寻常型天疱疮自身抗体检测及治疗方法的进展

寻常型天疱疮（pemphigus vulgaris,PV）是一累及皮肤与黏膜的自身免疫性皮肤病,属于表皮内大疱病,临床上较常见,可表现为广泛的红斑、松弛的水疱及糜烂、结痂面,尼氏征阳性。患者血清中存在针对桥粒黏蛋白1（desmoglein1,Dsg1,相对分子质量160 000）和（或）抗桥粒黏蛋白3（desmoglein3,Dsg3,相对分子质量130 000）的IgG型抗体［1］,该抗体和自身抗原结合,导致细胞间连接破坏,棘层松解发生和表皮内水疱形成［2］。根据PV患者血清中存在的自身抗体类型,可以分为黏膜主导型（mucosal dominant）与皮肤黏膜型（mucocutaneous）……     

桥粒芯糖蛋白1、3在HaCaT细胞、A431细胞及原代角质形成细胞中的表达

【摘要】 目的 测定不同类型角质形成细胞（KC）桥粒芯糖蛋白（DSG）1、DSG3及DSG1 mRNA、DSG3 mRNA表达。 方法 不同KC（HaCaT细胞、A431细胞及原代KC）培养后,直接免疫荧光观察细胞DSG1、DSG3的表达,定量聚合酶链反应（qPCR）测定DSG1 mRNA、DSG3 mRNA相对表达水平。 结果 DSG1与DSG3表达于三种角质形成细胞,荧光强度显示,A431细胞高于HaCaT细胞,HaCaT细胞高于原代KC。三种细胞均表达DSG1、DSG3 mRNA,原代KC DSG1与DSG3 mRNA的相对表达量分别为HaCaT细胞的291.7%及237.4%,差异有统计学意义（P < 0.01）;A431细胞DSG1 mRNA为HaCaT细胞的0.1%、DSG3 mRNA则为HaCaT细胞的18.8%,差异有统计学意义（P < 0.05）。 结论 三种KC均可用于对DSG1、DSG3的相关研究;相对而言,正常培养的A431细胞表面DSG1、DSG3表达较HaCaT细胞及原代KC高;原代KC DSG1、DSG3 mRNA水平较HaCaT细胞及A431细胞高。     

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。     

胆碱能受体激动剂逆转天疱疮棘层松解的机制研究

目的 研究胆碱能受体激动剂对天疱疮棘层松解的逆转作用及其机制。 方法 将HaCaT细胞与寻常型天疱疮IgG（PV-IgG）共培养建成天疱疮细胞模型后,再加入胆碱能受体激动剂卡巴胆碱共培养,以PV-IgG诱导的天疱疮细胞模型作为对照,通过细胞解离实验定量分析卡巴胆碱对棘层松解的逆转情况,用免疫荧光方法定性观察桥粒蛋白变化;分别用RIPA和Triton X-100裂解细胞,得到总蛋白和胞质蛋白,用蛋白免疫印迹灰度值定性分析HaCaT细胞表面与黏附相关的桥粒芯蛋白3（Dsg3）、桥斑珠蛋白（PG）的变化,不同时间点p38丝裂原活化蛋白激酶（p38 MAPK）、表皮生长因子受体（EGFR）的磷酸化水平;用定量聚合酶链反应（qPCR）检测上述细胞表面蛋白在mRNA水平的变化;通过免疫共沉淀方法定性分析Dsg3与PG相互作用的变化情况。 结果 PV-IgG组细胞碎片数为46.67 ± 2.03,卡巴胆碱组为18.67 ± 2.52,两组比较,t = 11.22,P < 0.01;免疫荧光实验发现,卡巴胆碱可以逆转PV-IgG所致的桥粒分子内化。在天疱疮细胞模型中,细胞总的Dsg3和PG含量下降,非桥粒部分的Dsg3下降,非桥粒PG含量增加,且Dsg3与PG的相互作用减弱,加入卡巴胆碱后可逆转上述变化。卡巴胆碱也可使Dsg3 mRNA的相对表达量（2-△△Ct）由1.428 ± 0.215增加至4.974 ± 0.948（t = 3.65,P = 0.01）,PG mRNA的相对表达量由1.563 ± 0.247增加至13.420 ± 1.715（t = 6.85,P < 0.01）。磷酸化实验中,卡巴胆碱可以抑制EGFR磷酸化,而对p38 MAPK磷酸化无明显影响。 结论 胆碱能受体激动剂卡巴胆碱具有逆转棘层松解的作用,这种逆转作用的机制可能包括：抑制Dsg3和PG内化并增加其表达,增强Dsg3与PG的相互作用,抑制棘层松解关键信号EGFR的磷酸化。     

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。     

桥粒芯糖蛋白3刺激下寻常型天疱疮患者外周血T淋巴细胞增殖检测分析

目的 观察桥粒芯糖蛋白3（Dsg3）对寻常型天疱疮（PV）患者外周血T淋巴细胞增殖的影响。方法 PV患者12例,正常人22例,提取外周血单一核细胞（PBMC）,采用流式细胞仪技术分别检测Dsg3刺激下PBMC中T淋巴细胞亚群变化及T淋巴细胞增殖情况,分析PV患者与正常人之间存在的差异。结果 PV患者PBMC在Dsg3刺激下Th2型T淋巴细胞所占比例为12.17% ± 5.32%,Th1型T淋巴细胞所占比例为4.08% ± 1.50%;未经Dsg3刺激的阴性对照组分别为9.84% ± 5.41%和3.91% ± 1.38%。PV患者PBMC在Dsg3刺激和无Dsg3刺激时的Th2型T淋巴细胞所占比例均显著高于正常人（P < 0.05）;Dsg3刺激下PV患者T淋巴细胞发生增殖,CD4+ T淋巴细胞增殖率为4.65% ± 3.28%,显著高于正常人（P < 0.05）。结论 Dsg3可诱导PV患者CD4+ T淋巴细胞发生特异性增殖,且主要为Th2优势型。     

寻常型天疱疮患者及一级亲属抗体IgG亚型检测

目的观察寻常型天疱疮(pemphigus vulgaris,PV)患者和一级亲属血清中PV自身抗体(PV-IgG)亚型及其与桥粒芯糖蛋白(desmoglein,Dsg)反应性,探讨PV-IgG检测的临床意义和PV的发病机制。方法用间接免疫荧光法(indirect immunofluorescence,IIF)检测27例PV患者、40例一级亲属和20例正常对照者血清PV-IgG,并用免疫印迹法(Westernblot,WB)检测PV-IgG亚型及其与Dsg1、Dsg3反应性。结果PV患者的PV-IgG阳性率和平均抗体滴度明显高于其一级亲属者(P0.001),活动期患者的PV-IgG阳性率和平均抗体滴度也明显高于缓解期(P0.01);PV患者中PV-IgG阳性率(92.9%)明显高于一级亲属(17.5%)和正常对照组(0%)(P0.0001,0.001),PV患者中Dsg3反应性IgG4水平明显高于一级亲属(P0.01),且其在活动期病例中水平也明显高于缓解期(P0.05)。结论本病的发生与PV-IgG水平有关;Dsg3是PV的主要自身抗原;IgG4是PV的主要致病抗体。     

安石榴苷对中波紫外线诱导HaCaT细胞光损伤的预防作用研究

目的 探讨安石榴苷对中波紫外线（UVB）诱导角质形成细胞损伤的保护机制。 方法 培养的HaCaT细胞分为空白对照组、安石榴苷组、UVB组、安石榴苷 + UVB组。噻唑蓝（MTT）法检测细胞增殖能力,Hoechst/碘化丙锭（PI）染色和流式细胞仪检测细胞凋亡,RT-PCR法测定金属基质蛋白酶1（MMP1）及其组织抑制因子1（TIMP1） mRNA表达水平,Western印迹检测丝裂原活化蛋白激酶（MAPK）通路相关蛋白P38、JNK、ERK的磷酸化水平变化。 结果 MTT试验示,10 ～ 40 μmol/L安石榴苷对UVB诱导的HaCaT细胞损伤有较佳的预保护作用。UVB组HaCaT细胞强Hoechst和强PI双染细胞较空白对照组增多,安石榴苷 + UVB组较UVB组减少。流式细胞仪分析,UVB组凋亡细胞百分率（9.82% ± 0.11%）高于空白对照（1.24% ± 0.91%,P < 0.01）,而安石榴苷（10、20、40 μmol/L） + UVB组凋亡细胞百分率（分别为6.38% ± 0.14%、5.24% ± 0.17%、3.77% ± 0.11%）较UVB组低,差异有统计学意义（均P < 0.01）。UVB组MMP1 mRNA相对表达量（12.376 ± 0.602）高于空白对照组（1.007 ± 0.147,P < 0.01）,而TIMP1 mRNA相对表达量（0.103 ± 0.006）低于空白对照组（1.006 ± 0.139,P < 0.01）,安石榴苷组MMP1及TIMP1 mRNA与空白对照组比较,差异无统计学意义（均P > 0.05）。安石榴苷预处理的HaCaT细胞经30 mJ/cm2 UVB照射后MMP1 mRNA相对表达量较UVB组降低（均P < 0.01）,而TIMP1 mRNA较UVB组升高（均P < 0.01）。Western印迹示,经UVB照射后,HaCaT细胞p-ERK、p-JNK及p-p38表达升高（均P < 0.01）。安石榴苷组HaCaT细胞p-ERK、p-JNK及p-p38表达没有明显改变（P > 0.05）,而安石榴苷 + UVB组有不同程度下降（均P < 0.01）。 结论 安石榴苷对UVB引起HaCaT细胞损伤有一定的预防作用。     

抗构象表位桥粒芯蛋白抗体和乙酰胆碱受体抗体滴度与寻常型天疱疮病情活动度的相关性研究

【摘要】 目的 研究寻常型天疱疮患者血清中相关抗体滴度与病情严重程度和病情活动度的相关性。方法 收集2012—2015年于中国医学科学院皮肤病医院首次就诊的24例活动期寻常型天疱疮（PV）患者,评估患者活动期和稳定期天疱疮疾病面积指数（PDAI）,并采集血清标本。采用酶联免疫吸附实验（ELISA）检测血清标本中具有致病作用的抗构象表位桥粒芯蛋白（Dsg）抗体滴度、总Dsg抗体滴度和乙酰胆碱受体（AChR）抗体滴度。计量资料比较采用t检验,计数资料比较采用Fisher 精确检验法,相关性比较采用Pearson分析。结果 活动期患者的Dsg1抗体滴度（611.4 ± 136.8）与抗构象表位Dsg1抗体滴度（585.5 ± 134.7）差异无统计学意义（t = 0.13,P = 0.89）,Dsg3抗体滴度（708.6 ± 130.7）高于抗构象表位Dsg3抗体滴度（297.2 ± 54.4,t = 2.90,P < 0.01）。活动期患者的Dsg1抗体滴度及抗构象表位Dsg1抗体滴度与PDAI评分均呈正相关（r = 0.54、0.54,均P < 0.01）;Dsg3抗体滴度与PDAI评分无相关性（r = 0.11,P = 0.62）,抗构象表位Dsg3抗体滴度与PDAI评分呈正相关（r = 0.53,P < 0.01）。20例稳定期患者血清中Dsg1抗体与抗构象表位Dsg1抗体滴度与首次就诊时比较均明显下降。Dsg3抗体滴度仅7例明显下降;13例仍存在较高滴度的Dsg3抗体,其中6例抗构象表位Dsg3抗体滴度明显下降,5例AChR抗体滴度由阳性转为阴性。结论 Dsg1抗体及抗构象表位Dsg1抗体滴度都可以反映病情活动度。部分患者病情活动度与Dsg3抗体滴度不一致,抗构象表位Dsg3抗体或者AChR抗体可能有助于反映病情活动度。     

寻常型天疱疮患者血清骨桥蛋白的检测北大核心CSCD

目的 探讨寻常型天疱疮（PV）患者血清中骨桥蛋白在PV发病中的作用.方法 用酶联免疫吸附试验对31例寻常型天疱疮患者和35例健康对照组血清骨桥蛋白的浓度进行测定.用SPSS17.0及Excell统计软件对数据进行处理.结果 PV患者血清骨桥蛋白的表达水平显著高于健康对照组（P＜0.01）.皮肤黏膜同时受累患者血清骨桥蛋白水平高于仅皮肤受累组（P＜0.05）.合并感染组血清骨桥蛋白水平高于未合并感染组（P＜ 0.01）; PV患者的血清骨桥蛋白水平与抗Dsg3抗体呈正相关（rs=0.489,P＜ 0.01）.结论 骨桥蛋白在PV患者血清中高表达,特别是皮肤黏膜同时受累的患者,且与天疱疮抗体滴度呈正相关.     

Clues to diagnosis for unusual mucosal pemphigus demonstrating undetectable anti‐desmoglein 3 serum antibodies by routine tests

Pemphigus is an autoimmune blistering disease caused by immunoglobulin (Ig)G autoantibodies against desmogleins (Dsg). In mucosal‐dominant pemphigus vulgaris (PV), anti‐Dsg3 antibodies play a critical role in acantholysis. We followed two mucosal‐dominant PV cases who suffered from refractory oral mucosal erosions. In these cases, anti‐Dsg3 serum antibodies were not detected by indirect immunofluorescence and enzyme‐linked immunosorbent assay (ELISA). However, direct immunofluorescence showed the intercellular IgG deposition in the epidermis and histopathological findings revealed suprabasal acantholysis. In order to analyze the pathomechanisms in these cases, we first examined the Dsg3 expression patterns in lesional sites and compared them with those of typical mucosal‐dominant PV cases. In typical PV cases, the alteration of Dsg3 distribution was observed in lesional sites by immunostaining. The aggregation of Dsg3, which is the characteristic change in PV mucosal lesions, was observed as the initial change prior to acantholysis. In our cases, a clustering of Dsg3 was observed at mucosal lesions, and the expression levels of Dsg3 in acantholytic lesions were decreased, as observed in typical mucosal‐dominant PV cases. Although anti‐Dsg3 serum antibodies could not be detected by routine tests, anti‐Dsg3 serum antibodies were detected by Dsg3 ELISA using 10‐times more concentrated sera (highly sensitive ELISA). Moreover, purified and concentrated PV IgG showed high pathogenicity when examined by dissociation assay. In conclusion, the detection of morphological changes in Dsg3 distribution and highly sensitive ELISA method could be useful for the early diagnosis of PV recurrence.     

No activation of urokinase plasminogen activator by anti-desmoglein 3 monoclonal IgG antibodies in cultured human keratinocytes

BACKGROUND: Although pemphigus vulgaris (PV)-IgG has been shown to activate urokinase plasminogen activator (uPA) in cultured keratinocytes, activation of uPA is thought to have no primary role in PV-acantholysis, because PV-IgG is still pathogenic in uPA- and tissue-PA-knockout mice. OBJECTIVE: To determine if PV-IgG-induced uPA activation is due to specific antibody against Dsg3, we examined whether or not pathogenic monoclonal anti-Dsg3 antibody can activate uPA, because PV-IgG is thought to contain antibodies against unknown antigens besides Dsg3. METHODS: We stimulated cultured normal human and DJM-1 keratinocytes with monoclonal anti-Dsg3 IgG1 antibodies (pathogenic AK23, AK19 and nonpathogenic AK18, AK20), negative control monoclonal mouse IgG1 and positive control PV-IgG. Cells were treated with IgGs over a time course of 24h, and uPA-protein content and activity in the culture medium were determined by enzyme-linked immunosorbent assay (ELISA) and chromogenic assay, respectively. RESULTS: The uPA-protein content in samples treated with or without pathogenic, nonpathogenic, control monoclonal mouse IgG1s and PV-IgGs increased continuously up to 24h, with no differences between samples, suggesting a spontaneous secretion. In contrast, uPA activity in the culture medium of cells treated with PV-IgG increased dramatically, whereas that of cells treated with all AK-IgGs and control monoclonal mouse IgG1 did not increase at all. CONCLUSION: These results suggest that PV-IgG-dependent uPA activation is not related to anti-Dsg3 antibody activity, which is an essential factor in PV-IgG acantholysis, and that it may be due to other antigens than Dsg3 or unknown factors contained in PV-IgG fraction.     

Detection of antibodies against the non-calcium-dependent epitopes of desmoglein 3 in pemphigus vulgaris and their pathogenic significance

Background Antidesmoglein (anti‐Dsg) 3 serum antibody titres are usually correlated with the disease activity of pemphigus vulgaris (PV), but some patients retain high titres even in remission. Objectives The aim of our study was to determine whether anti‐Dsg3 antibodies in PV sera recognized calcium (Ca²⁺)‐dependent or non‐Ca²⁺‐dependent epitopes, and to evaluate their pathogenicity. Methods Dsg3 baculoprotein‐coated enzyme‐linked immunosorbent assay (ELISA) plates were treated with 0·5 mmol L^?1 ethylenediaminetetraacetic acid (EDTA). The binding ability of anti‐Dsg3 monoclonal antibodies (mAbs) was analysed. Eight of the 83 patients with PV who were screened had elevated Dsg3 ELISA index values > 100 in remission. The binding ability of these PV sera was analysed. We evaluated the pathogenicity of anti‐Dsg3 serum antibodies against the non‐Ca²⁺‐dependent epitopes using a dissociation assay. Results The reactivity of pathogenic anti‐Dsg3 mAbs against the Ca²⁺‐dependent epitopes diminished markedly in the EDTA‐treated ELISA, whereas no such reduction was observed in mAbs against the non‐Ca²⁺‐dependent epitopes. The sera of all the patients contained antibodies against both Ca²⁺‐dependent and non‐Ca²⁺‐dependent epitopes. In six out of the eight patients, the ratio of antibodies against Ca²⁺‐dependent to non‐Ca²⁺‐dependent epitopes decreased in remission. EDTA‐treated Dsg3 baculoproteins adsorbed anti‐Dsg3 serum antibodies against the non‐Ca²⁺‐dependent epitopes, but the remnant PV antibodies retained the ability to induce acantholysis in the dissociation assay. Conclusions We have established an assay to measure indirectly the titres of anti‐Dsg3 serum antibodies against the Ca²⁺‐dependent epitopes, based on the differences between EDTA‐untreated and EDTA‐treated ELISA index values, as a routine laboratory test to reflect the pathogenic anti‐Dsg3 serum antibody titres more accurately.     

Internalization of constitutive desmogleins with the subsequent induction of desmoglein 2 in pemphigus lesions

Acantholytic blisters in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are caused by a dissociation of desmosomes mediated by autoantibodies against desmoglein (Dsg) 3 and Dsg 1, respectively. The blistering occurs at the suprabasilar level in PV and at the subcorneal level in PF, which corresponds to the distribution of target antigens in the epidermis: there is a more prominent expression of Dsg 1 in the upper layer, whereas Dsg 3 is more prominent in the lower layer. To elucidate the histogenesis of acantholysis, we studied the alterations of the desmosomal components and the expression pattern of Dsg isoforms in the lesional and perilesional epidermis of pemphigus patients. The results demonstrated an internalization of the desmosomes in the lower epidermis of PV, PF and pemphigus vegetans. A similar phenomenon was induced in monolayers of keratinocytes cultured with PV sera. However, little change was observed in E-cadherin expression until acantholysis became manifest. This internalization occurred prior to overt acantholysis, and was frequently associated with the induction of Dsg 2 expression in the basilar or lower layers of the epidermis. These findings indicate an alteration of Dsg isoform expression in subclinical pemphigus lesions, which might be related to the characteristic acantholytic patterns: the suprabasilar layer in PV and the upper epidermis in PF.     

Serum and salivary desmoglein 1 and 3 enzyme‐linked immunosorbent assay in pemphigus vulgaris: correlation with phenotype and severity

Background To the best of our knowledge there is only one report about salivary desmoglein (Dsg) 1 and 3 enzyme‐linked immunosorbent assay (ELISA) in pemphigus vulgaris (PV), whereas several studies have been performed on serum. Aims To find the sensitivity of serum and salivary anti‐Dsg1 and 3 antibodies in the diagnosis of PV, and to determine the relationship between disease severity and phenotype with antibody levels. Methods Fifty new patients with PV were included in this study. The diagnosis of PV was confirmed by histopathology and direct immunofluorescence. Demographical data, disease severity and phenotypes were recorded on questionnaire sheets. Dsg1 and Dsg3 ELISA were performed on serum and salivary samples of patients and controls. Results Thirty‐seven patients had mucocutaneous phenotype; whereas mucosal dominant and cutaneous dominant phenotypes were seen in 11 and 2 patients respectively. The sensitivities of serum anti‐Dsg3 and anti‐Dsg1 were 94% and 72% respectively. The sensitivities of salivary anti‐Dsg3 and anti‐Dsg1 antibodies were accordingly 94% and 70%. Compared with mucosal phenotype, serum and salivary anti‐Dsg1 antibodies were significantly higher in the patients with mucocutaneous phenotype. Serum Dsg1 antibodies were related with cutaneous and serum Dsg3 antibodies with mucosal severity scores. Salivary Dsg1 antibodies were significantly correlated with mucosal severity (P = 0.00); however there was no correlation between this antibody and cutaneous severity (P = 0.07). Salivary Dsg3 antibodies were not correlated with mucosal severity (P = 0.16). Conclusion Saliva Dsg ELISA could be used for diagnosis of PV. Salivary Dsg1 antibodies had a significant correlation with mucosal severity.     

Differences in the expression of pemphigus antigens during epidermal differentiation

Epidermal desmogleins with molecular weights of 130/140kDa (Dsg3 or PVA) and 150/160 kDa (Dsg1 or DGI) are recognized by autoantibodies from patients with pemphigus vulgaris (PV) and pemphigus foliaceus (PF), respectively. In order to understand the histogenesis of both types of pemphigus, we studied the expression patterns of Dsgl and Dsg3 during stratification of cultured keratinocytes. Monolayers of cultured normal human keratinocytes demonstrated uniform inter cellular staining with PV sera. The staining pattern was distinct from the focal staining with PF sera observed only in the stratified areas. Both Dsgl and Dsg3 proteins and their mRNA were expressed by the monolayers, whereas no production of Dsg2 (HDGC) mRNA was found. The relative ratio of Dsg3 to the total desmogleins, as determined by density on immunoblotting, decreased as the cultured keratinocytes stratified. In the completely stratified keratinocytes cultured on collagen membrane, Dsgl became predominant, with subsequent reduction of PV antigen expression. The relative decrease of Dsg3 (PVA) during epidermal differentiation might be responsible for the induction of suprabasal acantholysis in PV.     

Simultaneous occurrence of anti‐BP180 mucous membrane pemphigoid and mucosal‐dominant pemphigus vulgaris

We describe the simultaneous initial coexistence of mucous membrane pemphigoid (MMP) with blepharosynechia and anti‐bullous pemphigoid (BP)180 antibodies and pemphigus vulgaris (PV) with lesions limited to the oral mucosa, with the presence of anti‐desmoglein (Dsg)3 antibodies. A 75‐year‐old woman had severe oropharyngeal erosions and ulcers with blisters, and blepharoconjunctivitis and blepharosynechia of the left eye. Histopathological examination of the oral mucosa found acantholysis. Indirect immunofluorescence revealed IgG antikeratinocyte cell surface antibodies, and ELISA disclosed anti‐Dsg3 antibodies. Immunoblotting found positive reactivity with recombinant proteins of both the BP180‐NC16a domain and the BP180 C‐terminal domain. To our knowledge, the simultaneous initial occurrence of MMP and PV has never been reported previously. We present a rare case of concurrent PV and anti‐BP180 MMP, the diagnosis of which was confirmed by ELISA and immunoblotting assays.     

Low pathogenicity of anti‐desmoglein 3 immunoglobulin G autoantibodies contributes to the atypical clinical phenotypes in pemphigus

The clinical phenotypes of pemphigus can be explained by the desmoglein (Dsg) compensation theory. However, some atypical cases such as cutaneous pemphigus vulgaris (cPV), in which patients have anti‐Dsg3 antibodies without oral erosions, do not conform to this theory. To explain the discrepancy between clinical phenotypes and anti‐Dsg antibody profiles, the pathogenic strength of immunoglobulin (Ig)G autoantibodies against Dsg3 must be taken into consideration. We analyzed the epitopes and blister‐inducing pathogenic strength of the sera from three patients having IgG against Dsg3 without oral erosions with domain‐swapped recombinant proteins and dissociation assay using cultured normal human epidermal keratinocytes. The results showed that all sera contained IgG directed against the amino terminal EC1 domain of Dsg3, as is found in most PV sera. However, dissociation assays revealed that the pathogenic strength of the anti‐Dsg3 antibodies in all three cases was extremely lower than that of typical PV cases with mucosal involvement. In conclusion, when anti‐Dsg3 IgG antibodies are not sufficient to inhibit the expression of Dsg3 in the oral mucosa, but can inhibit the expression in the skin, skin blisters can result. Therefore, the pathogenicity of anti‐Dsg3 antibodies should be regarded as a key factor contributing to the clinical phenotype in pemphigus patients with conflicting antibody profiles.     

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.