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Background: The current standard in the serologic diagnosis of autoimmune bullous diseases (AIBD) is a multistep procedure sequentially applying different assays. In contrast, the BIOCHIP Mosaic technology combines multiple substrates for parallel analysis by indirect immunofluorescence., Methods: Sera from 749 consecutive, prospectively recruited patients with direct immunofluorescence-positive AIBD from 13 international study centers were analyzed independently and blinded by using (1) a BIOCHIP Mosaic including primate esophagus, salt-split skin, rat bladder, monkey liver, monkey liver with serosa, recombinant BP180 NC16A, and gliadin GAF3X, as well as HEK293 cells expressing recombinant desmoglein 1, desmoglein 3, type VII collagen, and BP230 C-terminus and (2) the conventional multistep approach of the Department of Dermatology, University of Lübeck., Results: In 731 of 749 sera (97.6%), specific autoantibodies could be detected with the BIOCHIP Mosaic, similar to the conventional procedure (725 cases, 96.8%). The Cohen κ for both serologic approaches ranged from 0.84 to 1.00. In 6.5% of sera, differences between the 2 approaches occurred and were mainly attributed to autoantigen fragments not present on the BIOCHIP Mosaic., Limitations: Laminin 332 and laminin γ1 are not represented on the BIOCHIP Mosaic., Conclusions: The BIOCHIP Mosaic is a standardized time- and serum-saving approach that further facilitates the serologic diagnosis of AIBD., (Copyright © 2020 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

Although T cells are considered as the central component in immune-mediated diseases, supportive evidence has demonstrated that B cells also contribute to the progression of these diseases. B cells are divided into various subsets according to their secreted cytokines. Different B cell subsets play diverse roles in immune-mediated dermatoses. Regulatory B cells (Bregs) are defined functionally by their ability to secrete IL-10, which has been revealed to contribute to immunological tolerance. Drugs that deplete B cells, such as rituximab, are now used for the treatment of several immune-mediated dermatoses. In this review, we present and discuss the current knowledge on the roles of B cells in several immune-mediate dermatoses including psoriasis, pemphigus, bullous pemphigoid, and dermatomyositis, atopic dermatitis., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Paraneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies., Objectives: To develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera., Methods: We produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates., Results: A2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values., Conclusions: Our novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Bullous pemphigoid and pemphigus constitute two major autoimmune blistering diseases (AIBD) with complicated disease pathomechanisms involving a multitude of cytokines and immunological pathways. The purpose of our literature review of the cytokines and chemokines involved in these AIBDs was to allow for a meta-analysis of studies detailing differential cytokine and chemokine changes in these conditions. Elucidation of inflammatory pathways could lead to more targeted therapies, several of which specific monoclonal antibodies already exist and are used safely for other autoimmune diseases. A systematic review of the Pubmed/Medline database was performed for articles characterizing cytokines/chemokines involved in BP and pemphigus. Further, a meta-analysis was carried out using standardized methods, including assessment for heterogeneity. The results of our analysis demonstrated numerous inflammatory alterations in these AIBDs. Significant alterations included serum levels of IL-5, IL-6, IL-8, IL-17, CCL-17, and CCL-26 in patients with BP, and increased blister fluids levels of IL-5, IL-6, IL-8, CCL11, and TNF-α. Blister fluid levels of IL-1α are decreased in BP. In pemphigus, we identified significantly increased serum levels of IL-10, IL-17, and CCL17. We have additionally summarized all studies excluded from meta-analysis to provide a comprehensive summary of cytokine/chemokine alterations in these two conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

IgG3 is the IgG subclass with the strongest effector functions among all four IgG subclasses and the highest degree of allelic variability among all constant immunoglobulin genes. Due to its genetic position, IgG3 is often the first isotype an antibody switches to before IgG1 or IgG4. Compared with the other IgG subclasses, it has a reduced half-life which is probably connected to a decreased affinity to the neonatal Fc receptor (FcRn). However, a few allelic variants harbor an amino acid replacement of His435 to Arg that reverts the half-life of the resulting IgG3 to the same level as the other IgG subclasses. Because of its functional impact, we hypothesized that the p.Arg435His variation could be associated with susceptibility to autoantibody-mediated diseases like pemphigus vulgaris (PV) and bullous pemphigoid (BP). Using a set of samples from German, Turkish, Egyptian, and Iranian patients and controls, we were able to demonstrate a genetic association of the p.Arg435His variation with PV risk, but not with BP risk. Our results suggest a hitherto unknown role for the function of IgG3 in the pathogenesis of PV.

Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are autoimmune blistering diseases, and substantial numbers of CD163 + tissue-associated macrophages (TAMs) are detected in both diseases. PV and BP possess different subsets of helper T cells, suggesting that the cytokine profiles of PV and BP might be different. The purpose of this study was to investigate the microenvironment of lesional skin and serum of PV and BP patients, focusing on the immunomodulatory factors related to TAMs, such as periostin (POSTN), chemokines, cytokines and matrix metalloproteinases (MMPs). We first performed immunohistological staining of POSTN in PV and BP lesions. POSTN was prominent in the superficial dermis in both PV and BP lesions. Next, to validate the activation of CD163 + TAMs in PV and BP patients, we examined the serum levels of soluble (s)CD163. The serum sCD163 levels in PV and BP patients are significantly higher than in healthy controls. To further elucidate the molecular mechanisms of the effects of POSTN on CD163 + TAMs in PV and BP, we examined chemokines, MMPs and cytokines selected by DNA microarray database. The serum CXCL5 levels from PV patients are significantly higher than those in BP patients and healthy controls. The IL-36γ expression on infiltrating macrophages was prominent only in the lesional skin of PV, while the MMP12 deposition was detected in both PV and BP lesions. Our results shed light on the novel pathogenesis of PV through CD163 + TAMs., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: A novel chemiluminescent enzyme immunoassay (CLEIA) was recently developed to quantify autoantibodies specific for desmogleins (Dsgs) and BP180, the target antigens of pemphigus and pemphigoid. This assay is automated and highly accurate and efficient., Objective: To validate the use of the CLEIA for detection of autoantibodies during the clinical courses of patients with pemphigus and pemphigoid., Methods: To define cut-off values for Dsg1, Dsg3, and BP180, we evaluated 47 serum samples from patients with pemphigus foliaceus (PF), 59 from those with pemphigus vulgaris (PV), 52 from those with bullous pemphigoid (BP), and 995 from healthy individuals. We also evaluated any fluctuations in CLEIA titers according to disease activity during the clinical course of 10 cases each of PF, PV, and BP. We used clinical symptom scores, the pemphigus disease area index (PDAI) and the bullous pemphigoid disease area index (BPDAI), to evaluate disease activity., Results: The cut-off values for the CLEIA titers determined by the Youden index were 15.4U/mL for Dsg1, 14.9U/mL for Dsg3, and 16.8U/mL for BP180. CLEIA titers fluctuated in parallel with the PDAI/BPDAI scores in 28 of the 30 cases with PF, PV, or BP. Although the CLEIA and enzyme-linked immunosorbent assay (ELISA) values in the same samples differed substantially in some cases, the concordance rates of positive/negative results between the CLEIA and ELISA were 96% for Dsg1, 97% for Dsg3, and 96% for BP180., Conclusion: The CLEIA, a newly developed, highly effective autoantibody detection system, is as reliable as ELISA for evaluation of the clinical courses of pemphigus and pemphigoid., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: Pemphigus and bullous pemphigoid are two autoimmune diseases that have similar pathogenesis. Both have a genetic predisposition, which promotes the production of auto antibodies targeted against different components of the epidermal desmosome and hemidesmosome. Antiphospholipid antibodies (aPL) are heterogeneous group of antibodies found in patients with autoimmune diseases and inflammatory conditions and are associated with thrombotic events., Objective: We sought to determine the expression profile of eight non classical aPLs in ABD patients., Methods: A cohort of 266 serum samples of patients with pemphigus, bullous pemphigoid and controls was screened for the presence of eight aPL antibodies, using the Bio-Rad BioPlex™ 2200 system., Results: Phosphatidylserine-beta-2-glycoprotein-I (aPS-β2GPI) and anti prothrombin complex (aPT-PT) serum profiles were significantly more prevalent among ABD patients; 20.7% patients with ABD compared to 5.9% of control patients were positive for aPS-β2GPI IgM. In addition, aPT-PT IgM was more prevalent among ABD patients (31% vs. 14.8%)., Conclusion: aPL auto antibodies are more prevalent in ABD. Any clinical association should be further determined.

Pemphigus and bullous pemphigoid are two autoimmune diseases that have a similar pathogenesis. Both have a genetic predisposition which promotes the production of auto-antibodies targeted against different components of the epidermal desmosome and hemidesmosome. Environmental factors play an important role in the pathogenesis of this autoimmune disease. Among these, the role of infectious agents was debated as a causative factor. We sought to determine a possible connection between various infectious agents and autoimmune bullous disease (ABD). A cohort of 148 serum samples of patients with pemphigus, bullous pemphigoid and controls was screened for evidence of a prior infection with HBV, HCV, EBV, CMV, Helicobacter pylori, Toxoplasma gondii and Treponema pallidum, utilizing the Bio-Rad BioPlex 2200 system as well as ELISA assays to complete the panel. HBV, HCV, H. pylori, T. gondii and CMV were demonstrated to have significantly higher prevalence of antibodies in patients with pemphigus and bullous pemphigoid in comparison to controls. Among them, we found a novel association between H. pylori and ABD. Our study suggests a contributing role for HBV, HCV, H. pylori, T. gondii and CMV in inducing ABD in the genetically susceptible host., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The main autoimmune blistering skin disorders are pemphigus vulgaris (PV) and bullous pemphigoid (BP). They differ in the inflammatory infiltrate, which is more intense in BP. Inflammation is known to activate coagulation in several disorders. Local and systemic activation of coagulation was evaluated in BP and PV. We studied 20 BP patients (10 active and 10 remittent), 23 PV patients (13 active and 10 remittent) and 10 healthy subjects. The coagulation markers prothrombin fragment F1+2 and D-dimer were measured by enzyme-immunoassays in plasma. The presence of tissue factor (TF), the main initiator of blood coagulation, was evaluated immunohistochemically in skin specimens from 10 patients with active PV, 10 patients with active BP and 10 controls. Plasma F1+2 and D-dimer levels were significantly high in active BP (P = 0.001), whereas in active PV the levels were normal. During remission, F1+2 and D-dimer plasma levels were normal in both BP and PV. TF immunoreactivity was found in active BP but neither in active PV nor in normal skin. TF reactivity scores were higher in active BP than in controls or active PV (P = 0.0001). No difference in TF scores was found between active PV and controls. BP is associated with coagulation activation, which is lacking in PV. This suggests that BP but not PV patients have an increased thrombotic risk. The observation that thrombotic complications occur more frequently in BP than in PV further supports this view.

Aim: Recent insights on the pathogenesis of autoimmune blistering diseases have pointed out the opportunity of new diagnostic tools, such as enzyme-linked immunosorbent assay (ELISA) for Desmogleins 3 and 1 (Dsg3, Dsg1), and bullous pemphigoid (BP) 180 antigen auto-antibodies. The aim of the present prospective study was to evaluate the diagnostic values of these tests in blind with histopathology and direct immunofluorescence (DIF), the assessment of correlation with clinical presentation and severity of disease, as well as eventual modifications of serum auto-antibodies titres in course of treatment., Methods: From June 2005 to June 2007, all consecutive patients with clinically blistering diseases presenting to the Dermatology Department of Cagliari were enrolled in the study. Biopsy specimens were performed in all cases and sent for histopathological examinations including haematoxylin-eosin stain and DIF to the Unit of Pathological Anatomy of the same University. Serum samples were tested with Dsg3, Dsg1 and BP180 ELISA in the internal laboratory of the Dermatology Department, and results were worked out many days before histopathology reports. Final diagnosis was established on clinical, histological and immunopathological findings. A selected sample of patients with active autoimmune blistering disease underwent repeated immunosorbent assays at 1-2-6 months from first diagnosis and treatment introduction., Results: Forty-two patients (23 men, 19 women) were enrolled in the study and divided into three groups: pemphigus (N=17), pemphigoid (N=19) and other diseases (OD; N=6), depending on the final diagnosis assessed by histological, immunopathological and serological examinations. In pemphigus group ELISA showed circulating antibodies against Dsg3 in all patients (100%) and against Dsg1 in 13 patients (76.5%). In the pemphigoid group, 16 of 19 sera showed positive scores above the cut-off value (84.2%), but sensibility was higher if considering only the bullous pemphigoid final diagnosis (16/17). None of the other bullous diseases (0%) exceeded the cut-off value for Dsg1, Dsg3 and BP180 ELISA. Correlation with histopathology and direct immunofluorescence was excellent for pemphigus and very good for pemphigoid. Eight patients (6 P; 2 BP) underwent a serial measurement of the autoantibodies levels: two patients (1 PV and 1 BP) showed an ELISA antibodies titres decrease after two months of treatment, in parallel with an excellent clinical response. Whereas in six cases (5 PV and 1 BP) the ELISA titres overstayed high at I and II month. Clinically the disease was active in all six patients, and a treatment adjustment was performed (increased corticosteroid dosage and/or azathioprine initiation in all cases, high dose intravenous immune globulin in one case). At month VI, a decrease on ELISA antibody levels was documented in three patients (3 PV), parallel to a clinical remission. Whereas in other three patients (2 PV, 1 BP), persistent high Dsg3 ELISA titres were related to a still active disease: although clinically improved, blisters flared up at any attempt to taper drugs dosage., Conclusion: Dsg3, Dsg1 and BP ELISA is a sensitive, easy and quick reading tool for the diagnosis of the main autoimmune blistering diseases: pemphigus and bullous pemphigoid. More over, autoantibodies titre correlate with disease severity, and is useful to monitor treatment response.

Background: Autoantibodies of bullous pemphigoid (BP) patients react with two components of the hemidesmosome of stratified epithelia: the BP antigen 230 (BP230) and the BP antigen 180 (BP180). Recently, strong evidence has been provided that autoantibodies to BP180 play a key role in subepidermal blister formation in BP patients, and NC16A contains an important antigen determinant of BP., Objective: To study the role of BP180NC16a enzyme-linked immunosorbent assay (BP180NC16a-ELISA) in the diagnosis of BP in China., Methods: Sera from BP patients (n = 42) and control subjects (normal controls, n = 24; pemphigus patients, n = 18) were measured by BP180NC16a-ELISA. All BP sera were obtained at presentation from patients who had not received previous systemic treatment. The values of immunoglobulin G (IgG) antibody levels measured by ELISA were compared with those measured by indirect immunofluorescence (IIF) (gold standard for the diagnosis of BP) on salt-split skin., Results: Using BP180NC16a-ELISA, 41 of the 42 BP sera were positive, whereas only one of the serum samples from 24 normal controls was positive and all the pemphigus sera showed a negative result. Thus, the sensitivity and specificity of BP180NC16a-ELISA were both 97.62%. There was no correlation between the mean ELISA values and IIF titers. The ELISA and IIF results were further compared and analyzed using a 2 x 2 contingency table, which showed that they were not significantly different., Conclusions: It is suggested that BP180NC16a-ELISA is a useful tool for the diagnosis of BP.

Background: B cells have been demonstrated to have critical roles in developing autoimmune bullous diseases. Recently identified tumor necrosis factor-like molecules, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) are essential molecules for B cell development, survival, and proliferation. Although the functions of APRIL have not been fully evaluated, recent studies suggest that circulating levels of APRIL are increased in various autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis., Objectives: To determine serum APRIL levels in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), and compare those with clinical findings and laboratory findings., Patients/methods: Sera from 15 PV patients, 43 BP patients, and 15 normal controls were subjected to ELISA assays to measure serum APRIL, BAFF, Dsg3, and BP180 levels., Results and Conclusions: Circulating APRIL levels were significantly elevated in BP patients but not in PV patients, and correlated with serum BAFF levels. Our study revealed that serum APRIL levels tended to be increased in the quite early stage of disease. In conclusion, circulating APRIL levels may be a useful marker for early activation of autoimmune diathesis, and furthermore, an effective therapeutic target molecule in patients with BP.

Background: Autoimmune bullous diseases are characterized by autoantibodies against specific adhesion molecules of the skin and/or mucous membrane. While these autoantibodies are known to play a primary role in the disease manifestation, it remains unknown how disease-specific autoreactive B cells and autoantibodies are induced. Recent studies have indicated the importance of the CD40 and CD40 ligand (CD40L) receptor-ligand pair in the immunopathogenesis of autoimmune diseases. CD40L circulates in soluble form, and some reports suggest that serum soluble CD40L (sCD40L) levels are increased in various autoimmune diseases., Objectives: To determine serum sCD40L levels in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), and to determine their correlation with clinical findings and laboratory findings., Patients and Methods: Sera from 10 PV patients, 35 BP patients and 12 normal controls were subjected to ELISA assays to measure serum levels of sCD40L, anti-desmoglein-3 antibody and anti-BP180 antibody., Results and Conclusions: Circulating sCD40L levels were significantly elevated in BP patients, but not in PV patients. Serum sCD40L levels increased in the early stage of disease onset and recurrence in BP patients. In conclusion, circulating sCD40L levels may be a useful marker for early activation of autoimmune diathesis and, furthermore, an effective therapeutic target in patients with BP., (Copyright 2007 S. Karger AG, Basel.)

Background: Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by eosinophilia and high serum IgE levels. The accumulated evidence suggests that various cytokines are involved in the lesional skin of patients with BP. Recently, thymus and activation-regulated chemokine (TARC/CCL17), a CC chemokine, was identified as a selective chemoattractant for CC chemokine receptor 4 (CCR4)-expressing cells., Objective: In this study, we examined the involvement of TARC in patients with BP., Methods: We determined the fluid and serum TARC levels in patients with BP by enzyme-linked immunosorbent assay and compared the serum TARC levels with the eosinophil numbers in peripheral blood. We also compared the serum TARC levels in five patients with BP before and after they were treated. Moreover, we examined TARC, CCR4 and CXC chemokine receptor 3 (CXCR3) expression in the lesional skin of patients with BP by immunohistochemical procedures. Furthermore, we measured CCR4 positivity in CD4+ CD45RO+ cells of peripheral blood mononuclear cells (PBMCs) in patients with BP and healthy control subjects., Results: The fluid TARC levels in patients with BP were significantly higher than those in blisters from burn patients or suction blisters of healthy control subjects. The serum TARC levels in patients with BP were also significantly higher than those in pemphigus vulgaris (PV) patients and healthy control subjects, and decreased after the treatment. The serum TARC levels in patients with BP significantly correlated with the eosinophil numbers in peripheral blood (r = 0.72, P < 0.002). Immunohistochemistry showed a strong reactivity of TARC in the epidermal keratinocytes (KCs) of BP. Moreover, both CCR4 and CXCR3 were expressed on the dermal infiltrating CD4+ T cells mainly beneath the bullae of patients with BP. Fluorescence-activated cell sorting analysis showed a higher percentage of CCR4 positivity in CD4+ CD45RO+ cells of PBMCs in patients with BP than that in healthy control subjects, while there was no significant difference of CXCR3 positivity in CD4+ CD45RO+ cells of PBMCs between patients with BP and healthy control subjects., Conclusions: These findings strongly suggest that TARC may be one of the important chemokines that are involved in the pathogenesis of BP.

Mucous membrane pemphigoid (MMP), also known as cicatricial pemphigoid, is a rare vesiculobullous disease of mucosal tissues, which involves the oral, ocular, and other mucous membranes. We have studied a group of patients with histologically and immunopathologically proven pemphigoid disease involving predominantly the conjunctiva and oral mucosa in addition to other mucosae. The purpose of our study was to (i) demonstrate the specific binding of autoantibodies present in the sera of patients with MMP to normal human oral mucosa by indirect immunofluorescence (IIF) and (ii) to study the role of these autoantibodies in the pathogenesis of subepithelial blister formation using normal human buccal mucosa in organ culture. Serum and IgG fractions from MMP patients showed homogeneous smooth linear binding along the basement membrane zone (BMZ) of the normal buccal mucosa on IIF. Serum from pemphigus vulgaris patients showed intercellular or keratinocyte cell surface staining. BMZ separation developed at 48 h after incubation of normal human buccal mucosa in organ culture, with serum or IgG from patients with MMP but not after addition of normal human serum. Addition of pemphigus vulgaris serum to the in vitro culture of normal human buccal mucosa showed acantholysis. This preliminary report suggests that circulating autoantibodies may have an important role in the pathogenesis of MMP. This in vitro organ culture model will facilitate enhancing our understanding of various molecular events during the process of blister formation in MMP and in the study of other mucosal diseases., (Copyright 2000 Academic Press.)

Interleukin 10 (IL-10) is an immunoregulatory cytokine produced by T lymphocytes and macrophages. Recently, it has been suggested that IL-10 may be involved in the pathogenesis of various inflammatory and autoimmune diseases. Using an ELISA we investigated the presence of IL-10 in the serum and blister fluid of pemphigus vulgaris (PV) patients with active disease and those in prolonged clinical remission compared with normal controls. Sera from patients with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), oral pemphigoid (OP) and blister fluid from five patients with BP were also studied. Increased levels of IL-10 were detected in the sera of 87.5% of patients with active PV and were statistically significant (P=0.0003) when compared with levels in normal human serum. Lower levels of IL-10 were detected in 12.5% PV patients in remission and were statistically significant (P=0.0001) when compared with levels in patients with active disease. Levels of IL-10 were detected in sera of 4.6% (1 of 24) of the normal controls. The levels of IL-10 were approximately four times higher in blister fluids than levels in the serum in the same PV patients. This difference was highly statistically significant (P=0.0008). A correlation was observed between serum levels of IL-10 and titres of pemphigus autoantibodies and with disease severity. Elevated level of IL-10 was detected in the blister fluid from five BP patients. Levels of IL-10 in the sera of patients with BP, OCP and OP were not significantly increased. These preliminary data suggest that IL-10 in concert with other cytokines may play an important role in the pathogenesis of PV and BP., (Copyright 2000 Academic Press.)

Hyperglycemia is observed in some patients with autoimmune bullous diseases complicated by diabetes mellitus or treated with systemic corticosteroids. High concentrations of glucose can react with various proteins and change their structural and functional properties. We previously reported that nonenzymatic glycosylation of antibody can impair antigen-antibody binding. We ascertained whether glycosylation of autoantibody decreases the autoantibody titer by examining 30 sera from patients with pemphigus and pemphigoid. Nonenzymatic glycosylation in the physiological range was induced by incubation of sera with 1650 mM D-glucose at 4 degrees C for 7 days. The titers of sera were determined by indirect immunofluorescence (IIF). In all cases, the immunofluorescence intensity of glycosylated sera was weaker than that of nonglycosylated sera. Glycosylated sera showed a lower antibody titer by 1 doubling dilution in 18 out of 30 cases, compared with nonglycosylated sera. The ten BP patients' sera were also analyzed by immunoblotting for reactivity with the BP180-GST fusion proteins, S delta 1 and 4575. All BP sera reacted with S delta 1, and 5 out of 10 BP sera reacted with both S delta 1 and 4575. In all the sera that reacted only with S delta 1, the glycosylated sera showed a 1 doubling dilution decrease in autoantibody titer. Interestingly, in 4 out of 5 sera that reacted with both S delta 1 and 4575, there were no differences in the antibody titer between glycosylated and nonglycosylated sera. These results indicate the possibility of a false decrease in autoantibody titers of sera from patients with autoimmune bullous diseases complicated with hyperglycemia. Although the false decrease in titers of autoantibodies induced by nonenzymatic glycosylation is not dramatic, it must be considered in order not to underestimate the disease activity of pemphigus in such cases.

Background: Recently, we reported that soluble E-selectin (sE-selectin), an isoform of the cell membrane E-selectin, an adhesion molecule synthesized only by endothelial cells, is significantly increased in sera of the patients with bullous pemphigoid (PB) or pemphigus vulgaris. A significant correlation was also found between the serum sE-selectin levels and the number of skin lesions, suggesting the possible use of this molecule to gauge disease intensity before therapy. One of the sE-selectin inducers is tumor nerosis factor-alpha (TNF-alpha), that is also able to enhance vascular endothelial growth factor (VEGF), a strong endothelium activator., Objective: On the basis of these observations, the present study was conducted to analyze the serum levels of VEGF, sE-selectin, and TNF-alpha in 8 patients with BP (age: 82, range 54-87, 7 males, 1 female) and in 6 patients affected affected with PV (age: 55, range 44-65; 5 males, 1 female) and to verify possible correlations between these variables and the disease activity, In addition, serum sE-selectin levels were measured over time and compared with the serum anti-epithelium antibodies titers., Methods: The sE-selectin, VEGF and TNF-alpha levels were measured in the samples by means of commercially available ELISA kit. The same samples were also employed to measure the anti-epithelium antibody titers., Results: Serum VEGF, sE-selectin and TNF-alpha levels were significantly correlated each other (p at least < 0.01). All three variables were also significantly correlated with the number of lesions (p at least < 0.01). Serum VEGF levels were found increased (median = 178 pg/ml, range 37-595) as compared to 28 healthy controls (median = 135 pg/ml, range 18/269, p < 0.05). Also serum TNF-alpha levels were found increased (median = 5.5 pg/ml, range < 0.1-41.0) as compared to 28 healthy controls (median < 0.1 pg/ml, range < 0.1-5.3), p < 0.01). When the patients were observed over time, serum sE-selectin levels highly correlated with the disease intensity in both dermatoses, although with different regression curves., Conclusions: These data further underline the endothelium involvement in these bullous dermatoses and stress the possibility of employing sE-selectin as a non-specific follow-up marker of both BP and PV.

Soluble E (sE)-selectin represents the soluble isoform of cellular E-selectin, an adhesion molecule synthesized only by endothelial cells. As a consequence, it may be considered a marker of endothelial activity. The aim of this study was therefore to evaluate the serum levels of sE-selectin in nine patients affected with pemphigus vulgaris (PV) and in 15 patients with bullous pemphigold (BP). Higher amounts of sE-selectin, median 40.3 ng/mL, range 30-109.6 were found in the patients when compared with 20 healthy individuals, median 28.5 ng/mL, range 6.4-48; P < 0.01, matched for sex and age. These levels were also significantly correlated with the number of detectable lesions (r = 0.63, P < 0.001) when the patient data were considered at the time of the first observation. Thirteen subjects were followed over time for a maximum of 3 months (from three to seven observations). During therapy, the number of lesions and the serum sE-selectin values decreased concomitantly. Differently from sE-selectin, the serum soluble intercellular adhesion molecule-1 (sICAM-1) values were not significantly different in the patients from the controls and showed no correlation with the serum sE-selectin concentrations or with the number of lesions. The data presented point to the possible use of sE-selectin determinations as a non-specific follow-up marker, suitable to gauge disease intensity over time and emphasize that endothelial activation is present in BP as well as in PV.

Among 13 patients with pemphigus or bullous pemphigoid, high titers of anti-interferon-alpha (IFN-alpha) antibodies were present in all serum samples of one patient suffering from pemphigus foliaceus. This patient was characterized by a relatively benign course of the disease. The IFN antibodies were of oligoclonal or polyclonal origin, predominantly of the IgG subtype, and displayed a broad spectrum of specificity including various natural and recombinant IFN-alpha subtypes as well as recombinant IFN-omega 1. In vitro, these antibodies neutralized both the antiviral and antiproliferative activities of the respective IFN types. Recognition of the patient's endogenous IFN-alpha demonstrated their autoantibody nature. The IFN antibodies were present at diagnosis and resistant to continued immunosuppressive treatment. Despite clinical remission, the IFN antibodies persisted, suggesting that they were not pathogenically related to the skin manifestations of the pemphigus. There were no sings of immune complex-mediated organ damage. IFN antibodies constitute a new class of autoantibodies that may occur in conjunction with pemphigus and likely interfere with the endogenous IFN system.

Serum dehydroepiandrosterone sulphate (DHEAS) levels and diagnostic autoantibody titers were measured in patients with pemphigoid/pemphigus [n = 46/4; 21 men and 29 women, 42 to 93 years of age (mean 79)]. Twenty-four patients were either on peroral Prednisolone (n = 11), or topical treatment with betamethasone (n = 13), and the other 26 were either receiving non-steroidal drugs or were untreated. Their DHEAS levels were compared to those of 20 patients with psoriasis, and to 23 patients with secondary osteoarthritis (OA). Assessing the patients by group, the mean DHEAS level was markedly lower in the pemphigoid/pemphigus than in the psoriasis and OA patients (geometric mean 600 vs. 2130 and 2100 nmol/l, respectively; p < 0.001). This difference was independent of steroid treatment. No correlation was found between the DHEAS levels and antibody titers. The low levels found in pemphigoid/pemphigus are concordant with those reported for systemic lupus erythematosus, rheumatoid arthritis and polymyalgia rheumatica/giant cell arteritis. DHEAS deficiency is a permanent feature in these autoimmune diseases, and may contribute to their etiology and/or pathophysiology.

To develop a new method to evaluate autoantibodies in various autoimmune bullous skin diseases, we examined reactivity of bullous pemphigoid (BP) and pemphigus vulgaris (PV) patients' sera with partial bacterial fusion proteins of the 230 kD BP antigen (BPAG1) and PV antigen, respectively, by an enzyme-linked immunosorbent assay (ELISA), and compared the results with those of immunoblotting. We used two fusion proteins derived from the mouse BPAG1 and a fusion protein derived from the amino-terminus (EC1-2) of PV antigen. For both BP and PV sera, the ELISA scores were well correlated with the reactivities on immunoblot assays. The present study indicates that ELISA using recombinant antigen proteins in various autoimmune bullous skin diseases will be a new useful technique to detect the autoantibodies. However, development of recombinant proteins with the entire molecule and correct conformation will be necessary to establish a perfect ELISA system in the future.

It is still unclear what kinds of mechanisms are involved in blister formation after antibodies bind to the antigens in pemphigus and bullous pemphigoid. The effects of IgGs from pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid sera on intracellular calcium concentration ([Ca++]i) and inositol 1,4,5-trisphosphate were examined in a human squamous cell carcinoma cell line (DJM-1 cells) and in cultured human keratinocytes to clarify whether signal transduction via calcium is involved. IgGs were purified with protein A affinity column from the sera of five pemphigus vulgaris patients, three pemphigus foliaceus patients, eight bullous pemphigoid patients, and 14 normal volunteers. Keratinocytes were cultured in Eagle's minimum essential medium containing 1.8 mM Ca++ and loaded with fura-2/AM, followed by addition of the IgGs. Subsequently, [Ca++]i was determined by measuring the fluorescence ratio (F340/F360) with videomicroscopy. Pemphigus IgGs (seven of eight cases) induced a rapid and transient increase in [Ca++]i in both the cells, whereas a [Ca++]i increase was caused by very few IgGs from bullous pemphigoid (one of eight cases) and normal sera (two of 14 cases). The pemphigus IgG-induced transient [Ca++]i increase was not affected by chelating extracellular Ca++ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid. In addition, monoclonal antibodies acid. In addition, monoclonal antibodies against 180-kD and 230-kD antigens did not exert this change. Pemphigus IgGs that caused a [Ca++]i increase induced rapid and transient production of inositol 1,4,5-trisphosphate, peaking at 20 seconds. These findings suggest that IgG from pemphigus induces Ca++ mobilization by inositol 1,4,5-trisphosphate from internal stores, and that mechanisms of antibody-transmitted signaling in pemphigus may differ from those in bullous pemphigoid.

Importance of kallikreinogenases in acantholysis and epidermo-dermal separation was shown, when the kallikreinkinin patterns were studied in blood plasma and blister fluids of 118 patients with pemphigus, of 78 patients with pemphigoid as well as of 224 guinea pigs. Under conditions of pemphigoid, as distinct from pemphigus, serine proteinases were mainly produced by active immunocytes responsible for destruction of epidermal basement membrane; activation of kinins was observed in skin impairments.

Radioimmunoassays of some hypophyseal, adrenal, thyroid, pancreatic, and sexual gland hormones, carried out in patients with pemphigus and bullous pemphigoid, have revealed increased levels of ACTH and hydrocortisone in their blood sera, vs. the reference values. Glucocorticoid therapy has normalized the ACTH level, and hydrocortisone content has proved to be below the norm. The blood plasma triiodothyronine level has been elevated, whereas insulin and glucagon lowered in the patients with pemphigus vs. those with bullous pemphigoid. In females of a menopausal age, suffering from pemphigus, increased levels of estradiol and reduced ones of testosterone have been detected, as compared to those suffering from bullous pemphigoid. These data evidence a varying functional activity of the endocrine glands in these dermatitides.

Specific antibodies present in the sera of patients with bullous pemphigoid or pemphigus vulgaris were detected in an enzyme-linked immunosorbent assay (ELISA) employing a squamous carcinoma cell line, SCaBER, as substrate. Bullous pemphigoid sera bound preferentially to permeabilized cells, suggesting that the antigens are largely intracellular. The assay may prove to be a useful addition to current methods of detecting circulating antibodies in these patients.