Autoimmune blistering diseases: Histologic meaning

Clinics in Dermatology (2011) 29, 377–388

Autoimmune blistering diseases: Histologic meaning Jaka Radoš, MD ⁎ Department of Dermatology and Venereology, University Hospital Center Zagreb, School of Medicine, University of Zagreb, Salata 4, 10000 Zagreb, Croatia

Abstract The histologic picture of intraepidermal and subepidermal autoimmune bullous dermatoses is presented. Histologic changes are described according to the temporal evolution of lesions, with special reference to crucial elements of the histologic differential diagnosis. The diagnosis of autoimmune bullous dermatoses is complex, mostly requiring additional immunofluorescence assays along with histoclinical correlation to detect the antibodies or target antigen by the methods of molecular biology or immunohistochemistry. Additional tests to reach an accurate diagnosis in various autoimmune bullous dermatoses are briefly described, emphasizing the need of proper integration of all clinical and laboratory data. Although frequently inadequately specific, the histologic finding provides a link between clinical findings and target molecular studies in autoimmune bullous dermatoses. © 2011 Elsevier Inc. All rights reserved.

Introduction Bullous dermatoses are a heterogeneous group of skin diseases manifested by different clinical pictures, occasionally with overlapping features, but always with the occurrence of bullae as their major characteristic.1-14 The classification of bullous diseases is based on three morphologic characteristics: 1. anatomic level of the fissure—subcorneal, within the malpighian layer, suprabasal, subepidermal; 2. the mechanism responsible for the occurrence of bulla— spongiosis, acantholysis, ballooning degeneration of keratinocytes; and 3. inflammatory infiltration—density and composition of the infiltrate.1-14 Autoimmune bullous dermatoses are a heterogeneous group of diseases characterized by antibodies to structural ⁎ Corresponding author. E-mail address: [email protected] 0738-081X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2011.01.007

components of the skin and mucous membranes.1-14 The diagnosis is derived from the clinical picture (occasionally with signs typical for a particular bullous dermatosis)1-14 and histopathologic studies, 1-14 but it also requires detection of antibodies in tissues by direct immunofluorescence (DIF)1-15 or in circulation by indirect immunofluorescence (IIF),1-14,16 of the specific target antigen by enzyme-linked immunosorbent assay (ELISA)1-14,17,18 or Western blot.1-14,18,19 The new sophisticated ultrastructural and molecular techniques17-20 have enhanced the knowledge and understanding of the pathogenesis of bullous dermatoses with the description of new entities (eg, anti-p200 pemphigoid). These novel methods are not performed routinely but only at specialized laboratories. The classification of bullous dermatoses in general and of autoimmune bullous dermatoses still relies on histologic criteria, primarily according to the localization of bullae; so, intraepidermal and subepidermal bullous dermatoses are distinguished. The presence of acantholytic keratinocytes was considered a key element for the histologic diagnosis of

378 pemphigus. Acantholytic cells themselves, however, are neither pathognomonic for a particular disease nor an exclusive feature of intraepidermal bullous diseases. A significant clue in subepidermal bullous dermatoses is the proportion of inflammatory cells, such as neutrophils and eosinophils, located intralesionally or in adjacent dermis. Yet, morphologic lesions may not always be fully reliable; therefore, supplementary methods should be used. The involvement of similar molecular targets as target antigens for autoantibodies explains the morphologic and biologic overlapping between different bullous diseases, for example, bullous pemphigoid (BP) and linear immunoglobulin (Ig) A dermatosis. Definitive differentiation among various immunobullous dermatoses is highly important for different therapeutic modalities and disease prognosis.

J. Radoš such as herpes virus, but also as an effect of neutrophilreleased enzymes upon keratinocytes. 7 This effect is observed in subepidermal vesicular dermatitides where neutrophils prevail as well as in intraepidermal pustular dermatitides such as impetigo, pustular psoriasis, dermatophytoses, and suppurative folliculitides. Acantholytic cells are seen in acanthoses that are not strictly inflammatory such as genodermatosis (Darier disease), and its histopathologic simulators such as epidermal nevus, warty dyskeratoma, and the histologic variant of squamous cell carcinoma known as pseudoglandular squamous cell carcinoma. Accordingly, acantholytic cells are not a finding exclusive for pemphigus.7 The cytologic Tzanck test is used for fast demonstration of acantholytic epidermal cells.5

Pemphigus vulgaris

Intraepidermal bullous dermatoses Intraepidermal bullous diseases are characterized histopathologically by the occurrence of intraepidermal bullae or pustules.1,3,5-9 A great number of etiopathogenetically diverse diseases can produce intraepidermal bullae and thus present similar or even identical histologic findings.1,3,5-9 These diseases include hereditary intraepidermal bullous dermatoses (Hailey-Hailey disease, incontinentia pigmenti, and bullous ichthyosis), bacterial intraepidermal diseases (staphylococcal scalded skin syndrome [SSSS], and bullous impetigo), viral intraepidermal diseases (herpes simplex, herpes zoster, varicella, and hand-foot-and-mouth disease), various intraepidermal bullous dermatoses (dyshidrotic dermatitis, frictional blister, hydroa vacciniforme), and miliaria crystallina.1,3,5-7 Desmosomes as adhesion complexes may be damaged by various secondary phenomena, for example, after severe edema, either intercellular (spongiosis) or intracellular (ballooning degeneration in various viral infections).1,3,5-7 A histologic finding of intraepidermal bullae with the presence of acantholysis is the main histopathologic feature of autoimmune intraepidermal diseases from the pemphigus group; therefore, the diagnosis and differentiation of pemphigus from other intraepidermal bullous diseases mentioned will depend on the knowledge of appropriate clinical information and the results of immunofluorescence assays. The autoimmune intraepidermal bullous diseases include five defined entities from the pemphigus family: pemphigus vulgaris, pemphigus foliaceus (PF), pemphigus herpetiformis (PH), IgA pemphigus, and paraneoplastic pemphigus (PNP).1,5 In pemphigus vulgaris (PV), bullae occur due to the loss of keratinocyte cohesion (acantholysis, Greek akantha, thorn, and lysis, dissolution, decomposition), most probably consequential to interference to the desmosomal protein adhesion function by antidesmoglein antibodies.1,3,5-9,21,22 In pemphigus, acantholytic cells are not only present as an Ig and complement effect, or due to some infective agent

The fully developed PV lesions are characterized by the occurrence of an intraepidermal vesicle mostly located immediately above the basal layer of the epidermis, suprabasally (Figure 1).1,3,5-8 In some patients, however, bullae are also found in lower levels of the spinous layer.1,3,5-8 The location of PV bullae at these sites correlates well with desmoglein 3 (Dsg3) distribution in the epidermis.1,3,5-8 Acantholytic cells are round and have intensively eosinophilic cytoplasm, a pyknotic nucleus, and a perinuclear halo. Because there are no desmosomes in the basal membrane zone, basal keratinocytes remain attached distally to the basal membrane, but dividing laterally, thus forming a characteristic histologic picture known as “rows of tombstones.” The bullae contain serum and acantholytic keratinocytes. In PV, acantholysis frequently involves adnexal epithelium as well. Perivascularly and interstitially located mild to moderately dense lymphocyte infiltrates with eosinophils or neutrophils, or both, are found in the dermis. In mucous lesions, plasma cells are seen in the infiltrates.1,3,5-8

Fig. 1 Pemphigus vulgaris (hematoxylin-eosin stain, original magnification × 40): intraepidermal bulla containing acantholytic keratinocytes and dermal papillae are coated with a basal cell layer.

Autoimmune blistering diseases: histologic meaning In the early stage of bullae formation, vacuoles and small areas of acantholysis among keratinocytes are seen in the basal layer and lower levels of the spinous layer; their confluence results in the occurrence of fissures and then bullae.1,3,5-8 In the very early stage of PV, acantholysis is absent; however, eosinophilic or neutrophilic spongiosis is pronounced.1,3,5-8 Eosinophilic and neutrophilic spongiosis is not specific of PV because it is also found in various conditions, among them acute contact dermatitis, PF, BP, herpes gestationis (HG), drug eruption, and spongiotic reaction to insect bites.5 With lesion aging, the intralesional inflammatory infiltrate consists of neutrophils, lymphocytes, macrophages, and eosinophils. Erosions and ulcerations may occur due to the instable cover of the bullae. Older lesions may have several layers of keratinocytes at the base of the bulla due to keratinocyte migration and proliferation. Eventually, socalled villi are observed.1,3,5-8 The histopathology of pemphigus vegetans, a subtype of PV, is similar to that of PV; however, because it is chronic variant of PV, papillomatosis, acanthosis, and occasionally intraepidermal eosinophilic abscesses are observed. It usually occurs on the face and in the intertriginous areas, morphologically in the form of crusts, free from visible bullae.3,5-8 In case of mucosal involvement with PV, the lesions are histologically identical to skin changes. Any mucous membrane can be involved. Oral lesions are always present and can be the first manifestation of the disease.1,3,5-8 Evaluation of patients with exclusively oral lesions may frequently be hampered because intact bullae are difficult to obtain due to trauma or mastication, so biopsy specimens will only show erosions or ulcerations; therefore, biopsy specimens should preferably be obtained from the margin of a denuded area, where specific histologic alterations can then be detected.5 In patients with only oral lesions, the specimen should be obtained from intact oral mucosa for DIF assay, which is more sensitive than routine light microscopy study.5,23 Histologic study alone is not sufficient to make a precise diagnosis of PV. The diagnosis should be confirmed by DIF, IIF assay, or ELISA using Dsg1 and Dsg3 recombinant fusion proteins.1,3,5,8,15-19,24 The bullae in pemphigus rupture easily; therefore, it is important to obtain a biopsy specimen from an early lesion to make an accurate diagnosis.3 A biopsy specimen should also be obtained from a very small lesion (a bulla as a whole) so that the epidermis remains attached to the dermis. Punch biopsy should not be performed due to the shear moment and possible epidermis detachment from the dermis. Previous application of a cooling spray is recommended if a punch technique is used. A bulla can be excised with a scalpel. If no fresh bullae are available, then an old bulla can be moved into the adjacent skin by applying gentle perpendicular pressure. The newly formed fissure will then reveal early and specific histologic alterations.5

379

Pemphigus foliaceus The bullae in PF are very superficial and thus extremely fragile; therefore, an intact lesion is difficult to obtain in the biopsy specimen. Patients usually show erosions without bullae, thus leaving no clinical suspicion of bullous dermatosis. A fully developed PF lesion is characterized histopathologically by the occurrence of intraepidermal bulla within the granular layer and highest parts of the spinous layer.1,3,5-8 This feature correlates well with the intraepidermal distribution of Dsg1.1,3,5-8,25 The bullae contain serum, acantholytic keratinocytes, and occasionally, neutrophils may be present in abundance. Acantholytic cells are generally present in low numbers, which frequently requires a careful and thorough histologic study (Figure 2). A finding of dyskeratotic keratinocytes in the granular layer may prove useful in reaching the diagnosis.5 Diagnostically, the bullae or erosion lesions in PF may be very difficult and occasionally even impossible to differentiate from bullous impetigo or SSSS. Paradoxically, although superficial pemphigus is a bullous rather than a pustular dermatosis, numerous neutrophils can be found in the subcorneal space, thus contributing to the false impression of a basically pustular dermatosis.6 PV, eosinophilic, or neutrophilic spongiosis likewise may be present in the early stage of PF.1,3,5-8 The histopathology of pemphigus erythematosus is identical to that of PF.1,3,5-8 Fogo selvagem is an endemic form of PF in rural regions of South America, Brazil in particular.26 The term “superficial pemphigus” has been used

Fig. 2 Pemphigus foliaceus (hematoxylin-eosin stain, original magnification × 40): only a few acantholytic and dyskeratotic cells are seen on the surface of the eroded epidermis.

380

Fig. 3 Paraneoplastic pemphigus (hematoxylin-eosin stain, original magnification × 20) presents with a planus-like lesion, vacuolar degeneration of the basal layer, necrotic keratinocytes, and lichenoid infiltrate.

J. Radoš erythema exudativum multiforme (EEM) and lichen planus (Figure 3), shows interface dermatitis with a thinned or mildly hyperplastic epidermis and vacuolar degeneration of the basal membrane zone, frequently hypergranulosis, necrotic keratinocytes, and lymphocytes lined along the basal zone or individually in the epidermis, with dense, almost bandlike lymphocyte infiltrates in the upper dermis.1 The target molecules for autoantibodies in PNP generally include desmoglein and the plakin family proteins, including envoplakin, periplakin, plectin, desmoplakin 1 and 2, BP230, and as yet an incompletely characterized 170-kDa protein.19 The diagnosis of PNP cannot be established by histopathologic findings alone. A biopsy specimen showing some acantholysis and interface alterations appears to be relatively nonspecific. A definitive diagnosis requires a high level of suspicion by both the pathologist and the clinician, a positive search for tumors, and confirmation by IF and immunoblot studies.1,3,5-7,9,15-19

IgA pemphigus for the triad consisting of PF, pemphigus erythematosus, and fogo selvagem.7 Whether these three conditions are only variants of one pathologic process remains unclear.7 The lesions cannot be differentiated by conventional microscopy. As in PV, the diagnosis cannot be reached by histopathology alone, therefore, requiring IF assay and ELISA using Dsg1 and Dsg3 recombinant fusion proteins.1,3,5-8,15-19 PF may transform to PV, with modification in the clinical picture, histopathology, and antidesmoglein profile.27

IgA pemphigus is characterized by intercellular IgA deposits and clinically presented by pustules rather than

Paraneoplastic pemphigus Histologic features are variable in PNP, which clinically manifests by painful bullae on mucous membranes and ulcerations with polymorphous cutaneous lesions.1,3,5-9,28 When specimens obtained from skin lesions are evaluated, one must be aware that lesions of different clinical appearance will yield a different histologic picture. Overlapping of histologic patterns is seen in most lesions. The lesions may show a combination of features similar to erythema multiforme, lichen planus, pemphigus vulgaris, and pemphigoid.5,7 Histopathologically, PNP is a basically interface dermatitis associated with necrotic keratinocytes and only few acantholytic cells.3,29 A combination of vacuolar lesion of the basal layer and acantholysis is important for making the diagnosis.3,5-7,9 Three histopathologic patterns have been distinguished.1 In the first pattern, intraepidermal suprabasal acantholytic bullae are found, as in PV. The second pattern characteristic of PNP shows intraepidermal suprabasal acantholytic bullae combined with vacuolar degeneration of the basal layer. The third histologic pattern, otherwise not characteristic of PNP but potentially observed in other conditions, such as graft-versus-host disease (GVHD), drug eruption,

Fig. 4 Immunoglobulin A pemphigus (hematoxylin-eosin stain, original magnification × 40): a subcorneal deposit of neutrophils and acantholytic keratinocytes is seen in the bulla content and in the upper spinous layer.

Autoimmune blistering diseases: histologic meaning bullae or vesicles.1,3,5-8 Histologically, IgA pemphigus is characterized by the formation of intraepidermal bullae.1,3,5-8,30,31 According to the site of bulla occurrence, there are two variants of the disease: subcorneal pustular dermatosis (SPD; Figure 4) with the bullae located subcorneally,1,3,5-8 and intraepidermal neutrophilic IgA dermatosis, where bullae are located in the spinous layer.1,3,5-8 In both variants, the bullae contain acantholytic keratinocytes and a variable number of neutrophils. The epidermis is usually hyperplastic, with neutrophilic exocytosis. Perivascularly and interstitially located superficial infiltrates of lymphocytes, neutrophils, and occasionally, eosinophils are found in the upper dermis. As in PV, histologic pictures of neutrophilic spongiosis may be observed. Histologic pictures of IgA pemphigus are not diagnostic and can be seen in other diseases.1,3,5-8,30,31 Diagnosis should be confirmed by IF assay.1,3-7,29,30 Desmocolin 1 (Dsc1) is an autoantigen in the IgA pemphigus SPD variant. Dsc1 is limited to the granular layer and upper parts of the spinous layer and correlates well with the occurrence of bullae. In the intraepidermal neutrophilic variant, the autoantigens are Dsg1 and Dsg3.1

Pemphigus herpetiformis PH is a pemphigus variant with clinical characteristics similar to herpetiform dermatitis,1,3,5-8 although corresponding to pemphigus, usually foliaceus, by its histologic and IF findings.5 This variant has been differentiated primarily owing to its clinical picture, in which erythematous, pruriginous, papular, or vesicular lesions frequently show herpetiform distribution. Biopsy findings may be frequently variable and therefore nonspecific.32 Although eosinophilic spongiosis is most typical for this variant, neutrophilic spongiosis33 or mixed neutrophilic-eosinophilic spongiosis33 may also be found. Eosinophilic and neutrophilic spongiosis is also seen in early urticarial lesions.1 Intraepidermal vesicles or pustules, also of variable composition, as well as dermal papillary neutrophilic microabscesses, are frequently found. Acantholytic cells are usually but not necessarily identified. Multiple biopsy specimens may often be needed to reach the diagnosis.3 Fully developed PH lesions show intraepidermal bullae, in most cases located subcorneally. Occasionally, the bullae may be located suprabasally or in the spinous layer. As with other autoimmune diseases, diagnosis is made by correlating the clinical picture and histopathologic finding with an IF assay and ELISA results.1,3,5-8,32,33

381 membrane proteins, for example, epidermolysis bullosa, acquired autoimmune diseases, such as BP, cellular immunity-mediated disorders like erythema multiforme, and toxic epidermal necrolysis (TEN) metabolic disorders such as porphyria, and bullae that occur due to marked subepidermal edema, as seen in insect bites and acute dermal inflammatory processes (eg, Sweet syndrome).2,4-7 Unlike inborn subepidermal dermatoses, where there is no inflammatory infiltrate in the dermis, such infiltration is usually present in autoimmune bullous dermatoses.2,4-7 Subepidermal bullae may occur within the lamina lucida (LL) (eg, BP) or deep in the lamina densa (eg, epidermolysis bullosa acquisita [EBA]).2,4-7 Although the classification of subepidermal dermatoses is based on the new concepts from immunoelectroscopy and molecular studies, such techniques need not necessarily be used in the routine daily approach to patients with bullous dermatoses. The autoimmune subepidermal bullous dermatoses include BP, HG, lichen planus pemphigoides, cicatricial pemphigoid (CP), dermatitis herpetiformis, linear IgA disease, EBA, and anti-p200 pemphigoid.5 The pemphigoid group of autoimmune bullous diseases includes BP, pemphigoid gestationis, CP, linear IgA dermatosis, and lichen planus pemphigoides.2,4-7,34 Dermatitis herpetiformis and EBA have been considered separate entities since their first descriptions.2,4-7 Anti-p200 pemphigoid is a new entity that some authors have already classified as being in the group of subepidermal autoimmune bullous dermatoses.5

Subepidermal bullous dermatoses These diseases are characterized histopathologically by the occurrence of subepidermal bullae by a variety of mechanisms that include mutational defects of basal

Fig. 5 Bullous pemphigoid (hematoxylin-eosin stain, original magnification × 40): eosinophilic spongiosis with the formation of eosinophil intraepidermal abscess in the early stage of bullous pemphigoid development.

382

Bullous pemphigoid The histologic characteristics of BP depend to a certain extent on the age of the biopsied lesion. The specimens of early erythematous and urticarial lesions generally display dermal edema and a perivascular lymphohistiocytic infiltrate that contains a high number of eosinophils. Eosinophilic spongiosis may occasionally be present (Figure 5), and flame figures may be seen if eosinophils are present in abundance. Mild interface alterations can also be observed in early stages, along with vacuolar degeneration of basal cells.2,4-7 The fully developed BP lesion is characterized by the occurrence of subepidermal bullae2,4-7,10 that usually contain plasma, fibrin, and inflammatory cells (lymphocytes, eosinophils, and occasionally neutrophils).2,4-7,10 Cell-poor pemphigoid (noninflammatory forms of the disease with scant inflammatory infiltrate) may occasionally be observed in biopsy specimens obtained from the bullae located on noninflammatory altered skin. Such cases pose a differential diagnostic problem, especially if clinical data and IF results are not available.4 The bullae are frequently accompanied by moderately dense to dense infiltrates of lymphocytes, numerous eosinophils, and some neutrophils (cell-rich BP). In some cases, cell-rich BP neutrophils may predominate in the infiltrates and occasionally are found along the dermoepidermal junction, where vacuolar degeneration is observed. Neutrophils may also accumulate in the apices of dermal papillae, resulting in the formation of papillary neutrophilic abscesses as in dermatitis herpetiformis.2,4-7 The reason why some bullae are accompanied by dense inflammatory infiltrates remains obscure, but others are almost free from inflammatory cells.7 The classification of BP among subepidermal bullous dermatoses is only partially correct because the bullae in BP, and also in HG, may occasionally be found in the epidermis due to extensive spongiosis (Figure 6). 7 Irrespective of the eosinophil count in the early urticarial

Fig. 6 Bullous pemphigoid (hematoxylin-eosin stain, original magnification × 20): intraepidermal and subepidermal bullae are seen.

J. Radoš stage of BP, intraepidermal eosinophils are accompanied by spongiosis, which may be slight or very severe, with progression to vesicles. Briefly, BP and HG may represent intraepidermal or subepidermal bullous dermatosis.7 Intraepidermal bullae can also be found in older bullae (late biopsy) due to epidermal regeneration and epithelial migration.5 The epidermis covering the bulla usually is flat and may be necrotic. The bullae in BP are located in the LL area.2,4-7,10,35 Histopathology alone is inadequate to make an accurate diagnosis of BP, because similar histologic alterations can also be seen in other diseases, in particular, drug allergies and in response to insect bites. Cell-poor pemphigoid may occasionally be difficult to differentiate from EBA, porphyria cutanea tarda, and bullae caused mechanically such as by suction.7 By their typical presentation, BP and HG are relatively easily differentiated from herpetiform dermatitis (DH) and linear IgA dermatosis (LAD). The early and well-developed BP and HG lesions are predominated by eosinophils, whereas neutrophil infiltrates predominate in DH and LAD. In old DH and LAD bullae, however, eosinophils may prevail over neutrophils in dermal papillae and subepidermal bullae. A BP or HG bulla is difficult to differentiate from DH or LAD bulla in these situations.36 A finding of neutrophil nuclear dust seen on the bulla edges in DH and LAD but not in BP and HG can help.7 To reach an accurate diagnosis of BP, the histopathologic finding should be correlated with the results of IF assay with additional blister mapping and Western blot analysis.37 In NaCl split perilesional skin, IgG deposits are usually seen on the epidermal side of the fissure. Circulating antibodies to BP antigen (BPAG) 1 and BPAG2 can be detected by ELISA. On immunoblotting, these antibodies recognize BPAG1 in most cases, and BPAG2 less frequently.2,4-6,10,15-19

Herpes gestationis HG is an autoimmune bullous dermatosis from the bullous pemphigoid group that occurs in the second or third trimester of pregnancy and in puerperium.38 Current data suggest an autoimmune pathogenesis where hormones are also involved. The synonym for HG is pemphigoid gestationis.11 BP cannot be differentiated from HG in the urticarial or in bullous stage of the disease.4 The histopathology of HG is similar to cell-rich BP, showing subepidermal bullae with lymphocyte and eosinophil infiltrates.2,4-7,39 There is severe papillary dermal edema.5,40 The bullae usually contain plasma, fibrin, and inflammatory cells, including lymphocytes, eosinophils, and occasionally, neutrophils. The epidermis covering the bulla is flat or necrotic. Urticarial lesions in HG are histopathologically comparable with those in BP, showing perivascular and interstitial lymphocyte infiltrates with numerous eosinophils and some neutrophils on occasion.

Autoimmune blistering diseases: histologic meaning On blister mapping with antibodies to type IV collagen, the bulla is found in the LL region.2 Histopathology alone is inadequate to reach an accurate diagnosis; therefore, it should be confirmed by blister mapping, DIF, complement fixation assay by detection of the HG factor, which is actually an IgG-class antibody capable of complement activation by the classic pathway (ie, complement fixing anti-basement membrane antibody), ELISA (uncovering circulating IgG antibodies to BPAG 1 or BPAG2), and Western blot (circulating IgG antibodies to 180-kDa or 230-kDa protein bands).2,4-6,11,15-19 The routine test set includes histopathology and DIF. Dubious cases require additional testing such as IIF, ELISA, and immunoblotting.41 Clinical and laboratory criteria should both be used to make the diagnosis.41

383 changes typical for lichen planus, including a compact horny lesion, acanthotic epidermis, hypergranulosis, and bandlike lymphocyte infiltrate (Figure 7).43 Histopathology alone is not sufficient to make an accurate diagnosis of LPP. The diagnosis should be correlated with the clinical picture and the results of antigen mapping using antibodies to type IV collagen with bullae within LL and DIF (linear IgG deposits along the basement membrane (BM) zone in perilesional intact human skin and IgG deposits on the epidermal side of the bulla in perilesional salt-split skin), ELISA (circulating IgG antibodies to BPAG2), and Western blot (circulating IgG antibodies to 180-kDa protein bands).2,4,5,11,15-19 LPP may occur in childhood, and the histopathologic finding does not differ from the finding recorded in adults.44

Lichen planus pemphigoides Lichen planus pemphigoides (LPP) is a heterogeneous condition characterized by basal membrane antibodies to numerous antigens.4 The etiology of this bullous dermatosis remains obscure.42 It has been postulated that damage to basal keratinocytes in lichen planus might reveal hidden antigen determinants that lead to the occurrence of autoantibodies and bullous lesions. 42 LPP should be differentiated from vesicles that may occasionally appear in lichen planus due to severe hydropic degeneration of the basal layer (lichen planus vesiculosus).2,4-7,42 Histopathologic finding in LPP depends on the lesion obtained by the biopsy and mostly resembles lichen planus.5,42 If a specimen is obtained from a lesion on clinically healthy skin, it will show a subepidermal bulla with perivascular and interstitial lymphocyte and eosinophil infiltrate.2 The infiltrate may occasionally be scant, as in cell-poor BP.2 If a specimen is obtained from a bulla on a lichenoid lesion, there will be a subepidermal bulla with

Fig. 7 Lichen planus pemphigoides (hematoxylin-eosin stain, original magnification × 20): subepidermal bulla and changes characteristic of lichen planus are seen in a biopsy specimen obtained from a bulla on a lichenoid papule.

Cicatricial pemphigoid CP has numerous relatively well-differentiated clinicopathologic variants that have developed subsequent to autoimmune disease directed toward many different BM antigens.4 CP is a rare disease predominated by mucous lesions and characterized by scar formation.2,4-6 Ocular and oral lesions are most common; thus, many patients present to specialists of oral or dental surgery, or to ophthalmologists, rather than to dermatologists.2 A DIF finding of antibodies in perilesional skin is the gold standard for the diagnosis of CP with involvement of ocular mucosa. Definitive diagnosis is made by combining the clinical picture and a positive DIF result.45 In case of concurrent eye and skin involvement, a skin biopsy specimen should be obtained first.45 No mucosal lesions are present in the Brunsting-Perry variant, but areas with recurrent eruption of bullae that heal with atrophic scar formation are seen on the head and neck skin. This variant is characterized histopathologically by fibrosis.5 There is no histopathologically unique CP pattern.5 Three histologic patterns have been mentioned.2 In the first, there are subepidermal bullae and inflammatory infiltrate mostly composed of lymphocytes, along with the possible presence of eosinophils and neutrophils. Plasma cells are found in mucosal lesions (oral and genital). The second pattern shows subepidermal bullae and fibrosis that varies from mild to moderate, but the inflammatory cell infiltrates are of lower density compared with the first pattern. The third pattern includes subepidermal bullae, fibrosis, and bandlike lymphocyte and eosinophil infiltrates with a variable presence of neutrophils and plasma cells.2 Epithelial lesions resemble those seen in lichen planus.2 Histopathologically, CP may frequently be indistinguishable from BP and was considered a BP variant in the past.2,4,5 Some authors, however, note clinical, histopathologic, immunologic, and biologic differences between BP and CP.7 Clinically, CP frequently involves the mucous membrane, whereas histopathologically, the bullae extend

384 along the epithelial structures. Inflammatory infiltrates reveal more neutrophils than eosinophils, and fibroplasia is observed in the upper dermis, in contrast to BP, which is characterized by eosinophil predominance, without involvement of the epithelial structures and fibrosis.7 Unlike, BP, CP may be a devastating disease leading to blindness46 or gastrointestinal tract strictures. The autoantigen responsible for CP has not yet been identified. The known autoantigens are epiligrin (also known as laminin 5), α6β4 integrin-β4 subunit, BPAG2, and BPAG1. All of these antigens are found within LL.5 CP esions are not diagnostically specific by histopathology and can also be seen in other diseases,2 and therefore, CP cannot be diagnosed by histopathology alone. To confirm the diagnosis, histologic finding should be correlated with the clinical picture, IF results (linear IgG deposits along BM zone in intact perilesional skin, and IgG deposits along the epidermal, dermal or both sides of the fissure in split skin), blister mapping (bulla within LL), Western blot, immunoprecipitation (circulating antibodies to several antigens), and ELISA (circulating antibodies to BPAG1 or BPAG2).2,4-6,15-19 CP is not currently considered a disease entity but is a term unifying a number of diseases with a similar clinical phenotype and cicatrices, and with predilection for mucous membranes and conjunctiva, and for the occurrence of subepidermal bullae. Many cases described in early literature as CP without appropriate immunologic studies should better be classified as LAD or EBA that primarily involve mucous surfaces.5

Dermatitis herpetiformis Since the very first description of the disease by Duhring, dermatitis herpetiformis (DH) has been described as a separate disease entity.2 In his book published in 1925, Kyrle was the first to describe and draw typical histopathologic DH lesions with a characteristic description of neutrophils in dermal papillae and subepidermal spaces.7 Classic finding includes neutrophil deposits47 or leukocytoclasia in dermal papillae with the formation of subepidermal fissure. If subepidermal cleft is not overtly pronounced, the finding is only suggestive of DH.48 Early DH lesions are characterized by superficial perivascular and interstitial lymphocyte and neutrophil infiltrates, and edematous papillary dermis. Neutrophils are also present in the dermis and along BM zone, with visible vacuolar degeneration. Neutrophil nuclear dust may also be observed. With progression of the lesion, neutrophils are collected in some dermal papillae, with the occurrence of fibrin in papillary tips. Greater neutrophil accumulation results in abscesses in dermal papillae, usually with subepidermal fissures above them (Figure 8). Eosinophils can also be found in inflammatory infiltrates as well as in papillary abscesses. With further lesion progression, subepidermal fissures transform to

J. Radoš

Fig. 8 Dermatitis herpetiformis (hematoxylin-eosin stain, original magnification × 20): subepidermal bulla, neutrophil abscesses are seen in the tips of dermal papillae and subepidermal clefts.

subepidermal bullae, which represent the histopathologic finding of a fully developed lesion. The bullae generally contain plasma, fibrin, neutrophils, and eosinophils. The epidermis over the bulla is flat and may be necrotic. Lymphocyte, neutrophil, and eosinophil infiltrates are present in the dermis. The number of eosinophils increases with lesion age.2,4-7,14 Because the lesions are pruritic, they frequently are excoriated, thus the histologic finding being nonspecific.49 Therefore, an early papule, papulovesicle, or a small bulla with healthy appearing skin around it should be used for histopathologic study. Histopathology is inadequate for making the diagnosis because similar histopathologic changes are also found in other bullous dermatoses such as linear IgA, EBA, or bullous form of systemic lupus erythematosus.2,4-7,14 Diagnosis should be confirmed by DIF, which reveals granular IgA deposits in dermal papillae. Linear IgA deposits along BM zone are seen in some patients. The serum test for endomysial or tissue transglutaminase antibodies, or both, is positive.2,4-7,15-19 In DH, there is no evidence for circulating antibodies to keratinocyte or BM zone proteins on IIF.2

Linear IgA disease Childhood linear IgA disease (chronic bullous dermatosis of childhood) is identical to bullous dermatosis in adults (adult type linear IgA dermatosis); however, there are differences in clinical presentation and therefore should be described as separate entities. Similar to the clinical picture, the histopathologic picture of linear IgA disease is also heterogeneous.2,4-7,12,50-52 Histologic characteristics are similar if not even identical to dermatitis herpetiformis.5 According to some authors, there is a low tendency to the formation of papillary

Autoimmune blistering diseases: histologic meaning

385 Histopathologic study is not of diagnostic value, because similar lesions are also seen in other conditions. Perilesional skin or mucosa DIF reveals linear IgA deposits along BM zone, whereas circulating IgA antibodies to BM proteins are demonstrated by IIF. Using NaCl split skin, the antibodies bind to the epidermal side of the fissure in most cases. Studies using Western immunoblot indicate that LAD is a heterogeneous condition. Dermal antigens include 285-kDa and 250-kDa proteins and type VII collagen. The epidermally bound antibodies react with BP230, BP180, and 200/280-kDa antigens, which are different from BP antigens.2,4-6,12,15-19 The 120-kDa (LAD1) and 97-kDa antigens described in early literature are proteolytic products of BP180.4

Fig. 9 Linear immunoglobulin A dermatosis (hematoxylin-eosin stain, original magnification × 20): subepidermal bulla, neutrophils, eosinophils, and fibrin are seen in the bulla content; marginally, there is a linear arrangement of neutrophils along the dermoepidermal junction and vacuolar degeneration of the basal layer.

microabscesses and high tendency to uniform neutrophil infiltrates along the dermoepidermal junction.7 Fully developed lesions show subepidermal bullae with lymphocyte, eosinophil, and neutrophil infiltration. The infiltrate is superficial, perivascularly and interstitially located, and visible at the base and edges of the bulla. In most cases, neutrophils prevail over eosinophils in the infiltrate. Neutrophils are usually found in linear deposits along BM zone, which shows vacuolar degeneration (Figure 9). Neutrophils are also seen in the epidermis and in some dermal papillae, in particular, adjacent to bullae. They may form papillary neutrophilic abscesses as in DH. Occasionally, only neutrophil abscesses and subepidermal fissures over them are present. The abscesses may contain eosinophils. In some cases of fully developed LAD lesions, eosinophils predominate in the infiltrates. In the mucous forms of LAD, lesions similar to those found in chronic forms are present, however, usually with a greater number of plasma cells. Ocular LAD lesions cannot be differentiated histologically from CP.2 In early stages, there is vacuolar degeneration of BM zone with neutrophils lined along the zone. With disease progression, subepidermal bullae and eventually fibrosis occur. The early urticarial LAD lesions show perivascular and interstitial lymphocyte infiltrates with some neutrophils and eosinophils. Neutrophils usually prevail, however, eosinophils may occasionally predominate. Vacuolar degeneration of BM zone is also present. Eosinophilic or neutrophilic spongiosis (or their combination) may be seen.2,4-7,12 The most typical histologic picture is the occurrence of subepidermal bulla with a mixed neutrophil and eosinophil infiltrate in dermal papillae, with a characteristic subepidermal neutrophil deposition in the form of elongated line in some areas.51

Epidermolysis bullosa acquisita EBA is a rare, chronic, bullous dermatosis characterized by numerous clinical presentations; thus, it may be mistaken for some other dermatosis. The diagnosis of EBA has for years been based on the exclusion of all other recognizable bullous dermatoses including PCT, BP, DH, pemphigus, EM, and bullous drug reactions.2,4-7,13 Histopathologically, fully developed EBA lesions are characterized by the occurrence of subepidermal bullae.2,4-7,13 In the classic variant of EBA, the bullae are associated with scant perivascular and interstitial lymphocyte infiltration in the upper dermis. Some eosinophils or neutrophils are occasionally present (Figure 10). Fibrosis or fibrosis and milia are found in recurrent lesions (Figure 11). In the inflammatory variant of EBA, the bullae are associated with moderately dense lymphocyte infiltrate that also contains eosinophils and neutrophils. The infiltrate is visible at the base and edges of the bulla. In some patients, eosinophils predominate in inflammatory infiltrates; this EBA, which is similar to BP, is the most common form of EBA.5 In some other lesions, neutrophils predominate and may form papillary neutrophilic abscesses as in DH. Occasionally, infiltrates may consist mostly of lymphocytes. In the noninflammatory form, the bullae are free from inflammatory infiltrate at the sites of trauma (eg, elbows and knees). In the inflammatory form that occurs irrespective of trauma, the bullae are accompanied by inflammatory infiltrates with numerous neutrophils. Neutrophils or neutrophil bands or nuclear dust are sometimes present along the dermoepidermal junction or in dermal papillae, thus preventing differentiation from DH or LAD.7 The mucous form shows a similar histopathologic finding as that seen in the inflammatory form of EBA, only the infiltrate is usually denser and contains more plasma cells. The histopathologic finding of ocular EBA cannot be differentiated from ocular CP or ocular LAD.2 When antibodies to type IV collagen were used, the bullae were located in the sublamina densa in all EBA variants.2 Early urticarial lesions in the inflammatory forms of EBA cannot be differentiated histologically from urticarial

386

Fig. 10 Epidermolysis bullosa acquisita (hematoxylin-eosin stain, original magnification × 40): inflammatory form of the disease presents with medium-dense lymphocyte infiltrate and a few neutrophils and eosinophils.

lesions in other autoimmune bullous dermatoses and show perivascular and interstitial lymphocyte, neutrophil, and eosinophil infiltrates.2,4-7,13 Eosinophils and neutrophils may predominate in the infiltrates and be present along

Fig. 11 Epidermolysis bullosa acquisita (hematoxylin-eosin stain, original magnification × 40): a part of milia and adjacent fibrosis from recurrent lesion biopsy are seen.

J. Radoš BM zone, which in turn shows vacuolar degeneration.2 Eosinophilic or neutrophilic spongiosis may also be present.2 The histopathologic finding is not pathognomonic of EBA because it may also be recorded in many other diseases.2,4-7,13 The diagnosis of EBA can be verified in correlation with the clinical picture and results of IF and blister mapping.15-19 Identification of the target autoantigen by Western blot or immunoblot techniques, or by ELISA with the use of recombinant fusion proteins of type VII collagen, is currently available only in few laboratories.53 DIF demonstrates linear IgG deposits along the BM zone. Deposits of IgG, IgA, and IgM are sometimes present together. IgG deposits are found on the dermal side of perilesional NaCl split skin or mucosa, whereas autoantigen (type VII collagen) is present in anchoring fibrils extending from the lamina densa to the papillary dermis. In EBA, circulating IgG antibodies react in Western blot or immunoprecipitation with the 290-kDa protein band representing type VII collagen.53 Histopathologic criteria are neither reliable nor consistent. The diagnosis of EBA can only be verified by electron microscopy. The intensity of inflammation observed by the clinician usually correlates with the amount of inflammatory infiltrate in the dermis.54 The noninflammatory or inflammatory cell-poor forms of disease may be reminiscent of porphyria or other types of EB and inflammatory forms of DH or LAD. Briefly, when encountering subepidermal bullous dermatosis that is cell-poor or cell-rich with neutrophils, a histopathologist should consider EBA and be aware that reaching an accurate diagnosis still requires other methods, such as electron microscopy, and immunoelectron microscopy in particular,55, which is the diagnostic gold standard because the diagnosis cannot be reliably set by conventional microscopy.

Anti-p200 pemphigoid Anti-p200 pemphigoid is a separate subepidermal bullous dermatosis with clinical features of BP, dermatitis herpetiformis, or linear IgA bullous dermatosis.5 It was first described in 1996, with about 100 patients reported to date.19 Patients most commonly present with generalized eruptions of urticarial papules or plaques in combination with tight bullae strongly resembling BP.5 On histopathology, dermal papillary microabscess and some eosinophils are observed.56,57 Whether the presence of eosinophils is associated with the age of the bulla used for the specimen is unknown.5 Histopathologic lesions may be reminiscent of those seen in LAD or DH, with subepidermal bullae and superficial inflammatory infiltrate predominated with B neutrophils.56,57 IF reveals linear IgG and C3 deposits along the BM. 5,15-19 Antibodies are directed against 200-kDa glycoprotein in the LL.5,19 Target antigen in p200 remains unknown in spite of continuous efforts of the researchers. There is evidence for laminin γ1 to be the autoantigen in this disease.58 It is important to recognize this

Autoimmune blistering diseases: histologic meaning entity because it shares comparable histologic and IF features with EBA. Unlike EBA, anti-p200 pemphigoid has a limited course and resolves rapidly on immunosuppressant therapy, without leaving scars.5

Conclusions From the dermatopathologist's point of view, the diagnosis of bullous dermatoses is extremely complex. On making the diagnosis, the dermatopathologist should integrate all information on the clinical picture, patient age, and localization of lesions with history data and microscopy finding. On doing this, he or she should be aware of the limitations in reaching an accurate diagnosis due to the inadequately investigated pathogenesis of bullous dermatoses on the one hand and technical imperfections, such as inappropriate biopsy specimens, on the other hand. Identification of the mechanism of bulla formation (eg, spongiosis, acantholysis, subepidermal edema, basal layer vacuolization, etc.), anatomic localization of the fissure or bulla in the skin (intraepidermal or subepidermal), and analysis of the cellular inflammatory infiltrate in the dermis can be of considerable help in proper disease classification. In daily routine, various subepidermal bullous diseases can be differentiated by the nature of the dermal infiltrate. In PNP and lichen planus pemphigoides, there are subepidermal bullae with a predominant finding of lymphocytes in the inflammatory infiltrate. A finding of eosinophils in the inflammatory infiltrate is significant in BP and HG, and that of neutrophils in DHD, LAD, and EBA. Many bullous diseases, the subepidermal varieties in particular, can have a similar or even identical histologic finding, which poses a major diagnostic problem. In addition, histopathologic finding varies with time; therefore, it is of utmost importance to obtain biopsy specimen from a fresh lesion to choose an appropriate biopsy technique. In the case of epidermis regeneration or secondary alteration, such as infection or ulceration, reaching an accurate diagnosis is quite questionable. In some bullous dermatoses, the use of special techniques like DIF, split-skin immunofluorescence, or electron microscopy is necessary. If some key elements for setting the diagnosis are not available, histopathology can only point to a descriptive diagnosis, such as “subepidermal vesicular dermatitis with neutrophil predominance,” while in addition offering a differential diagnosis. With the new research techniques and sophisticated laboratory methods now available, great advances have been made in understanding the pathophysiology of bullous diseases, with implications in diagnostic accuracy and patient therapy. Histopathology will, however, always make a bridge between the patient and science (focusing on molecular studies) and provide a basis for the interdisciplinary approach to bullous dermatoses.

387

References 1. Megahed M. Intraepidermal blistering diseases. In: Megahed M, editor. Histopathology of blistering diseases with clinical, electron microscopic, immunological and molecular biological correlations. Berlin Heidelberg: Springer-Verlag; 2004. p. 49-153. 2. Megahed M. Subepidermal blistering diseases. In: Megahed M, editor. Histopathology of blistering diseases with clinical, electron microscopic, immunological and molecular biological correlations. Berlin Heidelberg: Springer-Verlag; 2004. p. 155-275. 3. Mc Kee PH, Calonje JE, Granter SR. Acantholytic disorders. In: Mc Kee PH, Calonje JE, Granter SR, editors. Pathology of the skin with clinical correlations. Boston: Elsevier Mosby; 2005. p. 139-71. 4. Mc Kee PH, Calonje E, Granter SR. Inherited and autoimmune subepidermal blistering diseases. In: Mc Kee PH, Calonje E, Granter SR, editors. Pathology of the skin with clinical correlations. Boston: Elsevier Mosby; 2005. p. 81-139. 5. Hong W, Brandling-Bennett HA, Harrist TJ. Noninfectious vesiculobullous and vesiculopustular disease. In: Elder D, Elenitsas R, Jaworosky C, Johnson B, editors. Lever's histopathology of the skin. 10th ed. Philadelphia: Lippincott-Raven; 2010. p. 246-67. 6. Weedon D. The vesiculobullous reaction pattern. In: Weedon D, editor. Skin pathology. 2nd ed. Edinburgh, London, New York: Elsevier Science, Churchill Livingstone; 2002. p. 129-91. 7. Ackerman AB, Chongchitnant N, Sanchez J, et al. Histologic diagnosis of inflammatory skin diseases: an algorithmic method based on pattern analysis. Baltimore: Williams & Wilkins; 1997. 8. Stanley JR. Pemphigus. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's Dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 459-68. 9. Anhalt GJ, Nousari CH. Paraneoplastic pemphigus. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 468-74. 10. Stanley JR. Bullous pemphigoid. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 475-85. 11. Shornick JK. Pemphigoid gestationis (herpes gestationis). In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 490-3. 12. Rao CL, Hall III RP. Linear immunoglobulin A dermatosis and chronic bullous disease of childhood. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 485-90. 13. Woodley DT, Chen M. Epidermolysis bullosa acquisita. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 494-500. 14. Hall III RP, Katz SI. Dermatitis herpetiformis. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 500-5. 15. Morrison LH. Direct immunofluorescence microscopy in the diagnosis of autoimmune bullous dermatoses. Clin Dermatol 2001;19:607-13. 16. Kanitakis J. Indirect immunofluorescence microscopy for the serological diagnosis of autoimmune blistering skin disease: a review. Clin Dermatol 2001;19:614-21. 17. Zillikens D. Diagnosis of autoimmune bullous skin diseases. Clin Lab 2008;54:491-503. 18. Harman KE. New laboratory techniques for the assessment of acquired immunobullous disorders. Clin Exp Dermatol 2002;27:40-6. 19. Schmidt E, Zillikens D. Modern diagnosis of autoimmune blistering skin disease. Autoimmun Rev 2010;10:84-9. 20. Ishiko A, Shimizu H. Electron microscopy in diagnosis of autoimmune bullous disorders. Clin Dermatol 2001;19:631-7. 21. Hertl M. Autoimmune bullous skin disorders. In: Hertl M, editor. Autoimmune disease of the skin. Pathogenesis, diagnosis, management. 2nd revised, enlarged ed. Wien, New York: Springer; 2005. p. 45-69.

388 22. Kitajima Y. Current and prospective understanding of clinical classification, pathomechanisms and therapy in pemphigus. Arch Dermatol Res 2003;295:S17-23. 23. Scully C, Mignogna M. Oral mucosal disease: pemphigus. Br J Oral Maxillofac Surg 2008;46:272-7. 24. Nousari HC, Anhalt GJ. Pemphigus and bullous pemphigoid. Lancet 1999;354:667-72. 25. Dasher D, Rubenstein D, Diaz LA. Pemphigus foliaceus. Curr Dir Autoimmun 2008;10:182-94. 26. Culton DA, Qian Y, Li N, Rubenstein D, Aoki V, Filhio GH. Advances in pemphigus and its endemic pemphigus foliaceus (fogo selvagem) phenotype: a paradigm of human autoimmunity. J Autoimmun 2008; 31:311-24. 27. Park SG, Chang JY, Cho YH, Kim SC, Lee MG. Transition from pemphigus foliaceus to pemphigus vulgaris: case report with literature review. Yonsei Med J 2006;47:278-81. 28. Seghal VS, Srivastava G. Paraneoplastic pemphigus/paraneoplastic autoimmune multiorgan syndrome. Int J Dermatol 2009;48:162-9. 29. Zhu X, Zhang B. Paraneoplastic pemphigus. J Dermatol 2007;34: 503-11. 30. Kopp T, Sitaru C, Pieczkowski F, et al. IgA pemphigus—occurrence of anti-desmocollin 1 and anti-desmoglein 1 antibody reactivity in an individual patient. J Dtsch Dermatol Ges 2006;12:1045-50. 31. Petropoulou H, Politis G, Panagakis P, Hatziolou E, Aroni K, Kontochristopoulos G. Immunoglobulin A pemphigus associated with immunoglobulin A gammopathy and lung cancer. J Dermatol 2008;35: 341-5. 32. Wosnitza M, Blazek C, Megahed M. Pemphigus herpetiformis. Hautarzt 2008;59:459-60. 33. Duarte IB, Bastazini Jr I, Barreto JA, Carvalho CV, Nunes AJ. Pemphigus herpetiformis in childhood. Pediatr Dermatol 2010;27:488-91. 34. Olasz EB, Yancey KB. Bullous pemphigoid and related subepidermal autoimmune blistering diseases. Curr Dir Autoimmun 2008;10:141-66. 35. Patricio P, Ferreira C, Gomes MM, Filipe P. Autoimmune bullous dermatoses: a review. Contemporary challenges in autoimmunity. Ann N Y Acad Sci 2009;1173:203-10. 36. Verdolini R, Cerio R. Autoimmune subepidermal bullous skin disease: the impact of recent findings for the dermatopathologist. Virchows Arch 2003;443:184-93. 37. Laffitte E, Borradori L. Bullous pemphigoid: clinical features, diagnostic markers, and immunopathologic mechanisms. In: Hertl M, editor. Autoimmune disease of the skin. Pathogenesis, diagnosis, management. 2nd ed (revised, enlarged). Wien, New York: Springer; 2005. p. 71-93. 38. Cobo MF, Santi CG, Maruta CW, Aoki V. Pemphigoid gestationis: clinical and laboratory evaluation. Clinics (Sao Paulo) 2009;64:1043-7. 39. Engineer L, Bhol K, Ahmed R. Pemphigoid gestationis: a review. Am J Obstet Gynecol 2000;183:483-91. 40. Bedocs PM, Kumar V, Mahon MJ. Pemphigoid gestationis: a rare case and review. Arch Gynecol Obstet 2009;279:235-8. 41. Semkova K, Black M. Pemphigoid gestationis: current insights into pathogenesis and treatment. Eur J Obstet Gynecol Reprod Biol 2009;145:138-44.

J. Radoš 42. Pittelkow MR, Daoud MS. Lichen planus. In: Wolff K, Goldsmith LA, Katz SI, editors. Fitzpatrick's dermatology in general medicine. 7th ed. New York, Chicago: McGraw Hill; 2007. p. 244-55. 43. Willsteed E, Bhogal BS, Das AK, Wojnarowska F, Black MM. Lichen planus pemphigoides: a clinicopathological study in nine cases. Histopathology 1991;19:147-54. 44. Cohen DM, Ben-Amitai D, Feinmesser M, Zvulunov A. Childhood lichen planus pemphigoides: a case report and review of the literature. Pediatr Dermatol 2009;26:569-74. 45. Scmidt E, Meyer-ter-Vehn T, Zillikens D, Geerling G. Mucous membrane pemphigoid with ocular involvement. Part I: Clinical manifestations, pathogenesis and diagnosis. Ophthalmologe 2008; 105:285-98. 46. Rashid S, Dana MR. Cicatrizing and autoimmune diseases. Chem Immunol Allergy 2007;92:195-202. 47. Egan CA, O'Loughlin S, Gormally S, Powell FC. Dermatitis herpetiformis: a review of fifty-four patients. Ir J Med Sci 1997;166: 241-4. 48. Alonso-Llamazares J, Gibson LE, Rogers III RS. Clinical, pathologic, and immunopathologic features of dermatitis herpetiformis: review of the Mayo Clinic experience. Int J Dermatol 2007;46:910-9. 49. Rose C, Zillikens D. Dermatitis herpetiformis Duhring. In: Hertl M, editor. Autoimmune disease of the skin. Pathogenesis, diagnosis, management. 2nd ed. (revised, enlarged). Wien, New York: Springer; 2005. p. 95-108. 50. Sobjanek M, Sokolowska-Woydylo M, Szaba-Kania M, BarañskaRybak W, Maciejewska A, Wlodarkiewicz A. Clinical and immunopathological heterogeneity of 22 cases of linear IgA bullous dermatosis. Eur Acad Dermatol Venereol 2008;22:1131. 51. Guide SV, Marinkovich MP. Linear IgA bullous dermatosis. Clin Dermatol 2001;19:719-29. 52. Dippel E, Orfanos CE, Zouboulis C. Linear IgA dermatosis presenting with erythema annulare centrifugum lesions: report of three cases in adults. J Eur Acad Dermatol Venereol 2001;15:167-70. 53. Ishii N, Hamada T, Dainichi T, et al. Epidermolysis bullosa acquisita: what's new? J Dermatol 2010;37:220-30. 54. Remington J, Chen M, Burnett J, Woodley DT. Autoimmunity to type VII collagen: epidermolysis bullosa acquisita. Curr Dir Autoimmunn 2008;10:195-205. 55. Chen M, Hallel-Halevy D, Nadelman C, Woodley D. Epidermolysis bullosa acquisita. In: Hertl M, editor. Autoimmune disease of the skin. Pathogenesis, diagnosis, management. 2nd ed (revised, enlarged). Wien, New York: Springer; 2005. p. 109-31. 56. Rose C, Weyers W, Denisjuk N, Hillen U, Zillikens D, Shimanovich I. Histopathology of anti-p200 pemphigoid. Am J Dermatopathol 2007;29:119-24. 57. Dilling A, Rose C, Hashimoto T, Zillirens D, Shimanovich I. Anti-p200 pemphigoid: a novel autoimmune subepidermal blistering disease. J Dermatol 2007;34:1-8. 58. Dainichi T, Koga H, Tsuji T, et al. From anti-p200 pemphigoid to antilaminin γ1 pemphigoid. J Dermatol 2010;37:231-8.