人表皮细胞单克隆抗体与天疱疮抗体的比较

抗人表皮细胞单抗H₁₄和H₁₉,用人皮肤作IIF检查,其荧光模式相同于天疤疮抗体所见.为此,对它们进行比较研究,结果如下:1.免疫荧光相互阻滞试验发现不能相互阻抑.2.抗原经不同溶液、温度和酶处理后,它们对荧光染色的反应各不相同.3. SDS-PAGE显示它们各沉淀不同分子量的抗原.上述结果提示它们是抗人表皮细胞膜上不同抗原成分的抗体.因此对表皮细胞示相同荧光染色,不一定代表是相同的抗体.H₁₉染色复层鳞状上皮和肠道、肾小管上皮细胞,不着染其它组织,此单抗在肿瘤病理学上的作用尚需进一步研究.     

Use of recombinant pemphigus vulgaris antigen in development of ELISA and IB assays to detect pemphigus vulgaris autoantibodies

Aim/Objective The objectives of this study are: (1) to measure the titers of pemphigus vulgaris (PV) autoantibody in the sera of patients with active disease, using three different assays: (a) Indirect immunofluorescence (IIF) using monkey esophagus as a substrate, (b) immunoblot (IB) and, (c) enzyme-linked immunosorbent assay (ELISA) using recombinant PV antigen (rPVA). (2) To compare the sensitivity of these three assays. Background The titer of PV autoantibodies and disease severity and extent do not always correlate. This could be due to the lack of consistency and specificity of the substrate. Different results are obtained using different substrates. A standard substrate with uniformly controlled source of antigen would be more useful and clinically beneficial. Methods In this study we studied 25 PV patients, six each with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), mucous membrane pemphigoid (MMP), and herpes gestationis (HG), and sera from 16 normal subjects. IIF was used to determine the PV autoantibody using monkey esophagus. IB assay was used according to standard protocol using normal human epidermis and rPVA as substrates. ELISA was performed using rPVA as antigens expressed in E. coli. Results Sera of all 25 PV patients showed binding to the rPVA, normal human sera and the sera from the six BP, six OCP, six MMP, and six HG patients did not show any binding. In addition, we used antisera from rabbits immunized with PVA peptides (Bos-1, Bos-6) which also showed binding to rPVA, whereas normal rabbit sera did not show any reactivity. ELISA and IB titers in all the patients were 2.5 to 160 times higher than with the conventionally used IIF assay. The titers of the PV specific autoantibody measured using the rPVA did not show statistically significant differences between the ELISA and IB assays. Conclusions IB and ELISA are superior to IIF in evaluating the antibody levels in PV patients. ELISA is more practical and is preferable to IB and is recommended for clinical use.     

Ultrastructural binding site of pemphigus foliaceus autoantibodies: comparison with pemphigus vulgaris

We studied in vivo binding sites of pemphigus foliaceus (PF) auto-antibodies by immuno-gold labelling technique, and compared them with those of pemphigus vulgaris (PV). In early acantholytic lesions of PF, the bound antibodies indicated by 5 nm protein A-colloiclal gold particles were observed on the surface of keratinocytes, with particular affinity for desmosomes and separated attachment plaques. Nondesmosomal cell surfaces were sparsely labeled with the gold particles. A similar binding pattern was seen in the epidermal sheets obtained from a PV patient utilizing the Nikolsky phenomenon. These findings indicate that both PF and PV antigen-antibody complexes are densely located on the desmosomal areas in early pemphigus lesions, suggesting the pathogenic importance of functional impairment of desmosomes by the autoantibodies.     

The pathogenic role of autoantibodies in pemphigus vulgaris

Pemphigus vulgaris (PV) is a severe autoimmune bullous disease involving both the skin and mucosal areas, and characterized by intraepithelial flaccid blisters and erosions. The pathogenesis of this disease is not yet completely understood, but novel insights into desmoglein biology and autoantibody pathogenesis have recently been published. Acantholysis in PV seems to result from a collective action of autoantibodies against various keratinocyte self antigens, of which desmogleins 1 and 3 are the most important. Additional antigens including desmocollins and nondesmosome components, such as the mitochondrion, might take part in disease activation. Recently, apoptosis was reported as a possible underlying mechanism of acantholysis. Furthermore, apoptolysis is believed to be the link between suprabasal acantholytic and cell-death pathways. We review the possible hypotheses of the pathogenesis of PV: the desmoglein compensation theory, the antibody-induced apoptosis theory, the basal-cell shrinkage hypothesis and the newly published apoptolysis theory.     

Herpetiform pemphigus showing reactivity with pemphigus vulgaris antigen (desmoglein 3)

We report a patient with herpetiform pemphigus(HP)who showed reactivity only with pemphigus vulgaris(PV)antigen but not with pemphigus foliaceus(PF)antigen. Direct and indirect immuno- fluorescence revealed keratinocyte cell surface staining in the lower layers of the epidermis, where desmoglein 3 (Dsg3) is expressed. Immunoblot analysis, using ethylenediamine tetra-acetic acid- separated human epidermal extracts, revealed that the patient's serum recognized only a 130-kDa polypeptide which co-migrated with Dsg3. By antigen-specific immunoadsorption studies, using desmoglein 1 (Dsg1) and Dsg3 recombinant protein produced by baculovirus expression system, immunoreactivity of the patient's serum was completely adsorbed by Dsg3 alone, but not by Dsg1. These results indicate that this HP patient produced only anti-Dsg3 autoantibodies and no other autoantibodies against components of the keratinocyte cell surface. HP could be a variant of PV.in addition to PF, with unique clinical and histological features.     

A case of neonatal pemphigus vulgaris with co-existing BP180 autoantibodies

A male neonate was born with blisters on the trunk to a 37-year-old primigravid woman with a past medical history of recurrent, painful, topical steroid-responsive oral blisters. The diagnosis of neonatal pemphigus was made after the neonate and mother were found to have elevated desmoglein 3 (Dsg3) antibodies in conjunction with histopathologic features of pemphigus vulgaris. Interestingly, both neonate and mother also had elevated levels of BP180 antibodies, classically seen in bullous pemphigoid. This case is unique in that it portrays neonatal pemphigus, an already rare condition, complicated by the presence of BP180 antibodies.     

Antibody-mediated rosette formation of epidermal cells with leukocytes in pemphigus vulgaris

Summary Human peripheral-blood leukocytes (PBLs) were found to form rosettes with cultured human epidermal cells or a mouse epidermal cell line (Pam cells), but not with fibroblasts, when preincubated with heat-inactivated sera from patients with pemphigus vulgaris. Increased rosette formation was observed, however, when untreated sera were used for passive sensitization. Complement was not required for this reaction, because purified IgG fractions from a protein-A/Sepharose column retained the ability to induce rosette formation. Blocking experiments with -methylmannoside suggested that a carbohydrate moiety is involved in the binding of sensitized cells to epidermal cells. Thus, human PBLs can adhere to cultured mouse epidermal cells in the presence of PV IgG antibodies.     

Paraneoplastic pemphigus with circulating antibodies directed exclusively against the pemphigus vulgaris antigen desmoglein 3

A patient with follicular B-cell lymphoma presented with erythroderma associated with cutaneous and mucosal blisters. Histologic and direct immunofluorescence analysis of lesional skin showed a typical pattern of paraneoplastic pemphigus (PNP). Interestingly, indirect immunofluorescence on rat bladder was negative and immunoblot analysis of the patient's serum on epidermal extracts demonstrated antiepidermal antibodies that only recognized the pemphigus vulgaris antigen desmoglein 3, with no antibodies directed against the different proteins of the plakin family. To our knowledge this has never been reported in the literature. It exemplifies the overlap between pemphigus vulgaris and PNP and the pathogenic role of anti-desmoglein 3 antibodies in PNP. Moreover, it underscores the need to consider clinical, histologic, and immunologic features for the diagnosis of PNP.     

Neonatal pemphigus vulgaris with extensive mucocutaneous lesions from a mother with oral pemphigus vulgaris

The clinical phenotype of pemphigus is well explained by the combination of desmoglein (Dsg) 1 and Dsg3 distribution pattern and antiDsg autoantibody profile (Dsg compensation theory). It has been reported that neonatal skin has a similar Dsg distribution pattern to adult mucosal epithelia. We describe a newborn girl with mucocutaneous pemphigus vulgaris (PV) from a mother with mucosal dominant PV. The mother had had painful oral erosions for at least 7 months. Histopathological examination and direct and indirect immunofluorescence studies confirmed the diagnosis of PV and neonatal PV in the mother and daughter, respectively. The mother had a high titre of anti-Dsg3 IgG and a low titre of antiDsg1 IgG, while the neonate had only a high titre of anti-Dsg3 IgG, but no detectable antiDsg1 IgG. AntiDsg3 IgG, which caused the oral dominant phenotype in the mother, induced extensive oral as well as cutaneous lesions in the neonate. Our case provides clinical evidence for the Dsg compensation theory in neonatal PV.     

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）……     

The anti-desmoglein 1 autoantibodies in pemphigus vulgaris sera are pathogenic

Pemphigus vulgaris and pemphigus foliaceus are two closely related, but clinically and histologically distinct, autoimmune skin diseases. The autoantigens for pemphigus vulgaris and pemphigus foliaceus are desmoglein 3 and desmoglein 1, respectively. The anti-desmoglein 1 antibodies in pemphigus foliaceus and anti-desmoglein 3 antibodies in pemphigus vulgaris are pathogenic as determined by immunoglobulin G passive transfer animal models. More than 50% of pemphigus vulgaris sera also contain anti-desmoglein 1 autoantibodies; however, the pathogenicity of the anti-desmoglein 1 autoantibodies in pemphigus vulgaris remains unknown. In this study, we used soluble recombinant extracellular domains of desmoglein 1 and desmoglein 3 to obtain affinity-purified anti-desmoglein 1 and anti-desmoglein 3 autoantibodies from pemphigus vulgaris sera and examined the pathogenicity of each fraction separately using the passive transfer mouse model. By immunoprecipitation, the purified anti-desmoglein 1 and anti-desmoglein 3 showed no cross-reactivity. The anti-desmoglein 1 autoantibodies in pemphigus vulgaris induced typical pemphigus foliaceus lesions in neonatal mice, whereas the anti-desmoglein 3 fraction induced pemphigus vulgaris-like lesions. In addition, the pathogenic anti-desmoglein 1 and anti-desmoglein 3 autoantibodies in pemphigus vulgaris had predominant IgG4 subclass specificity. These findings suggest that the anti-desmoglein 1 antibodies in pemphigus vulgaris are pathogenic.     

Antigenic specificity of fogo selvagem autoantibodies is similar to North American pemphigus foliaceus and distinct from pemphigus vulgaris autoantibodies

Fogo selvagem (FS) is clinically, histologically, and immunopathologically similar to sporadic pemphigus foliaceus (PF, as seen in North America and Europe), although the epidemiology of these 2 diseases differs markedly. It has been proposed that FS is identical to PF but, for some reason, occurs in an endemic focus in central Brazil. If this hypothesis is correct, the autoantibodies in FS and PF should have similar antigenic specificities. We studied sera from 13 patients with FS from central Brazil, and compared them with 20 sera from patients with PF from the United States. All these sera had circulating antibodies that bound the cell surface of epithelial cells in identical patterns by indirect immunofluorescence on monkey esophagus or normal human skin. In immunoprecipitation studies none of the 13 FS sera precipitated the pemphigus vulgaris (PV) antigen from radiolabeled extracts of cultured human keratinocytes. This is similar to the findings with PF sera of which 19 of 20 sera did not react with PV antigen, but in sharp contrast to the results with PV sera which, as previously reported, all immunoprecipitate the PV antigen. Immunoblotting performed on extracts of normal human epidermis demonstrated that 7 of 20 PF sera specifically and intensely bound an approximately 160 kD polypeptide, previously identified as desmoglein I, a desmosomal glycoprotein. Similarly, 3 of 13 FS sera specifically bound a 160 kD polypeptide. Thirteen normal sera from North America, 8 normal and disease control sera from central Brazil, 11 PV sera, and 12 bullous pemphigoid sera did not specifically bind this polypeptide. Two-dimensional gel electrophoresis confirmed that the 160 kD polypeptides identified by the subgroup of PF and FS sera were identical. These studies demonstrate that, although the exact molecular specificities of the majority of PF and FS sera remain to be determined, FS autoantibodies do not bind the PV antigen and a subgroup of FS autoantibodies have molecular specificity identical to that of a subgroup of PF autoantibodies.     

The location of binding sites of pemphigus vulgaris and pemphigus foliaceus autoantibodies: a post-embedding immunoelectron microscopic study

Pemphigus is a life-threatening autommune blistering discase of skin and mucous membranes that has two major subtypes based on clinical and histolgical features, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Autoantibodies against the PV antigen (desmoglein 3) and the PF antigen (desmoglein 1) are involved in the pathogenesis of blister formation. In the present study, the location of epitopes recognized by autoantibodies of patients with PV and PF was studied by postembedding immunogold electron microscopy. PV and PF autoantibodies were observed bound predominantly to the intercellular domains of desmosomes, but not to the non-desmosomal keratinocyte cell surface. The relationship between the location of PF antigen and other constitutive desmosomal proteins. desmocollin, desmoplakin and plakoglobin, in normal human skin was investigated using a double immunogold labelling techinique. It was observed that PF antigen and desmocollin co-localize within the intercellular domain of the desmosomes. In contrast, the antibodies against desmoplakin and plakoglobin bound predominantly to the intracellular desmosomal attachment plaque with the binding site of the antibody against plakoglobin closer to the desmosomal cell membrane than that of the antibody to desmoplakin. We show that the LR White postembedded immunogold electronmicroscopy technique is convenient and easily applied to studied to studies of autoimmune bullous skin diseases. We have used it to demonstrated the precise localization of the binding sites of PV and PF autoantibodies and their reltionship with other constitutive desmosomal proteins.     

Lichen planus pemphigoides with circulating autoantibodies against 200 and 180 kDa epidermal antigens

We describe a case of lichen planus pemphigoides with circulating autoantibodies against 200 and 180 kDa epidermal antigens. A 24-year-old man presented pruritic erythematous patches with occasional central blister formation on the face, neck, and trunk and erythematous hyperkeratotic patches on the elbows and feet for 10 months. Histopathological findings of the neck and plantar lesions were consistent with lichen planus. Direct immunofluorescence studies from both lesions showed a linear deposition of IgG and C3 and cytoid bodies along the basement membrane zone. Indirect immunofluorescence studies demonstrated circulating IgG antibodies deposited on the epidermal side of salt-split skin. Immunoblot analysis using epidermal extract showed that the patient's serum reacted with 180 kDa bullous pemphigoid antigen (BPAG2) and a 200 kDa antigen. The cutaneous lesions almost cleared with a low dose of prednisolone and acitretin.     

非桥粒芯糖蛋白抗体在寻常型天疱疮中的作用机制

既往发现寻常型天疱疮(pemphigus vulgaris,PV)发病机制与桥粒芯糖蛋白1(desmoglein,Dsg1)和Dsg3抗体有关,目前在PV患者中发现大量非Dsg自身免疫性抗体,包括桥粒胶蛋白1(desmocollin,Dsc1)和Dsc3、斑菲素蛋白1(plakophilin,PKP1)和PKP3、斑珠...     

Predominant IgG4 subclass in autoantibodies of pemphigus vulgaris and foliaceus

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1. We have previously developed ELISAs using recombinant Dsg3 and Dsg1 expressed by baculovirus as a diagnostic tool for pemphigus. In this study, we determined the frequency of coexistence of IgA class as well as distribution of IgG subclass. Two out of 49 PV and PF sera tested had anti-Dsg1 IgA in addition to anti-Dsg1 IgG. Interestingly, one of them showed prominent pustular formation. Among IgG subclass, IgG4 was predominant and found in all of the 30 PV and 19 PF sera tested, followed by IgG1, detected in 25 out of 30 PV and 12 out of 19 PF sera. Even though IgG2 and IgG3 were detected in 13 and one PV and 6 and 4 PF sera, respectively, the ELISA titers had barely exceeded the cutoff value in most of the cases. There was no IgG subclass shift during the course of the disease in seven cases examined. These findings indicate that IgG4 subclass is the predominant autoantibodies in both PV and PF, while IgG1 is also frequently found.     

Drug-induced pemphigus: autoantibodies directed against the pemphigus antigen complexes are present in penicillamine and captopril-induced pemphigus

Pemphigus is an autoimmune blistering disease characterized by circulating autoantibodies directed against the keratinocyte cell surface. The two variants, pemphigus foliaceus and pemphigus vulgaris, can be distinguished at the molecular level by immunochemical studies. The large majority of patients with pemphigus develop the disease spontaneously; however, there is a small group of patients who develop pemphigus after treatment with certain medications, of which penicillamine and captopril are the best documented. Most patients with drug-induced pemphigus have circulating and/or tissue bound epidermal cell surface autoantibodies; however, the molecular specificity of these autoantibodies has not been studied. We performed immunoprecipitation studies utilizing extracts of 125I-labeled suction blister epidermis and the sera of three patients with drug-induced pemphigus foliaceus (two due to penicillamine and one due to captopril) and one patient with captopril-induced pemphigus vulgaris. We found that the three patients with drug-induced pemphigus foliaceus had circulating autoantibodies that are directed against the pemphigus foliaceus antigen complex and that the one patient with drug-induced pemphigus vulgaris had circulating autoantibodies that are directed against the pemphigus vulgaris antigen complex. This study demonstrates that autoantibodies from drug-induced pemphigus patients have the same antigenic specificity, on a molecular level, as do autoantibodies from other pemphigus patients.