抗体滴度较术前下降，且与病情的好转成正比。比正常人皮肤及大鼠舌、食管及猴舌为底物行间接免疫荧光阳性；以大鼠支气管、心肌、肝脏、肾脏等组织为底物行间接免疫荧光阴性。免疫印迹示患者血清可识别人角质形成细胞提取物中的210000和190000抗原。结论 以鼠膀胱为底物的间接免疫荧光可作为副肿瘤性天疱疮的过筛试验，且可通过抗体滴度的改变监测病情的变化。副肿瘤性天疱疮更易累及粘膜部位上皮组织。免疫印迹结果可确诊4例患者为副肿瘤性天疱疮。     

大疱性类天疱疮患者血清抗BP180自身抗体识别抗原表位的研究

目的分析大疱性类天疱疮(BP)患者血清抗BP180IgG和IgE自身抗体所识别的抗原表位。方法利用柱亲和层析的方法从10例BP患者血清中提取抗BP180-NC16A自身抗体,用免疫印迹技术对所获IgG和IgE自身抗体分别进行抗原表位鉴定。结果所有10例患者的自身抗体均识别aa507-aa532区域内(NC16A-2和NC16A-2.5)至少一个抗原表位,而且来自同一患者的IgG和IgE自身抗体在该区域所识别的表位完全一致。BP自身抗体与NC16A-1、NC16A-3和NC16A-4区域的结合率较低,与NC16A-5片段不结合。结论BP致病性自身抗体所识别的主要抗原表位可能位于BP180分子NC16A片段的aa507-aa532区域内。     

正常包皮为底物的免疫印迹技术检测寻常型天疱疮抗体

目的建立以正常包皮为底物用免疫印迹技术检测寻常型天疱疮抗体的方法。方法环切的正常包皮分离表皮后提取天疱疮抗原,进行免疫印迹反应,对15份寻常型天疱疮(PV)患者血清进行检测,并与间接免疫荧光(IIF)比较。结果15份血清都检测到与表皮分离抗原结合的IgG抗体,且在约130KD抗原处出现条带,7份血清同时与分子量为160KD抗原结合,健康对照者中未检测出特异性天疱疮抗体。免疫印迹和间接免疫荧光结果差异无显著性意义(P=1)。结论免疫印迹技术在天疱疮的实验室诊断中具有重要的应用价值。     

大疱及疱疹性皮肤病

982191 免疫印迹技术检测天疱疮患者血清结合的天疱疮抗原/顾富祥（南京鼓楼医院皮肤科）…//中华皮肤科杂志.-1998,31（1）.-47～48 应用免疫印迹,以正常表皮提取物为抗原,检查18例天疱疮患者血清。结果显示4例寻常性天疱疮（PV）血清都与表皮提取物中130000分子反应,4例疱疹样天疱疮（PH）血清2例与表皮提取物中130000蛋白反应,另2例与表皮提取物中160000反应,1例落叶性天疱疮（PF）和9例红斑性天疱疮（PE）血清中有4例与表皮提取物160000蛋白反应。表明免疫印迹技术能准确地测定天疱疮患者血清中自身抗体所对应的自身抗原的分子量,从而准确地诊断天疱疮,并可进一步加以分型。4例PH患者血清反应不同,因为它既可以转变为PV,也可以转化为PF,说明免疫印迹对不典型的天疱疮亦能诊断。图2参5 （张小莲）     

大疱性类天疱疮免疫学研究进展

大疱性类天疱疮是一种好发于老年人的自身免疫性表皮下大疱病,血清中存在针对靶抗原BP230和BP180的自身抗体,自身抗体识别的靶抗原表位主要位于BP180分子胞外的非胶原编码区NC16A区段.此外,细胞因子与补体的活化亦参与皮肤损伤和水疱形成.BP可能存在遗传易感性,HLA-II类基因HLA-DR和DQ在BP的发生和发展过程中起一定作用.     

大疱性类天疱疮患者治疗前后血清抗BP180自身抗体检测

我们应用酶联免疫吸附试验（ELISA）的方法检测BP患者血清内抗BP180自身抗体，并对治疗前后的抗体滴度变化进行了比较。研究表明，大疱性类天疱疮抗原1（BPAG1，BP230）和BPAG2（BP180）是BP患者血清自身抗体所针对的主要靶抗原。BP230系胞浆蛋白表达在角质形成细胞的胞浆内，BP180暴露在细胞外的部分是自身抗体结合的主要位点，多数抗原表位位于非胶原编码区NC16a结构域内，因而针对BP180的自身抗体被认为是主要的致病性自身抗体，对BP180及其自身抗体的研究在探讨BP发病机制和治疗对策以及临床诊断和判断疾病严重程度等方面具有重要意义。     

伴限局性Castleman病的副肿瘤性天疱疮临床及实验室研究

目的：报告6例伴限局性Castleman病副肿瘤性天疱疮，从临床、组织病理和免疫学方面进行了研究，从而能早期诊断，早期治疗。方法：采用全面临床检查，包括CT检查，常规组织病理检查，间接免疫荧光检查，免疫印迹及酶联免疫吸附测定（ELISA）。结果：6例患者临床上均有严重的口腔黏膜糜烂或溃疡。工有形态各异及程度不等的皮损，CT检查均示有潜在单发的内脏肿瘤。组织病理检查示表皮内疱，基底细胞液化变性，表皮内坏死角质形成细胞及真皮浅层以淋巴细胞为主的浸润。免疫学检查示患者血清中含抗棘细菌间抗体，它们能与桥粒的envoplakin及periplakin发生特异结合。手术切除后病理检查均为Castleman病，术后皮损逐渐消退。结论：副肿瘤性天疱疮无论在临床，皮损的组织病理学改变，还是免疫学上都具有特征性，对于临床上口腔黏膜有广泛糜烂或溃疡，按天疱疮治疗无效的患者，应考虑副肿瘤性天疱疮的可能性。早期诊断，及时切除肿瘤或作相应治疗，患者的预后是良好。     

天疱疮易感性与HLA-II类基因

天疱疮是一种自身抗体介导的自身免疫性疾病。已证明天疱疮易感性与人类白细胞抗原(HLA) -D区多态性基因相关 ,此相关性有种族差异。自身抗体的产生多呈T细胞依赖性。在自身抗体产生过程中 ,HLA -II类抗原将天疱疮抗原 (桥芯蛋白 )呈递给自身反应性CD4 + T淋巴细胞 ,后者在识别外来或自身抗原的同时需识别HLA -II类分子。HLA -II类基因核苷酸序列的多态性决定免疫反应的特性 ,与自身免疫性疾病的发生密切相关     

天疱疮易感性与HLA-Ⅱ类基因

天疱疮是一种自身抗体介导的自身免疫性疾病。已证明天疱疮易感性与人类白细胞抗原(HLA)-D区多态性基因相关，此相关性有种族差异。自身抗体的产生多呈T细胞依赖性。在自身抗体产生过程中，HLA-Ⅱ类抗原将天疱疮抗原(桥芯蛋白)呈递给自身反应性CD4^ T淋巴细胞，后者在识别外来或自身抗原的同时需识别HLA-Ⅱ类分子。HLA-Ⅱ类基因核苷酸序列的多态性决定免疫反应的特性，与自身免疫性疾病的发生密切相关。     

Epitopes in the linker subdomain region of envoplakin recognized by autoantibodies in paraneoplastic pemphigus patients

Sera from paraneoplastic pemphigus (PNP) immunoprecipitate multiple antigens from human epidermal protein extract. In this study, we further characterized the autoantibodies in 12 PNP sera. Immunoblotting using recombinant linker subdomains of envoplakin, periplakin, desmoplakin, and bullous pemphigoid antigen I found that 11 of the 12 sera recognized linker subdomains of envoplakin and periplakin. We then synthesized 12 peptides covering the linker subdomain of envoplakin for ELISA. One of the peptides, peptide no. 8, was recognized by nine out of the 12 sera with a higher affinity. A method of ligand-receptor binding assay was designed and performed using this peptide labeled with fluorescence as the ligand. Peptide no. 8 bound to CD20+ cells in Castleman's tumors from the patients whose sera were positive to this peptide by ELISA. Our data suggest that the linker subdomain of plakin proteins may be one of the major areas recognized by PNP autoantibodies, and epitopes in the linker subdomain of envoplakin recognized by PNP autoantibodies with a high affinity are dispersed in several areas and are variable among PNP patients. We also demonstrate that B-lymphocyte clones specifically reacting to epidermal proteins exist in Castleman's tumors from PNP.     

Detection of anti-envoplakin and anti-periplakin autoantibodies by ELISA in patients with paraneoplastic pemphigus

Paraneoplastic pemphigus patients (PNP) develop a group of autoantibodies, among which those against envoplakin and periplakin are almost always found. Epitope mapping has indicated that the linker subdomains of the proteins harbor the major antigenic sites recognized by PNP sera. In order to detect specific autoantibodies for the diagnosis of PNP, we expressed recombinant proteins containing linker subdomains of human periplakin and envoplakin in a human kidney cell line, and used them as the antigens for ELISAs. We found that all of the sera from 16 PNP patients recognized these two recombinant proteins by ELISA, and sera from 20 pemphigus vulgaris (PV), 12 pemphigus foliaceus (PF), 20 bullous pemphigoid (BP), 2 Castleman's tumor without PNP and 20 normal controls showed negative results. We also expressed the extracellular domain of desmoglein 3 (Dsg3) in the cell line, and used this recombinant Dsg3 as the ELISA antigen. Only 11 of our 16 PNP sera were positive, and most PV sera were positive. Our findings indicate that ELISAs using the recombinant proteins containing linker subdomains of envoplakin and periplakin expressed in a human cell line as the antigens are highly sensitive and specific for the diagnosis of PNP.     

Autoantibody production from a thymoma and a follicular dendritic cell sarcoma associated with paraneoplastic pemphigus

BACKGROUND: Paraneoplastic pemphigus (PNP) is an autoimmune mucocutaneous disease. We previously reported that B cells in a Castleman tumour associated with PNP produced autoantibodies. However, it is uncertain whether the production of autoantibodies from the associated tumour is a common mechanism in PNP. OBJECTIVES: To investigate autoantibody production in a thymoma and a follicular dendritic cell sarcoma that were excised from two patients with PNP. METHODS: Tumour cells were cultured, and their surface markers were identified. Indirect immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using culture media from the tumours were used to detect PNP autoantibodies. RESULTS: B cells with markers (CD22+, surface membrane IgG+ and surface membrane IgM+) of mature B lymphocytes constituted a proportion of cultured tumour cells in both tumours. Western blot showed that the medium from both the thymoma and the follicular dendritic cell sarcoma cells recognized 190-kDa periplakin and 210-kDa envoplakin bands of human epithelial proteins as well as recombinant linker regions of periplakin, envoplakin, desmoplakin and bullous pemphigoid antigen 1. ELISA was positive for antidesmoglein 3 antibody. CONCLUSIONS: The presence and localization in tumours of B-lymphocyte clones against proteins of the plakin family and desmoglein 3 in skin may not be confined to PNP with Castleman disease, but is possibly a common mechanism in PNP associated with various tumours.     

Unique role for the periplakin tail in intermediate filament association: specific binding to keratin 8 and vimentin

Plectin, desmoplakin, and the 230-kDa bullous pemphigoid antigen (BPAG1), members of the plakin family of proteins, are multifunctional cytolinkers, connecting the cytoskeletal structures to the cell adhesion complexes. Envoplakin and periplakin are components of the cornified envelope, but less is known about their role in tissues other than the stratified epithelium. Our tissue-wide survey utilizing RT-PCR revealed that periplakin, like plectin and desmoplakin, has a wide tissue distribution, but envoplakin expression is limited to certain tissues only, and BPAG1 is clearly specific for epidermal keratinocytes. Plectin, desmoplakin and BPAG1 are known to bind to the intermediate filaments through their C-terminal domains. The short C-terminal domain of periplakin is composed only of the linker domain, a region highly homologous between the plakin proteins. Here we demonstrate, through the use of yeast two-hybrid assay, a specific interaction of the periplakin linker domain with keratin 8 and vimentin. Co-expression of each plakin linker domain with keratin 8 revealed that periplakin and BPAG1 linkers co-localize with keratin signals in HaCaT cells, plectin and desmoplakin linkers were detected both in the nucleus and in cytoplasm together with the overexpressed keratin 8, while envoplakin linker localized independently into the nucleus. These results suggest that, in spite of its high homology and structural similarity with envoplakin, periplakin is functionally closer to the well-characterized plakin proteins plectin and desmoplakin, and thus may function tissue-wide as a scaffolding protein in intermediate filament assembly.     

Paraneoplastic pemphigus sera react strongly with multiple epitopes on the various regions of envoplakin and periplakin, except for the c-terminal homologous domain of periplakin

Paraneoplastic pemphigus sera react with multiple plakin family proteins, among which only envoplakin and periplakin are constantly detected by immunoblotting using normal human epidermal extracts. Using bacterial expression vectors containing polymerase chain reaction-amplified cDNA, we have prepared variously truncated recombinant glutathione-S-transferase-fusion proteins of envoplakin and periplakin, which presented N-terminal, central and C-terminal domains of each protein, as well as the so-called C-terminal homologous domain of envoplakin and the junctional regions of these domains. By immunoblotting using these 11 recombinant proteins, we demonstrated that most of the 26 paraneoplastic pemphigus sera reacted very strongly with multiple recombinant proteins of envoplakin and periplakin, except for the C-terminal homologous domain of periplakin. We also examined the reactivity with these recombinant proteins of other blistering diseases, including pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid, and found that a few nonparaneoplastic pemphigus sera showed a weak reactivity with some of the recombinant proteins. Interestingly, some sera showed relatively strong reactivity with the C-terminal homologous domain of periplakin to which paraneoplastic pemphigus sera reacted less frequently. These results indicate that, although nonparaneoplastic pemphigus sera occasionally show a weak reactivity with envoplakin and periplakin, the pathogenicity and the mechanism of antibody production in these cases may be different from those in paraneoplastic pemphigus.     

Sensitivity and specificity of clinical, histologic, and immunologic features in the diagnosis of paraneoplastic pemphigus

BACKGROUND: Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease characterized by the production of autoantibodies mainly directed against proteins of the plakin family. An overlapping distribution of autoantibody specificities has been recently reported between PNP, pemphigus vulgaris (PV), and pemphigus foliaceus (PF), which suggests a relationship between the different types of pemphigus. OBJECTIVE: Our purpose was to evaluate the sensitivity and the specificity of clinical, histologic, and immunologic features in the diagnosis of PNP. METHODS: The clinical, histologic, and immunologic features of 22 PNP patients were retrospectively reviewed and compared with those of 81 PV and PF patients without neoplasia and of 8 PV and 4 PF patients with various neoplasms. RESULTS: One clinical and 2 biologic features had both high sensitivity (82%-86%) and high specificity (83%-100%) whatever the control group considered: (1) association with a lymphoproliferative disorder, (2) indirect immunofluorescence (IIF) labeling of rat bladder, and (3) recognition of the envoplakin and/or periplakin bands in immunoblotting. Two clinicopathologic and two biologic features had high specificity (87%-100%) but poor sensitivity (27%-59%): (1) clinical presentation associating erosive oral lesions with erythema multiforme-like, bullous pemphigoid-like, or lichen planus-like cutaneous lesions; (2) histologic picture of suprabasal acantholysis with keratinocyte necrosis, interface changes, or lichenoid infiltrate; (3) presence of both anti-epithelial cell surface and anti-basement membrane zone antibodies by IIF; and (4) recognition of the desmoplakin I and/or BPAG1 bands in immunoblotting. Interestingly, 45% of patients with PNP presented initially with isolated oral erosions that were undistinguishable from those seen in PV patients, and 27% had histologic findings of only suprabasal acantholysis, which was in accordance with the frequent detection of anti-desmoglein 3 antibodies in PNP sera. CONCLUSION: The association with a lymphoproliferative disorder, the IIF labeling of rat bladder, and the immunoblotting recognition of envoplakin and/or periplakin are both sensitive and specific features in the diagnosis of PNP.     

Comparative study of autoantigen profile between Colombian and Brazilian types of endemic pemphigus foliaceus by various biochemical and molecular biological techniques

BACKGROUND: Besides Brazilian endemic pemphigus foliaceus (EPF), we have described another focus of EPF in Colombia. Our previous study suggested that Colombian EPF seemed to react various plakin family proteins, such as envoplakin, periplakin and BP230. OBJECTIVE: To further characterize the Colombian EPF and study the difference from Brazilian EPF, we examined the antigen profile of the two types of EPF. METHODS AND RESULTS: Immunoblotting using normal human epidermal extracts revealed that 38% Colombian EPF sera and 25% Brazilian EPF sera showed IgG antibodies reactive with desmoglein (Dsg) 1, pemphigus foliaceus antigen. The sera of both types of EPF showed protein bands co-migrating with plakin family proteins, particularly periplakin. Immunoblotting analyses using recombinant proteins of various domains of envoplakin, periplakin and BP230 revealed that a considerable number of Colombian EPF sera reacted with recombinant proteins of periplakin, while only few Brazilian sera reacted with some of the recombinant proteins of any plakins. Enzyme-linked immunosorbent assay (ELISA) for Dsg1 and Dsg3 showed that Dsg1 was reacted by almost all sera of both types of EPF. However, unexpectedly, while none of Colombian EPF sera reacted with Dsg3, about half of Brazilian EPF sera reacted with Dsg3. CONCLUSION: These results suggested that the Colombian EPF is basically similar to Brazilian EPF in terms that major antigen is Dsg1, but there were some different antigen profiles between the two types of EPF.     

Paraneoplastic pemphigus

Paraneoplastic pemphigus (PNP) is a life-threatening autoimmune blistering skin disease. Clinically, it is characterized by severe mucosal erosions and various cutaneous lesions associated with lymphoproliferative neoplasmas. Suprabasal acantholysis and clefts with scattered necrotic keratinocytes are the unique histopathological features. PNP patient sera recognize multiple antigens, which have been identified as the plakin protein family that includes desmoplakin, bullous pemphigoid antigen I (BPAG1), envoplakin and periplakin, and desmogleins 1 and 3. Castleman's tumor, non-Hodgkin's lymphoma, thymoma, follicular dendritic cell sarcoma and chronic lymphocytic leukemia are the commonly associated neoplasmas in PNP. We have also demonstrated that the autoantibodies reacting to epidermal proteins are directly produced by the cells in the associated tumors. Bronchiolitis obliterans is frequently found in PNP and may cause respiratory failure and death. In our experience, the early detection and removal of the tumor and i.v. administration of immunoglobulin are critical for the treatment of PNP.     

The tryptic cleavage product of the mature form of the bovine desmoglein 1 ectodomain is one of the antigen moieties immunoprecipitated by all sera from symptomatic patients affected by a new variant of endemic pemphigus

Multiple antigens are recognized by sera from patients with pemphigus foliaceus (PF). Several have been identified including keratin 59, desmocollins, envoplakin, periplakin, and desmogleins 1 and 3 (Dsg1 and Dsg3). In addition, an 80 kDa antigen was identified as the N-terminal fragment of Dsg1 using as antigen source an insoluble epidermal cell envelope preparation. However, still unsolved was the identity of the most important antigenic moiety, a 45 kDa tryptic fragment which is recognized by all sera from patients with fogo selvagem, pemphigus foliaceus, by half of pemphigus vulgaris sera and by a new variant of endemic pemphigus in E1 Bagre, Colombia that resembles Senear-Usher syndrome. Here, we report the identification of the 45 kDa conformational epitope of a soluble tryptic cleavage product from viable bovine epidermis. To elucidate the nature of this peptide, viable bovine epidermis was trypsin-digested, and glycosylated peptides were partially purified on a concanavalin A (Con-A) affinity column. This column fraction was then used as an antigen source for further immunoaffinity purification. A PF patient's serum covalently coupled to a Staphylococcus aureus protein A column was incubated with the Con-A eluted products and the immuno-isolated antigen was separated by SDS-PAGE, transferred to a membrane, and visualized with Coomassie blue, silver and amido black stains. The 45 kD band was subjected to amino acid sequence analysis revealing the sequence, EXIKFAAAXREGED, which matched the mature form of the extracellular domain of bovine Dsg1. This study confirms the biological importance of the ectodomain of Dsg1 as well as the relevance of conformational epitopes in various types of pemphigus.