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。     

重组Dsg3EC1-2及其嵌合毒素蛋白的原核表达及纯化

目的：在大肠杆菌中高效表达并纯化重组天疱疮抗原桥粒芯糖蛋白3(Dsg3)的胞外结构域EC1-2片段及其与假单胞菌外毒素PE40的嵌合毒素Dsg3EC1-2PE40。方法：在大肠杆菌中对目的蛋白进行诱导表达，并用金属鏊合亲和层析法进行分离纯化，经Western Blot试验鉴定。结果：所得重组蛋白可以和寻常型天疱疮抗体IgG发生特异性结合，而与正常人IgG不发生反应，其纯化后纯度分别达85％及80%。结论：Dsg3EC1-2片段及其嵌合毒素Dsg3EC1-2PE40在原核细胞中得到成功表达，为天疱疮的治疗研究奠定了基础。     

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.     

天疱疮患者皮损局部B淋巴细胞及其产生特异性抗体的功能研究

目的 检测寻常型天疱疮患者皮损局部B淋巴细胞及其产生特异性抗体的功能。方法 寻常型天疱疮患者35例,健康对照22例。取健康对照皮肤和寻常型天疱疮患者初发水疱或糜烂皮损组织,分离获得单个核细胞。流式细胞仪检测患者皮损局部淋巴细胞、CD19+ B细胞比例,以及特异性识别桥粒芯糖蛋白1（Dsg1）和Dsg3的CD19+ B淋巴细胞比例。体外培养寻常型天疱疮患者皮损局部淋巴细胞,ELISA法检测培养上清液中抗Dsg1和Dsg3抗体滴度,采用受试者工作特征曲线（ROC）分析阳性率。结果 寻常型天疱疮患者皮损局部淋巴细胞、CD19+ B细胞比例分别为17.95% ± 3.85%、4.27% ± 1.13%,高于健康对照组（7.83% ± 1.29%、0.61% ± 0.31%）,差异有统计学意义（t = 2.49,U = 13.00,均P < 0.05）。天疱疮患者皮损局部CD19+ B淋巴细胞中,表达IgG的细胞比例为（38.33 ± 5.56）%,表面识别Dsg1与Dsg3的细胞比例分别为12.87% ± 1.267%、10.42% ± 1.243%。局部淋巴细胞体外培养6 d后,培养上清液中抗Dsg1与Dsg3抗体滴度分别为（4.89 ± 1.56） U/ml、（35.45 ± 13.03） U/ml,阳性率分别为85%（17/20）和95%（19/20）。 结论 寻常型天疱疮患者皮损局部有可与Dsg1及Dsg3特异性结合的B淋巴细胞聚集,体外培养后可产生特异性抗Dsg1与抗Dsg3自身抗体。     

天疱疮患者皮损局部异位淋巴结构的病理学特征

目的 探讨天疱疮患者皮损处浸润淋巴细胞病理学特征及其与外周血抗桥粒黏蛋白（Dsg）1/Dsg3抗体滴度的相关性。方法 对2014—2016年上海交通大学医学院附属瑞金医院皮肤科93例寻常型和落叶型天疱疮患者的组织病理片进行分析。计算每例 × 50镜下淋巴细胞总数,定义为淋巴细胞密集程度指数。采用酶联免疫吸附试验（ELISA）测定天疱疮患者血清中抗Dsg1/Dsg3抗体滴度。统计淋巴细胞密集程度指数与抗体滴度间相关性。对其中8例寻常型和8例落叶型天疱疮患者的皮损样本进行免疫组化染色,分析CD3+ T细胞、CD20+ B细胞、CD138+ 浆细胞的分布。结果 在93例病理切片中,Grade1、Grade2、Grade3淋巴细胞聚集出现概率分别为100.00%、68.09%、10.64%,寻常型（56例）和落叶型天疱疮（37例）比较,Grade1?3淋巴细胞聚集出现概率差异无统计学意义。淋巴细胞的密集程度在寻常型和落叶型天疱疮患者比较,差异无统计学意义,与天疱疮患者血清中特异性抗Dsg1和抗Dsg3 的抗体滴度无相关性。在16例天疱疮病例中,全部有CD3+ T细胞出现,15例有CD20+ B细胞,12例有CD138+ 浆细胞存在。16例切片中,Grade1?3淋巴细胞聚集区域中均含有大量的CD3+ T细胞,而含有CD20+ B细胞的淋巴细胞聚集占总数的52.80% ± 5.78%,含有CD138+ 浆细胞的淋巴细胞聚集占总数的34.59% ± 7.42%。CD3+ T细胞、CD20+ B细胞、CD138+ 浆细胞在寻常型（8例）和落叶型天疱疮（8例）分布差异无统计学意义。结论 天疱疮患者皮损处普遍出现不同程度的淋巴细胞浸润,可能形成异位淋巴结构,参与皮损的形成和加重。     

副肿瘤性天疱疮抗Dsg1和抗Dsg3抗体滴度及亚型和细胞因子的表达

目的对不同病期的副肿瘤性天疱疮(PNP)患者血清中抗桥粒芯糖蛋白(Dsg)1和抗Dsg3抗体的滴度、亚型及其外周血单个核细胞(PBMC)中细胞因子IFN-γ和IL-4水平进行观察,探讨PNP的特殊发病机制。方法采用ELISA法检测6例PNP患者不同病期抗Dsg1和抗Dsg3抗体滴度及其IgG1和IgG4抗体亚型;采用流式细胞术(FCM)检测上述患者PBMC中T细胞的IFN-γ和IL-4水平,并与10例寻常型天疱疮(PV)患者、10例落叶型天疱疮(PF)患者及20例正常对照进行比较。结果 PNP患者组在急性期抗Dsg1和抗Dsg3抗体滴度均升高,稳定期均明显降低,抗Dsg1和抗Dsg3抗体亚型IgG1/IgG4均以IgG1为主,与PV组相比,差异均有统计学意义(P均<0.05),与PF组患者的抗Dsg1抗体亚型IgG1/IgG4比较,差异也有统计学意义(P<0.01);PNP组患者CD8+T细胞及CD4+T细胞内IFN-γ水平明显高于PV组患者和PF组患者(P=0.001),且稳定期CD8+T细胞及CD4+T细胞内IFN-γ水平较急性期明显降低(P<0.05)。PNP组急性期患者的CD4+T细胞内IL-4水平显著低于PV组患者(P<0.05)和PF组患者(P<0.01),但在稳定期时,其水平与急性期比较,差异无统计学意义(P>0.05)。结论 PNP患者在抗Dsg1和Dsg3抗体的亚型、细胞因子等方面与其他类型的天疱疮存在明显差异。     

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…     

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.     

天疱疮患者外周血滤泡辅助性T细胞的表达及其与血清抗Dsg抗体滴度的相关性

目的探讨天疱疮患者外周血滤泡辅助性T细胞(TFH)的表达及其与血清抗桥粒芯糖蛋白(Dsg)1,3抗体滴度的相关性。方法收集寻常型天疱疮患者59例,红斑型54例,其中初发患者36例,稳定期患者51例,复发患者26例;健康对照25例。采用流式细胞术检测不同病期患者外周血CD4+T细胞及CD4+CD45RA-活化T细胞中的TFH的比例(CXCR5+PD-1+),和健康对照进行比较;分析部分患者治疗前后TFH的变化情况;并进一步对活化TFH和特异性抗Dsg1,Dsg3抗体滴度的相关性进行研究。结果初发、稳定期和复发的天疱疮患者外周血滤泡辅助性T细胞占CD4+T细胞及活化T细胞的比例均显著高于健康对照组(P0.000 1)。治疗后患者的TFH明显下降,在活化T细胞中的比例下降更加明显。天疱疮患者外周血CD4+CD45RA-活化TFH与抗Dsg3抗体滴度呈微弱正相关(P=0.004)。结论天疱疮患者外周血TFH明显升高,与疾病的活动度有关,活化T细胞中TFH的比例变化更为敏感,可作为疾病活动度和疗效的评判指标。     

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.     

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.     

Late development of antidesmoglein 1 antibodies in pemphigus vulgaris: correlation with disease progression

The coexistence of antidesmoglein 3 (Dsg3) and antidesmoglein 1 (Dsg1) autoantibodies is well described in patients with pemphigus vulgaris (PV); however, there is little evidence of sequential development of these two autoantibodies. Autoantibody responses to Dsg3 and Dsg1 were studied in seven PV patients over time by enzyme-linked immunosorbent assay, using baculovirus expressed recombinant fusion proteins. All patients had anti-Dsg3 IgG antibodies at presentation. Two patients developed anti-Dsg1 later in the course of the disease. The transition in autoantibody profile was associated with disease progression to generalized PV involving mucous membranes and skin in both patients; one patient initially presented with a predominantly mucosal phenotype, the other with herpetiform pemphigus-like features. These findings demonstrate that there is an extension of autoimmune response from anti-Dsg3 only to both anti-Dsg3 and anti-Dsg1 in some patients, which is associated with an alteration in clinical expression in PV.     

Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. OBJECTIVES: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. METHODS: Dsg3-specific B cells were isolated as CD3(-)IgD(-)PI(-)Dsg3E-tag(+) cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. RESULTS: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 10(5) PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. CONCLUSIONS: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.     

Tolerance induction by the blockade of CD40/CD154 interaction in pemphigus vulgaris mouse model

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). We have recently developed an active disease mouse model for PV by adoptive transfer of splenocytes from Dsg3(-/-) mice. The purpose of this study was to determine the role of CD40/CD154 interaction in the pathogenic antibody production and development of the disease in PV model mice. When anti-CD154 monoclonal antibody (mAb) was administered to recipient mice prior to adoptive transfer, anti-CD154 mAb almost completely blocked the anti-Dsg3 IgG production and prevented blister formation. The blockade of CD40/CD154 interaction induced tolerance against Dsg3 as the suppression of antibody production was observed through day 70, and it was maintained even after challenge by immunization with recombinant mouse Dsg3 or by adoptive transfer of immunized Dsg3(-/-) splenocytes. Furthermore, the tolerance to Dsg3 was transferable because cotransfer of splenocytes from anti-CD154 mAb-treated mice and na?ve Dsg3(-/-) splenocytes significantly suppressed anti-Dsg3 IgG production in recipient mice. In contrast, when anti-CD154 mAb was injected after the mice had developed the PV phenotype, no significant suppression of the production of anti-Dsg3 IgG was observed. These findings indicate that the CD40/CD154 interaction is essential for the induction of pathogenic anti-Dsg3 IgG antibodies and that antigen-specific immune-regulatory cells induced by anti-CD154 mAb would hold a therapeutic option for autoimmune diseases.     

Clinical phenotype and anti-desmoglein autoantibody profile in paraneoplastic pemphigus

BACKGROUND: Paraneoplastic pemphigus (PNP) has similar features to pemphigus vulgaris (PV), including circulating anti-desmoglein (Dsg) IgG as pathogenic autoantibodies. When PV is divided into mucosal dominant type and mucocutaneous type, mucosal dominant type has only anti-Dsg3 IgG, whereas the mucocutaneous type has both anti-Dsg3 and anti-Dsg1 IgG. OBJECTIVE: The purpose of this study was to determine whether there is a difference in anti-Dsg autoantibody profile between mucosal dominant PNP and mucocutaneous PNP. METHODS: Twenty-one patients with PNP were categorized as mucosal dominant and mucocutaneous types based on clinical information. Antibody titers against Dsg3 and Dsg1 were measured by enzyme-linked immunosorbent assay by means of recombinant Dsg1 and Dsg3. RESULTS: There were 9 cases of mucosal dominant type and 12 cases of mucocutaneous type. Eight of 9 cases of mucosal dominant type were positive for anti-Dsg3 IgG, but 3 of them were also positive for anti-Dsg1 IgG. All 12 cases of mucocutaneous type were positive for anti-Dsg3 IgG, whereas only 6 of them were positive for anti-Dsg1 IgG. CONCLUSION: There was no clear association between the clinical phenotype and anti-Dsg antibody profile in PNP as seen in PV. This finding suggests that besides anti-Dsg IgG other pathologic mechanisms such as lichenoid reaction or interface dermatitis may be involved in the blister formation in PNP.