Using this pattern recognition by direct immunofluorescence microscopy we discovered several cases of EBA which would otherwise have been erroneously diagnosed as a form of pemphigoid or linear IgA disease.

IntroductionGeneralized atrophic benign epidermolysis bullosa (GA-BEB) is a form of nonlethal junctional epidermolysis bullosa characterized by universal alopecia and atrophy of the skin. We report a deficiency of the 180-kD bullous pemphigoid antigen in three patients with GABEB from unrelated families. We screened specimens of clinically normal skin from nine junctional epidermolysis bullosa patients (3 GABEB, 4 lethal, 1 cicatricial, 1 pretibial) by immunofluorescence using monoclonal antibodies to the 180-kD and 230-kD bullous pemphigoid antigens (BP180 and BP230). In the skin of the three GABEB patients there was no reactivity with antibodies to BP180, whereas staining for BP230 was normal. In the skin of the other six, non-GABEB patients, included in this study the expression of BP180 and BP230 was normal. Immunoblot analysis of cultured keratinocytes from one of the GABEB patients also failed to detect BP180 antigen, whereas BP230 was present in normal amounts. In previous studies on the expression of the bullous pemphigoid antigen in JEB, sera from bullous pemphigoid (BP) patients were used, the specificity of which had not been analyzed by immunoblotting (6-9). The expression was found to be normal or variable. Later it became apparent that BP-serum is a mixture of polyclonal antibodies, which are very difficult to characterize, even after affinity-purification (10). Fortunately, reliable monospecific antibodies for each of the two major bullous pemphigoid antigens have now become available (1 1, 12).In this study we used monoclonal antibodies specific for BPI 80 and BP230 (11,12). In addition we studied the expression of the newly described 500-kD hemidesmosomal plaque protein HD1 (13)

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This study shows that in a considerable number of supposedly IgG-mediated pemphigus patients IgA to Dsg1 and Dsg3 is also present. In most cases the antigen specificity of the IgA follows the antigen specificity of the IgG, although in a small number of cases IgA is present against the Dsg not recognized by IgG.

BackgroundThe demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.MethodsIn the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.ResultsDirect IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).ConclusionsWe conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis.

Background-Wegener's granulomatosis (WG) is considered a pauci-immune systemic vasculitis based on the absence of immune deposits in renal biopsies of patients with active disease. In animal models of antineutrophil cytoplasmic antibody (ANCA) associated glomerulonephritis, immune deposits along the glomerular capillary wall are present at early stages of lesion development. These deposits are degraded rapidly, resulting in "pauci-immune" lesions. Objective-To test the hypothesis that immune deposits can also be detected in early lesions of patients with WG, thereby initiating an inflammatory reaction that, in time, is augmented in the presence of ANCA, resulting in pauci-immune lesions later on. Methods-The presence of immune deposits in skin biopsies taken within 48 hours of lesion development was investigated. Direct immunofluorescence was used to examine 32 skin biopsies for the presence of immune deposits (IgG, IgA, IgM, C3c). When possible, a comparison was made between the immunofluorescence findings in renal and skin biopsies taken at the same time. Results-Four of 11 biopsies taken at initial presentation and four of 21 biopsies taken at the onset of a relapse of WG showed IgG and/or IgA containing immune deposits in the subepidermal blood vessels. All nine renal biopsies showed pauci-immune glomerulonephritis, irrespective of the presence (n=5) or absence (n=4) of immune deposits in the skin biopsy. Conclusion-A substantial number of skin biopsies showed immune deposits during active disease. These results could support the hypothesis that immune complexes may trigger vasculitic lesions in WG.

The integrin alpha 6 beta 4 is a member of the integrin family of adhesion receptors. The integrin alpha 6 beta 4 is preferentially expressed in stratified squamous epithelia, where it is localized in hemidesmosomes. A reduced number of rudimentary hemidesmosomes is often found in skin from patients with junctional epidermolysis bullosa (JEB). In this study we have investigated the expression of alpha 6 beta 4 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 alpha 6 and beta 4 subunits. The intensity of IF staining of the integrin alpha 6 beta 4 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 alpha 6 beta 4 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 of 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 alpha 6 beta 4 receptor, whereas the integrin remains intact in salt-split skin. This suggests that the defect is in alpha 6 beta 4 itself or perhaps its ligand.

Many atopic dermatitis (AD) patients have exacerbations of their skin disease in winter. These exacerbations may be caused by non-immunological 'non-specific' factors, such as low sun exposure and low temperature. To date, the influence of season on non-specific skin reactivity in AD has not been studied. The aim of the present investigation was to assess the influence of season on two skin parameters which may be used as quantitative measures of non-specific skin reactivity in AD: (i) susceptibility to repeated epicutaneous irritant (sodium lauryl sulphate, SLS) exposure, and (ii) weal and flare responses to intracutaneous injection of bioactive agents (codeine, FMLP, histamine, methacholine, substance P, trypsin). Four of 16 AD patients had dermatitis which was more severe in November than in July. Susceptibility to SLS was increased in November, both in AD patients and in control subjects. AD patients were more susceptible to SLS than control subjects in both July and November. Pre-exposure barrier function and skin hydration were reduced in November. The increased irritant susceptibility in November may be attributed to reduced barrier function, reduced skin hydration, and/or absence of the beneficial effects of ultraviolet light on cellular targets beneath the stratum corneum. Flare responses to codeine, methacholine, substance P and trypsin were also increased in November compared with July, especially in AD patients. However, smaller flares were observed in AD patients than in control subjects, in both July and November. Flare values were negatively correlated with dermatitis severity, probably because of down-regulation. Weal responses did not show a clear seasonal variation. Hence, susceptibility to epicutaneous irritants and reactivity to intracutaneously injected bioactive agents are parameters which may be used to monitor season-dependent changes in non-specific skin reactivity.