大疱及疱疹性皮肤病

20053798重组嵌合毒素Dsg3EC1-2PE40对寻常型天疱疮患者T、B淋巴细胞的影响/翟志芳(三军大西南医院皮肤科),刁庆春,郝飞…∥中华皮肤科杂志.2005,38(9).536～539对重组嵌合毒素Dsg3EC1-2PE40进行表达,并分离纯化;分别以不同浓度重组嵌合毒素作用于寻常型天疱疮患者外周血淋巴细胞,通过ELISPOT法、MTT比色试验及3H-TdR掺入试验来观察重组嵌合毒素对T、B淋巴细胞的影响。纯化后的重组嵌合毒素纯度达80%以上,能够特异性与天疱疮抗体IgG结合,而与正常人IgG不结合。重组嵌合毒素可使寻常型天疱疮外周血产生抗体的B细胞数量减少约40%…     

重组嵌合毒素Dsg3EC_(1-2)PE40对寻常型天疱疮患者T、B淋巴细胞的影响

目的探讨重组嵌合毒素Dsg3EC_(1-2)PE40对寻常型天疱疮患者外周血淋巴细胞的影响。方法对重组嵌合毒素Dsg3EC_(1-2)PE40进行表达,并分离纯化；分别以不同浓度重组嵌合毒素作用于寻常型天疱疮患者外周血淋巴细胞,通过ELISPOT法、MTT比色试验及~3H-TdR掺入试验来观察重组嵌合毒素对T、B淋巴细胞的影响。结果纯化后的重组嵌合毒素纯度达80％以上,它能够特异性与天疱疮抗体IgG结合,而与正常人IgG不结合。重组嵌合毒素可使寻常型天疱疮外周血产生抗体的B细胞数量减少约40％左右；在重组嵌合毒素作用下,T细胞的增殖、代谢较重组Dsg3EC_(1-2)明显降低,两者间差异有统计学意义(P<0．01)。结论重组嵌合毒素Dsg3EC_(1-2)PE40可以抑制寻常型天疱疮外周血B细胞的抗体产生作用,对寻常型天疱疮外周血T细胞具有明显的抑制或杀伤作用。     

重组嵌合毒素Dsg3EC1-2PE40对寻常型天疱疮患者T、B淋巴细胞的影响

目的 探讨重组嵌合毒素Dsg3EC_1-2PE40对寻常型天疱疮患者外周血淋巴细胞的影响。方法 对重组嵌合毒素Dsg3EC_1-2PE40进行表达,并分离纯化;分别以不同浓度重组嵌合毒素作用于寻常型天疱疮患者外周血淋巴细胞,通过ELISPOT法、MTT比色试验及³H-TdR掺入试验来观察重组嵌合毒素对T、B淋巴细胞的影响。结果 纯化后的重组嵌合毒素纯度达80%以上,它能够特异性与天疱疮抗体IgG结合,而与正常人IgG不结合。重组嵌合毒素可使寻常型天疱疮外周血产生抗体的B细胞数量减少约40%左右;在重组嵌合毒素作用下,T细胞的增殖、代谢较重组Dsg3EC_1-2明显降低,两者间差异有统计学意义(P<0.01)。结论 重组嵌合毒素Dsg3EC_1-2PE40可以抑制寻常型天疱疮外周血B细胞的抗体产生作用,对寻常型天疱疮外周血T细胞具有明显的抑制或杀伤作用。     

寻常型天疱疮抗原Dsg3基因的真核表达

目的 探讨寻常型天疱疮自身抗原Dsg3EC1-EC5在哺乳动物细胞的克隆、表达及纯化,并以重组的Dsg抗原检测寻常型天疱疮患者及正常人血清中的天疱疮抗体.方法 根据基因库中的Dsg3基因序列分析,采用RT-PCR法扩增自身抗原Dsg3EC1-EC5多肽片段的cDNA,定向插入真核表达质粒pcDNA3.1TM/myc-His(-)B中,稳定转染人宫颈癌细胞系表达重组融合蛋白,并经Ni+亲和层析柱纯化.采用蛋白质印迹法鉴定蛋白表达产物.以重组Dsg3EC1-EC5蛋白为抗原用ELISA检测40份寻常型天疱疮患者及40份正常人对照组血清.结果 成功构建真核表达重组体pcDNA3.1TM/myc-His(-)B-Dsg3.纯化的Dsg3EC1-EC5蛋白能够与寻常型天疱疮患者血清中的天疱疮抗体发生反应.ELISA方法检测寻常型天疱疮患者血清,结果显示,天疱疮抗体的阳性率为95%.结论 真核表达的Dsg3EC1-EC5蛋白与寻常型天疱疮患者血清中的抗体发生特异性反应.     

大疱及疱疹性皮肤病

20072998寻常型天疱疮抗原Dsg3基因的真核表达/孙峥嵘(中国医大附二院卫生部小儿先天畸形重点实验室),耿龙,吉耀华…∥中华皮肤科杂志.-2007,40(9).-552~554根据基因库中的Dsg3基因序列分析,采用RT-PCR法扩增自身抗原Dsg3 EC1-EC5多肽片段的cD-NA,定向插入真核表达质粒pcDNA3.1TM/myc-His(-)B中,稳定转染人宫颈癌细胞系表达重组融合蛋白,并经Ni 亲和层析柱纯化。采用蛋白质印迹法鉴定蛋白表达产物。以重组Dsg3 EC1-EC5蛋白为抗原用ELISA检测40份寻常型天疱疮患者及40份正常人对照组血清。结果成功构建真核表达重组体pcDNA3.1…     

利用噬菌体随机9肽库筛选寻常型天疱疮抗原模拟表位

目的 利用噬菌体随机9肽库筛选寻常型天疱疮抗原桥粒芯糖蛋白3（desmoglein, Dsg3）模拟表位,加深对寻常型天疱疮发病机制的认识。方法 通过大肠杆菌表达Dsg3的胞外结构域1和2（extracellular domain, EC1-2） 和谷胱甘肽转移酶（GST）的融合蛋白,从寻常型天疱疮患者血清中纯化与EC1-2特异结合的多克隆抗体,对噬菌体随机线九肽库和环九肽库进行亲和筛选,阳性噬菌体展示肽经ELISA和竞争性ELISA验证。结果 经过两轮亲和筛选,与自身抗体结合的噬菌体明显富集,免疫筛查、验证后得到3个阳性噬菌体展示肽,ELISA检测显示它们与患者血清反应,而不与正常人血清反应,竞争性ELISA检测显示它们可以抑制寻常型天疱疮患者血清与重组蛋白EC1-2的结合。结论 利用噬菌体随机9肽库筛选到3个与寻常性天疱疮密切相关的模拟表位。     

大疱及疱疹性皮肤病

971487 重组大疱性类天疱疮抗原片段的表达、纯化及鉴定/李冠群…//中华皮肤科杂志.-1996,29（6）.-421 通过基因工程的方法,在体外对大疱性类天疱疮（BP）的自身抗原之一BPAG2片段的重组cDNA克隆进行了诱导表达。将重组质粒pKPL-BPAG2转化至大肠杆菌POP2136中,并经SDS-PAGE分析表达产物。结果发现,细菌表达出分子量为22500的非融合性BPAG2片段,表达产物进一步经凝胶扫描分析显示,重组BPAG2抗原片段占细菌总蛋白的18.1％;与对应的cDNA长度相符合;重组片段经SDS-PAGE分离后得到较纯的重组蛋白片段,未见杂带出现;重组片段与抗BPAG2血清相结合,不能与BP的另一种自身抗原BPAG1的抗血清结合,证实其保留了BPAG2特异的免疫活性。作者认为,该重组蛋白片段可进一步应用     

The expression level of acetylcholine receptor in the patients with pemphigus

目的:研究不同型别天疱疮患者体内乙酰胆碱受体的表达状况,初步探讨其与病情活动度和天疱疮分型的关系.方法:主要采用免疫组化和ELISA方法.结果:乙酰胆碱受体IgG (acetylcholine receptor IgG,AChR IgG) 在 30例天疱疮患者中有8例患者为阳性,7例为寻常型天疱疮,1例为落叶型天疱疮,与正常对照组比较,差异没有统计学意义(P ＞ 0.05);在AChR IgG阳性的患者中,病情分级较重,当病情得到有效控制后,外周血AChR IgG的吸光度(A)值下降.AChRM3亚型在寻常型天疱疮中褐色的颗粒仍然分布在基底层,但颜色加深,颗粒变大,且大小不一致.AChRM3亚型在落叶型天疱疮中的表达模式与正常对照组无统计学差异.AChRα亚基在寻常型天疱疮和落叶型天疱疮中褐色的颗粒仍然分布在全层,但颜色加深,颗粒变大,且大小不一致.结论:在落叶型天疱疮和寻常型天疱疮中,AChR抗体的表达状况不同.AChR抗体更易出现在桥粒芯糖蛋白(Dsg)3抗体阳性患者的外周血中,在Dsg3阳性患者皮损的组织切片中,除了AChRα蛋白之外,AChRM3蛋白的染色模式也发生了改变.这提示Dsg3抗体更易导致AChR途径的异常.     

寻常型天疱疮抗原EC1-2表位的致病性

目的：通过寻常型天疱疮新生鼠模型的建立研究寻常型天疱疮抗原（PVA）致病性表位及其相应抗体的致病作用。方法：构建重组PVA细胞外区（EC）1－2片段分子，经亲和层析纯化后，免疫家兔，得到兔抗PVA EC1－2融合蛋白的抗血清，将提纯IgG抗体成分，被动转移到BALB／c新生鼠，15－18h后对新生鼠皮肤，血清进行组织病理，电镜和免疫荧光检查。结果：在对实验组新生鼠的评价中，其组织病理示表皮内水疱形成，棘细胞间棘突消失，棘 层松解；电镜示棘细胞间距离增宽，桥粒分离，溶解，消失，DIF和IIF均示棘细胞间免疫荧光沉积，面对照组小鼠除了IIF示棘细胞间微弱的荧光沉积外，其余检查均正常。结论：通过寻常型天疱疮新生鼠模型的建立，证明PVA分子中EC1－2表位为致病性表位，其相应的抗体为致病性抗体，另外，PVA重组分子的构建，新生鼠模型的建立为天疱疮以及其它自身免疫性疾病的诊断和治疗提供了一个模型 。     

大疱及疱疹性皮肤病

20 0 3 3 2 71　纯化与未纯化重组 PV A表位 E C 1 -2在检测抗 PV A 抗体中的比较 /潘萌 (上海二医大瑞金医院 )…∥中国皮肤性病学杂志 .-2 0 0 3 ,1 7(2 ) .-79构建 EC 1 -2重组质粒 ,进行融合蛋白表达 ,并以亲和层析原理纯化融合蛋白 ,用免疫印迹法将纯化前后的 EC 1 -2融合蛋白应用于寻常型天疱疮 (PV)患者的血清学检测。结果 ,2 2例被测P V患者血清中 ,用纯化前、后 E C 1 -2融合蛋白检测的阳性率分别为 54 .5%、77.3 %。认为以纯化后的 E C 1 -2融合蛋白为抗原底物 ,其 PV A抗体检出率显著高于纯化前。图 2参 5　 (马慧群 )…     

Development of chimeric molecules for recognition and targeting of antigen-specific B cells in pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV. Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for targeting B cells. Dsg3-GFP, a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3deltaN1) and modified Pseudomonas exotoxin were produced in bacteria (Dsg3deltaN1-PE40-KDEL, PE3 7-Dsg3deltaN1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3deltaN1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3deltaN1-immunized mice, with a 60% reduction in cell number compared with Dsg3deltaN1 alone. Thus, specific recognition and targeting of antigen-specific B cells in PV was demonstrated; this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.     

IgG against extracellular subdomains of desmoglein 3 relates to clinical phenotype of pemphigus vulgaris

Abstract:  Pemphigus vulgaris (PV) is associated with autoantibodies against desmoglein (Dsg) 3 inducing epidermal loss of adhesion. The major pathogenic epitopes of Dsg3 are presumably dependent of their conformation. The aim of this study was to characterize the IgG reactivity of sera from a cohort of clinically well-characterized PV patients against presumably non-conformational subdomains of the Dsg3 ectodomain including recently described NH₂-terminal immunodominant epitopes. By ELISA, IgG reactivity against distinct subdomains of Dsg3 was related to disease activity and the clinical phenotype of PV patients. Our findings suggest that (i) autoantibody from PV sera react with non-conformational epitopes of Dsg3; (ii) IgG reactivity against the NH₂-terminus and the extracellular domains (EC) 2-4 of Dsg3 was associated with active PV, while IgG titres were not strictly correlated with disease activity and (iii) IgG reactivity against the EC1-4 was associated with mucosal dominant PV and was decreased in cutaneous dominant PV. The findings may help to define more refined serological disease markers of PV.     

A mouse model of pemphigus vulgaris by adoptive transfer of naive splenocytes from desmoglein 3 knockout mice

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by antidesmoglein3 (anti-Dsg3) IgG autoantibodies. Recently, we developed a PV mouse model by adoptive transfer of splenocytes from recombinant Dsg3-immunized Dsg3(-/-) mice to Rag2(-/-) immunodeficient mice that expressed Dsg3. OBJECTIVES: We determined whether the adoptive transfer of naive splenocytes from nonimmunized Dsg3(-/-) mice induces the anti-Dsg3 IgG production and the PV phenoytpe in recipient mice. METHODS: We adoptively transferred naive Dsg3(-/-) splenocytes into Rag2(-/-) mice and compared their PV phenoytpe with those mice receiving immunized Dsg3(-/-) splenocytes. The numbers of splenocytes and their subpopulations required for anti-Dsg3 IgG production were examined. RESULTS: Mice that received naive Dsg3(-/-) splenocytes produced anti-Dsg3 IgG, which bound to keratinocyte cell surfaces in vivo, and developed the PV phenotype, including oral erosions with suprabasilar acantholysis. Antibody production and the appearance of the PV phenotype were delayed by approximately 2 weeks in mice that received naive splenocytes compared with mice that received immunized splenocytes. However, once the PV phenotypes developed, there were no apparent differences in disease severity between the two models. Interestingly, the anti-Dsg3 IgG titres were significantly lower in mice that received naive splenocytes than in mice that received immunized splenocytes, suggesting that the former antibodies were more potent than the latter. The frequency of anti-Dsg3 IgG production depended on the number of transferred naive splenocytes. Both CD4+ T cells and B220+ B cells from naive Dsg3(-/-) mice were essential for the production of anti-Dsg3 IgG antibodies. CONCLUSIONS: Dsg3-specific naive lymphocytes in Dsg3(-/-) mice can be primed and activated by the endogenous Dsg3 in recipient mice to produce pathogenic anti-Dsg3 IgG without active immunization. This approach using naive lymphocytes provides a unique model to dissect immunological mechanisms of tolerance against peripheral autoimmune targets.     

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.     

IgG reactivity against non-conformational NH-terminal epitopes of the desmoglein 3 ectodomain relates to clinical activity and phenotype of pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune disease caused by immunoglobulin G (IgG) autoantibodies against the desmosomal adhesion molecules, desmoglein (Dsg)3 and Dsg1. The aim of the study was to relate IgG reactivity of 123 PV sera and 40 control sera against NH(2)-terminal non-conformational epitopes of Dsg3 and Dsg1 with disease activity and clinical phenotype by enzyme-linked immunosorbent assay. The results show that (i) the overall reactivity and the titres of IgG reactive with the Dsg3 ectodomain, Dsg3(1-566), significantly correlated with the disease activity of the PV patients; (ii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with active PV while there was no direct correlation between the IgG titres and the disease activity; (iii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with mucosal and mucocutaneous PV; (iv) IgG titres against a small stretch of the NH(2)-terminus of Dsg3, Dsg3(25-88), were associated with active PV; and (v) IgG in the PV sera detected non-conformational epitopes in addition to the previously identified conformation-dependent epitopes of the Dsg3 and Dsg1 ectodomains.     

Coexistence of IgA antibodies to desmogleins 1 and 3 in pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus

BACKGROUND: Pemphigus is a bullous mucocutaneous autoimmune disease characterized by IgG autoantibodies to desmoglein (Dsg) 1 and/or Dsg3. Occasionally direct immunofluorescence of pemphigus skin reveals IgA depositions with an intraepidermal intercellular pattern in addition to the IgG deposition. OBJECTIVES: To investigate if pemphigus patients, in addition to having IgG autoantibodies, also generate IgA antibodies to Dsg1 and/or Dsg3. PATIENTS/METHODS: Sera of 100 pemphigus patients and 36 bullous pemphigoid controls were tested by IgA enzyme-linked immunosorbent assay (ELISA) to the recombinant extracellular domains of Dsg1 and Dsg3. The patients were selected on clinical grounds and positive IgG ELISA index values for Dsg1 and/or Dsg3. They were divided into four groups: patients having IgG to only Dsg1 (n=34), patients having IgG to both Dsg1 and Dsg3 (n=31), patients having IgG to only Dsg3 (n=27) and patients who had paraneoplastic pemphigus (PNP) (n=8). RESULTS: IgA antibodies to Dsg1 were found in 13 (38%) of the patients with IgG to Dsg1, in five (16%) of the patients with IgG to both Dsg1 and Dsg3, in four (15%) of the patients with IgG to Dsg3 and in none of the PNP patients. IgA antibodies to Dsg3 were found in one (3%) of the patients with IgG to Dsg1, in 18 (58%) of the patients with IgG to both Dsg1 and 3, in 18 (67%) of the patients with IgG to Dsg3, and in four (50%) of the PNP patients. Immunofluorescence analysis demonstrated intraepidermal intercellular staining IgA antibodies in serum and intercellular IgA deposits in skin of IgA ELISA-positive patients, although to a lesser extent than by ELISA. CONCLUSIONS: This study shows that in a considerable number of supposedly IgG-mediated pemphigus patients IgA to Dsg1 and Dsg3 is also present. In most cases the antigen specificity of the IgA follows the antigen specificity of the IgG, although in a small number of cases IgA is present against the Dsg not recognized by IgG.     

The severity of cutaneous and oral pemphigus is related to desmoglein 1 and 3 antibody levels

BACKGROUND: Pemphigus vulgaris (PV) and foliaceus (PF) are characterized by antibodies to the desmosomal proteins desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1), respectively. Past studies using indirect immunofluorescence (IIF) as a measure of pemphigus antibody levels have failed to demonstrate consistently a relationship between disease severity and IIF titres. However, IIF is not able to measure separately Dsg1 and 3 antibodies, unlike enzyme-linked immunosorbent assays (ELISA), which utilize recombinant proteins. OBJECTIVES: To compare independently Dsg1 and 3 antibody levels with the severity of both cutaneous and oral involvement in PV and PF. Patients and methods Four hundred and twenty-four serum samples were analysed from 80 subjects with PV and 24 with PF. IgG antibodies to Dsg1 and 3 were measured by ELISA. For every sample analysed, the associated severity of skin and oral disease were graded from 0 to 3; quiescent, mild, moderate and severe. RESULTS: A relationship between Dsg1 antibodies and skin severity was demonstrated such that a 10-unit increase in Dsg1 ELISA value was associated with a 34% chance of having a higher severity score [95% confidence interval (CI), 25-45%, P < 0.0005]. This was observed in both PV and PF. Oral severity was associated with Dsg3 antibody levels and a 10-unit increase in the Dsg3 ELISA value was associated with a 25% chance of a higher oral severity score (CI 17-33%, P < 0.0005). We were unable to demonstrate a relationship between Dsg1 antibodies and oral severity, even after adjusting for the effect of Dsg3 antibodies. Similarly, there was no relationship between Dsg3 antibodies and skin severity. CONCLUSIONS: This study suggests that the clinical phenotype of pemphigus, in particular the balance of skin and oral disease, is determined principally by the quantities of Dsg1 and 3 autoantibodies, respectively.