Epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a subtype of pemphigoid that may present with scarring similar to hereditary dystrophic epidermolysis bullosa, of which the naming of EBA was derived. The clinical subtype of EBA with scarring is named mechanobullous EBA, because blisters are evoked by sudden mechanical trauma to the skin. The other subtype of EBA with erythematous lesions without scarring is named inflammatory EBA, and may look like bullous pemphigoid. The mucous membranes can be involved in both subtypes. The pathogenesis is mediated by IgG or IgA against type VII collagen, which is the component of anchoring fibrils below the lamina densa. Diagnosis is confirmed by detection of a u-serrated linear pattern of immune depositions by direct immunofluorescence microscopy of a skin biopsy. The pathogenesis of both clinical subtypes is unknown, and is not related to binding of a particular epitope of the auto-antigen. EBA is associated with systemic lupus erythematosus and colitis ulcerosa. The disease is relative refractory to treatment.</p

Bullous systemic lupus erythematosus

Bullous systemic lupus erythematosus (BSLE) is rare heterogeneous cutaneous manifestation in patient with systemic lupus erythematosus (SLE). BSLE encompasses a subepidermal autoimmune bullous disease with type VII collagen autoantibodies, leading to epidermolysis bullosa acquisita in patients with SLE. Alternatively, an acute generalized hemorrhagic vesiculo-bullous eruption may also occur in patients with SLE caused by the extensive inflammatory reaction without the presence of type VII collagen. Vesicular eruptions can also be seen in subacute cutaneous lupus erythematosus due to severe inflammatory reaction with subepidermal clefting, which in extreme cases may resemble erythema multiforme (Rowell syndrome) or toxic epidermal necrolysis.</p

How to take a biopsy

The skin is an organ that is easy to access for microscopy. Sections of the skin may reveal the split level of the blister, the type and distribution of inflammatory cells, and the presence, class and distribution pattern of autoantibodies. A biopsy should be taken from a location with smallest change of sample error, and the specimen specially fixated and transported for the requested microscopic techniques. Taking biopsies is the area of expertise of the dermatologist, even taking conjunctiva biopsies for immunofluorescence (IF) is possible in cases of suspicion of an autoimmune blistering disease of the eye.</p

Keratinocyte footprint assay discriminates antilaminin-332 pemphigoid from all other forms of pemphigoid diseases

Background Antilaminin-332 mucous membrane pemphigoid is a chronic severe pemphigoid disease characterized by autoantibodies to laminin-332. At present no commercial assay is available to demonstrate antilaminin-332 antibodies, and diagnosis relies on in-house techniques with limited sensitivities. Objectives In order to move, keratinocytes cultured in vitro secrete laminin-332 to attach to the culture dish. In that way, they leave behind a unique footprint trail of laminin-332. We aimed to develop a sensitive and specific laboratory assay to determine antilaminin-332 autoantibodies in patient serum based on binding of patient IgG to these unique footprints. Methods Normal human keratinocytes were grown on glass coverslips and incubated with patient or control serum for 1 h. The binding of IgG was then investigated by immunofluorescence. After validating the test for its ability to identify antilaminin-332 autoantibodies it was converted into a daily available test based on binding of IgG to dried coverslips that can be stored frozen. The staining patterns of sera from patients with antilaminin-332 pemphigoid were then compared with those of sera from patients with other autoimmune bullous diseases and normal human sera. Results IgG of all antilaminin-332 pemphigoid sera (n = 16) bound to laminin-332 footprints, while all normal human controls (n = 55) were negative. From the sera of patients with other diseases (n = 72) four sera tested positive. The footprint assay was also positive for sera that were negative by salt-split skin analysis, demonstrating that it is a very sensitive technique. Conclusions The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies. What's already known about this topic? Antilaminin-332 mucous membrane pemphigoid is a severe form of pemphigoid, and patients may have an increased risk of malignancies. The diagnosis of antilaminin-332 mucous membrane pemphigoid is complicated by the lack of specific commercial tests for antilaminin-332 antibodies and can be confirmed only in specialized laboratories. Keratinocytes in culture need laminin-332 for adhesion and migration and therefore deposit it on the bottom of the culture dish. What does this study add? The keratinocyte footprint assay detects antilaminin-332 autoantibodies in patient serum using the native laminin-332 produced by cultured keratinocytes. What is the translational message? The keratinocyte footprint assay is a fast and specific assay to confirm or rule out the presence of antilaminin-332 autoantibodies

Expression of Integrin α6β4 in Junctional Epidermolysis Bullosa

The integrin α6β4 is a member of the integrin family of adhesion receptors. The integrin α6β4 is preferentially expressed in stratified squamous epithelia, where it is localized in hemidesmosomes. A reduced number of rudimentary he- midesmosomes is often found in skin from patients with junctional epidermolysis bullosa (JEB). In this study we have investigated the expression of α6β4 in skin specimens of patients with junctional (one non-lethal, two lethal) and dystrophic (two) epidermolysis bullosa, using immunofluorescent (IF) staining with five different monoclonal antibodies against the α6 and β4 subunits. The intensity of IF staining of the integrin α6β4 and bullous pemphigoid antigen (BPA) was unreduced along the epidermal basement membrane zone (EBMZ) of all EB patients, compared to that in skin of healthy human controls. However, in the skin of two patients with lethal (Herlitz) JEB, who did not express GB3, IF staining of integrin α6β4 and BPA showed a “stitchy” discontinuous linear pattern along the EBMZ with interruptions at the borders of adjoining basal keratinocytes. The same results were obtained by immunoelectron microscopy. They corresponded with freeze-induced partial cell detachment from the basement membrane at the ultimate baso-lateral edge ot the basal keratinocytes in lethal JEB skin. The basal lamellipodia at that location almost completely lacked tonofilaments and hemidesmosomes. Furthermore, in JEB there was a split between the intra- and extracellular epitopes of the integrin α6β4 receptor, whereas the integrin remains intact in salt-split skin. This suggests that the defect is in α6β4 itself or perhaps its ligand

The art of the artifact

A 10-year old girl and her mother visited four dermatologists in several months because of erosions in her face, suspected for dermatitis artefacta. However, when previously confronted the patient denied this diagnosis. Immunofluorescence investigations were negative. After building a trusted physician-patient relationship and empathically confronting the mother,the mother herself was able to provide her daughter a narrow escape, which resulted in complete remission of her condition. Dermatitis artefacta is a spot diagnosis, however the treatment is challenging for both the physician and the patient. A trusting physician-patient relation is crucial and providing verbal and non-verbal support is essential. One way of doing this is asking the patient to keep a diary. In addition every patient deserves a thorough but compact investigation. Once internal causes have been excluded and a trustful relationship has been established one can precede to a narrow escape or confrontation. In this article we provide practical tricks on how to approach a patient with dermatitis artefacta.</p

Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative

The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis