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A 60-year-old man with extensive erosive lesions within the scalp and face, limbs, trunk, and nasal and oral mucous membrane involvement was admitted to the ward with worsening of symptoms in the course of pemphigus vulgaris. The exacerbation took place during an attempt to reduce doses of immunosuppressive drugs and glucocorticosteroids. Additional examinations, otorhinolaryngological and dental consultation did not show another cause of this condition. Biological therapy with a monoclonal antibody directed against the CD20 antigen, rituximab, was introduced. The study presents the treatment efficacy in the patient 2 weeks after the last drug dose and compares them with the latest available knowledge on rituximab therapy in pemphigus vulgaris.

Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal bullous disorder of the skin and mucous membranes. The disease results from the production of immunoglobulin G (IgG) antibodies against type-VII collagen, a major component of anchoring filaments in the dermal-epithelial junction. The disease has two major forms of presentation: the classical (non-inflammatory) type and the inflammatory type. Classical EBA is mainly characterized by the following features: development of non-inflammatory tense blisters on trauma-prone areas, multiple milia cysts, minimal or no inflammation findings on histopathology. Alternatively, inflammatory EBA is defined by widespread inflammatory blistering eruptions and a neutrophil-rich inflammatory infiltrate on standard histopathology. In both cases, specialized immunopathological findings are further required to establish an accurate diagnosis. In this article, we present an atypical case that shares features of both inflammatory and non-inflammatory forms of EBA. The case also serves to review and synthesize current concepts on the etiopathogenesis, diagnosis, and treatment of this extremely rare disease.

Pemphigus is an uncommon autoimmune mucocutaneousblistering disease, mediated by antibodies directedagainst proteins present on the surface of keratinocytesthat provide mechanical structure to the epidermis. Incidencesof the various forms of pemphigus vary fromcountry to country. Pemphigus vulgaris consists of the80% of pemphigus cases and it occurs 10 times morethan pemphigus foliaceus. There have been several recentadvances in the management of pemphigus. Treatmentof with systemic steroids and cytotoxic immunosuppressantscan be highly effective. This article will discussits epidemiology, pathogenesis, clinical variants and approachesto treatment. J Clin Exp Invest 2011; 2 (4): 468-473

The accurate diagnosis of autoimmune bullous diseases of the skin requires evaluation of clinical, histologic, and immunopathologic findings. There is generally a risk of serious side effects or complications in the treatment of autoimmune bullous diseases especially of pemphigus. Therefore definite diagnosis is essential for accurate and effective treatment. Fortunately, there are invaluable laboratory methods such as immunofluorescence tests in confirming a diagnosis that is suspected by clinical examination. In this article, the application techniques of these tests briefly and particularly the immunofluorescence findings that could be obtained in autoimmune bullous diseases were discussed.

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6α and K6β, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6α/K6β null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.

We describe a patient with severe generalized dystrophic epidermolysis bullosa (EBD) and a novel combination of compound heterozygous mutations in the COL7A1 gene. The maternal mutation was an A-to-G transition (425-A --> G) at position -2 of the donor splice site within exon 3 that causes aberrant splicing of two abnormal transcripts. One includes intron 3, and one excludes both exon 3 and intron 3. Both splice variants contained a premature termination of the translation. The paternal mutation is a 25-bp deletion in exon 20 (2638de125) that leads to a frameshift and a premature termination codon 133 bp downstream from the site of deletion. This combination of mutations allowed expression of collagen VII mRNA. Immunofluorescence staining of the patient's skin and cultured keratinocytes with domain-specific collagen VII antibodies, however, demonstrated markedly reduced levels of alpha1(VII) polypeptides, and no stable collagen VII protein could be extracted from the patient's cells. Electron microscopy showed severely hypoplastic fibrils below the lamina densa, without evidence of normal anchoring fibrils. The clinically unaffected parents were heterozygous for the mutations, suggesting that both COL7A1 gene defects were recessively inherited disease-causing mutations that are "silent" in heterozygous carriers but in combination can severely interfere with the dermal-epidermal adhesion and lead to severe EBD.

Cytokines are key regulators of physiological inflammatory responses, while aberrant cytokine expression contributes to pathogenesis of autoimmune diseases. We noted increased IL-6 levels in human and murine epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune bullous dermatoses (AIBD) induced by autoantibodies to type VII collagen (COL7). In contrast to rheumatoid arthritis, blockade of IL-6 led to strikingly enhanced experimental EBA, while treatment with recombinant IL-6 was protective. This was due to classical IL-6 signalling and independent of IL-6 trans-signalling, as treatment of mice with sgp130Fc had no impact on EBA manifestation. Induction of EBA in mice led to increased IL-1ra levels in skin and serum, while blockade of IL-6 completely inhibited IL-1ra expression induced by autoantibodies to COL7. In line, treatment of mice with EBA with recombinant IL-6 induced IL-1ra concentrations exceeding those of untreated animals with EBA, and IL-1ra (anakinra) administration significantly impaired experimental EBA induction. We here identified a novel anti-inflammatory pathway in an organ-specific autoimmune disease. Modulation of this IL-1ra pathway by classical IL-6 signalling demonstrates anti-inflammatory and protective activities of IL-6 in vivo. (C) 2012 Elsevier Ltd. All rights reserved.

The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca ++ ] i ) and inositol 1,4,5-trisphosphate (1P 3 ) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca ++ ] i , IP 3 PA secretion, and cell-cell detachment in DJM-1 cells. [Ca ++ ] i and IP 3 contents were determined with or without 30-mm pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxy-methylester and a specific IP 3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell- cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca ++ ] and IP 3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 µM), but not with U73343 (10 µM), dramatically reduced the pemphigus IgG-induced increases in [Ca + + ] i , IP 3 , and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 cause no effects on cell viability and IgG binding to the cell surface. These results suggest that phopholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.

O pênfigo foliáceo endêmico (PFE) é uma afecção onde fenômenos auto-imunes são freqüentemente relacionados com sua patogênese. Abordaram-se neste estudo, alguns testes laboratoriais freqüentemente alterados em outras doenças auto-imunes. Foram estudados em 20 pacientes com PFE a presença ou alterações de fator antinúcleo (FAN), fator reumatóide (FR), proteína C-reativa (PCR), velocidade de hemossedimentação (VHS), eletroforese de proteínas e o número totalde leucócitos. Encontrou-se PCR positiva em mais de 65 % dos casos, leucocitose moderada na maioria dos pacientes, VHS aumentada e alterações discretas da análise de proteínas do sangue. O FAN e o FR apresentaram-se negativos em todos os casos. Embora estes exames sejam considerados inespecíficos, o seu estudo associado ao quadro clínico pode colaborar no acompanhamento do PFE.Endemic pemphigus foliaceus (EPF) has its pathogenesis frequently associated to autoimmune phenomena. In this paper, afew routine laboratory tests, usually disturbed in some autoimmune diseases, were taken in 20 patients with EPF, which were screened for antinuclear antibodies (ANA), rheumatoid factor (RF), C-reactive protein (CRP) and changes of eritrocyts sedimentation rate (ESR), serologic proteins electrophoresis and total leucocyte count. The CRP was found in 60% of cases, leucocitosis in 85%, high ESR in all of them and mild alterations in serologic proteins analysis. No ANA or RF was found. Although widely accepted as inespeciflc tests, we believe that an association of the laboratory routine tests with clinicalfindings, can prove to be helpful in the follow up care of these patients.

The diagnosis of autoimmune blistering skin disease is increasingly based on the skin antigen(s) that is the target of the autoimmune process. Some patients with bullous diseases have unusual basement membrane autoantibodies that react to antigens present in both the epidermal and dermal sides of skin split with 1 M NaC1. This combined staining pattern is due to antibodies directed in part to a 160-kilodalton (kD) basement membrane antigen present on the epidermal side of the split skin and to different antigen(s) present on the dermal side. The identity of the current antigen(s), and the significance of this unusual type of combined staining antibody response is not known. Sera of five patients with combined staining antibodies, 19 patients with other types of basement membrane antibodies (15 directed only to the epidermal and four only to the dermal side of split skin), and 23 patients without basement membrane antibodies were tested by Western immunoblot for reactivity to 4% sodium dodecyl sulfate + 2 M urea extracts of dermal and epidermal side of salt-split skin. Three (60%) of the combined staining sera had antibodies to an 82–84-kD antigen (p84) reproducibly present in dermal, but not epidermal, extracts of normal human skin. Antibodies to this antigen were absent in the 42 control sera. Western blot affinity-purified antibody to p84 bound only to the basement membrane, on the dermal side of 1 molar salt-split skin. By co-migration experiments, p84 differed from other basement membrane antigens including the major and minor bullous pemphigoid, cicatrizing pemphigoid, and epidermolysis bullosa acquisita antigens, types III and IV collagen, laminin, and epiligrin. Thus, combined staining antibodies are directed in part to an 82–84-kD antigen (p84), a normal but previously undescribed component of the basement membrane of human skin. This antigen differs from the basement membrane antigens recognized by autoantibodies in other subepidermal bullous diseases. The antibody response to p84 provides a specific marker for a novel autoimmune subepidermal blistering skin disease.

The porphyrias are disorders of porphyrin metabolism that result from inherited or acquired aberrations in the control of the heme biosynthetic pathway. Variegate porphyria is characterized by a partial reduction in the activity of protoporphyrinogen oxidase. In this study, we identified the first nonsense mutation in a family with variegate porphyria. The mutation consisted of a previously unreported G-to-T transversion in exon 5 of the protoporphyrinogen oxidase gene, resulting in the substitution of glutamic acid by a nonsense codon, designated E133X. Our investigation establishes that a nonsense mutation in the protoporphyrinogen oxidase gene is the underlying mutation in this family with variegate porphyria.

We reported previously that patients with psoriasis harbored at a very high frequency DNA sequences of the oncogenic human papillomavirus type 5 (HPV5) associated with epidermodysplasia verruciformis. Moreover anti-HPV5 antibodies were detected in 25% of the cases. Our aim was to find out whether keratinocyte hyperproliferation and/or autoimmunity could be responsible for HPV5 expression in psoriasis. We found that epidermal repair in patients with extensive second degree burns (n = 19) is frequently associated with the generation of anti-HPV5 antibodies. In patients with autoimmune bullous diseases (n = 118), a condition in which keratinocyte proliferation is involved in repair mechanisms, the prevalence of anti-HPV5 antibodies (15%-25%) was similar to that reported in psoriasis and significantly higher than that (5%) observed in individuals with no known history of human papillomavirus infection (n = 119). A high detection rate (57.9%) of HPV5 DNA was observed in patients with bullous diseases. Anti-HPV5 antibodies were found in patients with autoimmune connective tissue disorders with cutaneous involvement (n = 40) as frequently as in patients with bullous diseases. HPV5 DNA was detected in one of the 10 patients studied. In contrast, the prevalence of anti-HPV5 antibodies in patients with autoimmune neurological disorders (n = 47) and in patients with common warts (n = 28) or invasive carcinomas of the skin (n = 40) was as low as in the general population. It is worth stressing that a similar prevalence of antibodies against HPV1 was found in all groups studied. Our data strongly suggest that extensive keratinocyte proliferation is a major factor for the generation of anti-HPV5 antibodies and that autoimmunity may contribute to this phenomenon. It remains to be determined whether HPV5 and other human papillomavirus genotypes associated with epidermodysplasia verruciformis contribute to the hyperproliferation of keratinocytes occurring in epidermal repair and in psoriasis.

Pemphigus vulgaris and pemphigus foliaceus are cutaneous autoimmune diseases characterized by intraepithelial blisters and autoantibodies to desmosomal glycoproteins. The antigens recognized by pemphigus vulgaris and pemphigus foliaceus autoantibodies are desmoglein- 3 (Dsg3) and desmoglein-1 (Dsg1), respectively. Dsg3 and Dsg1 are members of the desmoglein subfamily of the cadherin supergene family of cell adhesion molecules. It has been well documented that a subset of pemphigus vulgaris sera have IgG reactivity to both Dsg1 and Dsg3, suggesting that Dsg1 may also participate in the autoimmune response of these patients. The cellular mechanisms of T cell autoimmunity in these patients, however, are completely unknown. In this study, we tested the proliferative responses of T lymphocytes from eight pemphigus vulgaris patients after incubation with Dsg3 and Dsg1 fusion proteins. The sera of four of these PV patients showed reactivity with both Dsg1 and Dsg3, whereas the remaining four reacted only with Dsg3. We found that T cells obtained from those patients that exhibited the combined Dsg1/Dsg3 autoantibody reactivity showed a proliferative response after exposure to either Dsg1 or Dsg3 fusion proteins. The cellular responses to both of these recombinant proteins were highly specific and restricted to the CD4-positive T cell population. T cells from pemphigus vulgaris patients with no anti-Dsg1 serum reactivity showed a proliferative response to Dsg3, but not to Dsg1. The Dsg1 fusion protein used in this study has minimal sequence homology with Dsg3. Thus, this study provides the first evidence that T cells from a subset of pemphigus vulgaris patients respond to both Dsg1 and Dsg3.

Indirect immunogold electron microscopy studies of cryofixed, freeze-substituted, and post-embedded normal human skin were performed to localize precisely the ultrastructural binding site of circulating autoantibodies from two groups of patients with cicatricial pemphigoid. One group of patients had circulating IgG autoantibodies that bound the dermal side of 1 M NaC1-split skin and immunoprecipitated epiligrin. The other group of patients had circulating IgG autoantibodies directed against the epidermal side of 1 M NaCl-split skin and showed no specific reactivity to any keratinocyte polypeptide by iminunoprecipitation. IgG autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid bound the lowermost aspect of the lamina lucida at its interface with the lamina densa; the greatest staining was seen beneath and beside hemidesinosomes, In contrast, IgG from cicatricial pemphigoid patients whose autoantibodies bound the epidermal side of 1 M NaCl- split skin localized to hemidesmosomes and the junction between hemidesmosoines and the plasma membranes of basal keratinocytes. Although the latter staining pattern is similar to that observed with anti-BPAG2 autoantibodies, sera from our patients with cicatricial pemphigoid did not bind BPAG2 in immunoprecipitation studies of radiolabeled human keratinocyte extracts or show immunoblot reactivity to a fusion protein corresponding to the immunodominant epitope of this polypeptide. These studies demonstrate the following: 1) Autoantibodies from patients with anti-epiligrin cicatricial pemphigoid consistently bind the lower lamina lucida at its interface with the lamina densa; and 2) other patients with the same phenotype may have IgG autoantibodies against yet-unknown epitopes in basal keratinocytes.

International audience; This study describes the presence of antibodies in sera from patients with cicatricial pemphigoid specific for a 168-kDa antigen expressed by buccal mucosa. Six cicatricial pemphigoid sera unreactive, with epidermal or dermal proteins in immunoblot assay were tested on mucosal protein extracts. Four of these sera labeled a mucosal 168-kDa antigen (M168) under reducing conditions. An additional cicatricial pemphigoid serum with circulating antibodies to 180-kDa bullous pemphigoid antigen (BPAg2) also labeled M168. None of these cicatricial pemphigoid sera reacted with the alpha, beta, or gamma subunits of laminin-5. Nitrocellulose elution studies showed that the M168 antigen is a basement membrane antigen and labeled the epidermal side of salt-split skin. Immunoaffinity-purified anti-M168 antibodies did not bind to the 230-kDa bullous pemphigoid antigen (BPAg1) or to the 180-kDa BPAg2. None of the control sera from healthy individuals or from bullous pemphigoid, pemphigus vulgaris, or pemphigus foliaceus patients reacted with Ml68. This study demonstrates the specificity of some cicatricial pemphigoid sera against a 168-kDa antigen that is different from the laminin-5 subunits and shares no epitopes with the antigens of bullous pemphigoid (BPAg1, BPAg2) or the epidermolysis bullosa acquisita.This study describes the presence of antibodies in sera from patients with cicatricial pemphigoid specific for a 168-kDa antigen expressed by buccal mucosa. Six cicatricial pemphigoid sera unreactive, with epidermal or dermal proteins in immunoblot assay were tested on mucosal protein extracts. Four of these sera labeled a mucosal 168-kDa antigen (M168) under reducing conditions. An additional cicatricial pemphigoid serum with circulating antibodies to 180-kDa bullous pemphigoid antigen (BPAg2) also labeled M168. None of these cicatricial pemphigoid sera reacted with the alpha, beta, or gamma subunits of laminin-5. Nitrocellulose elution studies showed that the M168 antigen is a basement membrane antigen and labeled the epidermal side of salt-split skin. Immunoaffinity-purified anti-M168 antibodies did not bind to the 230-kDa bullous pemphigoid antigen (BPAg1) or to the 180-kDa BPAg2. None of the control sera from healthy individuals or from bullous pemphigoid, pemphigus vulgaris, or pemphigus foliaceus patients reacted with Ml68. This study demonstrates the specificity of some cicatricial pemphigoid sera against a 168-kDa antigen that is different from the laminin-5 subunits and shares no epitopes with the antigens of bullous pemphigoid (BPAg1, BPAg2) or the epidermolysis bullosa acquisita.

Epidermolysis bullosa simplex (EBS) is a mechano-bullous disorder characterized by intraepidermal blistering within the basal keratinocytes as a result of trauma to the skin. As part of the DNA diagnostics program, our laboratory has analyzed a cohort of 57 patients with the initial referral diagnosis of EBS. Among these patients, 18 were found to harbor heterozygous mutations in the keratin 5 or keratin 14 genes, KRT5 and KRT14, respectively, whereas in 14 cases, the disease was associated with mutations in both alleles of the plectin gene. Among the keratin mutations, 12 were distinct and six were novel, and in most cases there was no family history of a blistering disease. Prenatal diagnosis of eight pregnancies with keratin gene mutations, at risk for EBS either because one of the parents was affected (three cases) or history of a previously affected child as a result of a de novo mutation (five cases), predicted two fetuses being affected and six being normal. No recurrence of the de novo mutations in these pregnancies was disclosed. Collectively, the data suggest that a significant number of cases diagnosed as EBS are due to plectin mutations, and many cases result from de novo mutations in KRT5 and KRT14 genes. These findings have implications for genetic counseling and prenatal diagnosis for EBS.

Two bullous pemphigoid antigens (BPAs) have been identified: a 230-kD intracellular hemidesmosome-associated molecule and a 180-kD transmembrane hemidesmosome-associated molecule, Although patients with bullous pemphigoid (BP) have been shown to have circulating antibodies directed against one or both BPAs, the antigenic specificity of tissue-bound BP autoantibodies has not been studied. Because these skin-bound antibodies may play an important role in disease initiation, we sought to determine their antigenic specificity. In situ-bound BP antibodies were eluted with glycine from salt-split perilesional skin biopsy specimens and subjected to immunoprecipitation. Ten of 13 patients had glycine-eluted antibodies that recognized the 230-kD BPA, whereas four of 13 patients had glycine-eluted antibodies that recognized the 180-kD BPA. When these glycine-eluted BP skin specimens were sequentially eluted with urea, we found either antibodies of the same specificity or loss of reactivity to one of the BPAs, but no reactivity to any BPAs previously undetected with glycine elution. Sequential elution with sodium dodecylsulfate revealed no detectable BP antibodies. The heavy- and light-chain isotypes of the circulating, tissue-bound, and eluted BP antibodies were very similar, suggesting that we are not eluting certain subsets of BP antibodies, Sera from these patients contained circulating antibodies that recognized either one or both BPAs. Our observations demonstrate that in situ-bound antibodies eluted from the skin of patients with BP are preferentially directed against the 230-kD BPA.