Dermatology: Practical and Conceptual Review | Dermatol Pract Concept. 2022;12(2):e2022116 1 Dermatology Practical & Conceptual Introduction The skin is the largest organ of the body, protecting us from the environment, regulating body temperature and permit- ting the sensation of touch. It is divided into 3 main lay- ers: the outermost epidermis layer, the middle dermis layer and the lower hypodermis layer [1]. The epidermis is mainly made up of keratin producing cells called keratinocytes. The protective barrier, regulation of epidermal temperature and nutrients and other functions of the epidermis are dependent upon the maintenance of stable connections between kerat- inocytes and other epidermal structures primarily mediated by adhesive desmosomal and hemidesmosomal proteins [2]. Desmosomes are specialized tight junctions critical to cellular adhesion (Figure 1). They are arranged on adja- cent sides of plasma membranes and can be seen in tissues Serological Biomarkers and Their Detection in Autoimmune Bullous Skin Diseases Ilana Heckler1, Michael Hong2,3, Animesh Amart Sinha3, Iswariya Venkataraman1 1 EUROIMMUN US Inc, 1 Bloomfield Ave, Mountain Lakes, New Jersey, United States 2 College of Medicine, Florida State University, Tallahassee, Florida, United States 3 Department of Dermatology, Jacobs School of Medicine, University at Buffalo, Buffalo, New York, United States Key words: diagnostics, pemphigoid, pemphigus, antibodies Citation: Heckler I, Hong M, Sinha A, Venkataraman I. Serological Biomarkers and Their Detection in Autoimmune Bullous Skin Diseases, Dermatol Pract Concept. 2022;12(2):e2022116. DOI: https://doi.org/10.5826/dpc.1202a116 Accepted: November 22, 2021; Published: April 2022 Copyright: ©2022 Heckler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing interests: None. Authorship: All authors have contributed significantly to this publication Corresponding author: Iswariya Venkataraman, Ph.D., EUROIMMUN US, 1 Bloomfield Ave, Mountain Lakes, New Jersey, United States. E-mail: iswariya.venkataraman@euroimmun.us Autoimmune bullous diseases (AIBDs) are a group of skin-related disorders that involve damage to structures maintaining cell-cell adhesion, such as desmosomes and hemidesmosomes. Key AIBDs include pemphigus related diseases, pemphigoid related conditions, acquired epidermolysis bullosa (EBA), and dermatitis herpetiformis (DH). Each group of conditions exhibits characteristic clinical lesion patterns and is associated with specific autoantibodies targeting epidermal and dermal struc- tures involved in cell-cell adhesion and skin integrity. Pemphigus diseases primarily target desmoglein (Dsg) 3 and Dsg1 proteins but several non-Dsg autoantibodies have also been linked to pemphigus. Pemphigoid diseases typically target bullous pemphigoid (BP)180 and BP230; EBA is associated with antibodies directed against anti-type VII collagen and DH by IgA autoantibodies against tis- sue transglutaminase and deaminated gliadin. Investigation into the serological biomarkers found in AIBDs have allowed the development of diagnostic assessments (i.e. tissue antibody detection and serological testing) based on the unique autoantibody profiles of a particular disease group. The meth- ods for the detection and quantification of disease-associated autoantibodies continue to evolve and improve. ABSTRACT 2 Review | Dermatol Pract Concept. 2022;12(2):e2022116 including cardiac muscle, gastrointestinal mucosa, and epi- thelia, all of which can be subject to significant mechanical stress during normal physiological and disease states [3]. Within desmosomes, there is a vast network of cadherin proteins (desmogleins, desmocollins and desmoplakins), linker proteins (eg plakoglobin, plakophilin) and keratin intermediate filaments, that connect as desmosome-inter- mediate filament complexes (DIFCs). Desmoplakin, which coordinates other cadherin proteins and keratin filaments, is the most prevalent protein within the desmosome [4]. Hemidesmosomes resemble tiny stud-shaped structures and are similar in shape to desmosomes. However, there are several differences between these 2 structural components. Hemidesmosomes attach keratinocytes to the extracellular matrix and utilize integrins rather than desmogleins and des- mocollins. Key hemidesmosomal-associated proteins include the cytoplasmic protein BP230, the transmembrane protein BP180, laminin 332 and collagen type VII [5]. Another fea- ture of hemidesmosomes is their role in signaling pathways, relevant for the migration of keratinocytes (Figure 1) [6]. Autoimmunity involves the presence of antibodies (pro- duced by B lymphocytes) and T lymphocytes that have escaped mechanisms of self-tolerance, both centrally and peripherally, that are reactive to one’s own self-antigens. When auto-reactive lymphocytes cause enough target tissue damage, autoimmune disease can occur. There are over 80 human autoimmune diseases in ex- istence, affecting over 20 million Americans [7]. Indicators of autoimmunity (ie antinuclear antibodies) suggest that the incidence of autoimmune disease has been increasing over the past few decades [8]. Collectively, autoimmune diseases present a tremendous, and likely under-estimated burden on healthcare costs: over $100 billion annually in the United States [9]. These costs Figure 1. Graphical representation of human skin. Desmosomes connect adjacent keratinocytes and are critical to cellular adhesion (top circle within desmosomes: there is a vast network of cadherin proteins [desmogleins, desmocollins and des- moplakins]), linker proteins [eg plakoglobin, plakophilin] and keratin intermediate filaments). Hemidesmosomes facilitate the adhesion of basal keratinocytes to the underlying basal lamina. Key hemidesmosomal-associated proteins include the cytoplasmic protein BP230, the transmembrane protein BP180, laminin 332 and collagen type VII. Review | Dermatol Pract Concept. 2022;12(2):e2022116 3 reflect a multitude of factors which are impacted by delays in diagnosis, poor or infrequent monitoring of disease activity leading to less than optimal disease management. Systemic autoimmune diseases affect multiple organs, whereas organ-specific diseases target a single organ such as the skin. Autoimmune diseases that affect the skin include vitiligo, scleroderma, lupus, psoriasis, vasculitis, and autoim- mune bullous dermatoses (AIBDs). AIBDs are a collection of autoimmune skin specific disease characterized by the pro- duction of autoantibodies against structural components of the skin including desmosomes and hemidesmosomes [10]. Such an autoimmune reaction interferes with intercellular connections within the epidermis in addition to the crucial linkage between the epidermis and the dermis. AIBDs man- ifest as skin layer separation and blistering and are divided into 4 main groups according to their target antigens and localization of the blisters: pemphigus diseases, pemphigoid diseases, acquired epidermolysis bullosa (EBA), and dermati- tis herpetiformis (DH) (Table 1) [10]. Clearly, there is need for a greater understanding of the epidemiology, pathophysiology, and natural history of AIBD. The continued evolution of methods for the reliable, acces- sible, and cost-effective detection of disease relevant auto- antibodies is an ongoing endeavor to improve our ability to diagnose and monitor autoimmune activity, with impact in clinical management and decision-making accurately and rapidly. Objectives Here, we discuss the key autoantigens in AIBDs and high- light how serological testing can be used in conjunction with clinical symptoms for diagnostic purposes. Pemphigus diseases Pemphigus diseases are commonly characterized by the pro- duction of autoantibodies primarily against the desmosomal proteins desmoglein (Dsg)3 and Dsg1 which results in the loss of epidermal cell-cell adhesion and subsequent blister formation [11]. Some patients experience only mucous mem- brane erosions with minimal skin blistering, others exhibit lesions on both mucosal as well as non-mucosal surfaces, while others still may only show skin involvement without mucous membrane involvement [11]. The clinical phenotype of pemphigus has been linked to defined Dsg3 and Dsg1 antibody profiles [12]. Additionally, differences in the nor- mal tissue distribution of Dsg1 and Dsg3 proteins (Dsg1 on the epidermal surface and Dsg3 in deep epidermal layers/ mucous membranes) may explain the varying clinical man- ifestations of different pemphigus forms [13]. For example, in pemphigus foliaceus (PF), IgG antibodies are only directed against Dsg1 and blistering is confined to the skin surface. On the other hand, in pemphigus vulgaris (PV), autoanti- bodies against both Dsg1 and Dsg3 can be observed, and Table 1. Target antigens in autoimmune bullous dermatoses AIBD Subtype Blister Location Target Antigena Ig Type Pemphigus PV (mucosal-dominant type) Intraepidermal Dsg3 IgG PV (mucocutaneous type) Intraepidermal Dsg3, Dsg1, Dsc1, Dsc2, Dsc3 IgG IgA Pemphigus Intraepidermal Dsg3, Dsg1, desmocollins PF Intraepidermal Dsg1 IgG PNP Intraepidermal Envoplakin, Dsg3, Dsg1, periplakin, epiplakin, plectin, desmoplakins, Dsc(1- 3), BP230, α2-macroglobulin-like 1 IgG Pemphigoid BP Subepidermal BP180, BP230 IgG MMP Subepidermal BP180, BP230, laminin332, integrin α6/ β4, and collagen VII IgG EBA Subepidermal Type VII collagen IgG DH Subepidermal Epidermal/tissue transglutaminase, endomysium, deamidated gliadin IgA/IgG Pemphigoid gestationis Subepidermal BP180, BP230 IgG Linear IgA bullous dermatosis Subepidermal Ectodomain fragment of BP180, BP230 IgA aMain target antigens are indicated in bold. AIBD = autoimmune bullous dermatoses; PV = pemphigus vulgaris; PF = pemphigus foliaceus; Dsc = desmocollins; PNP = paraneoplastic pemphigus; BP = bullous pemphigoid; MMP = mucous membrane pemphigoid; EBA = epidermolysis bullosa acquisita; DH = dermatitis herpetiformis; Dsg = desmoglein. 4 Review | Dermatol Pract Concept. 2022;12(2):e2022116 the degree of blistering and mucous membrane involvement varies based on the prevalence of either anti-Dsg1 and an- ti-Dsg3 [12]. This framework, correlating the clinical presen- tation of pemphigus to antibody profile, is known as the Dsg compensation hypothesis (DCH), featured prominently in dermatology textbooks and previous research studies [14]. As elegant as the hypothesis may be, however, recent studies have identified exceptions to this hypothesis [15,16]. PV accounts for 80% of all pemphigus cases and mainly affects middle-aged and elderly populations [17]. While Dsg3 (89% - 90% of patients) and Dsg1 (50% - 60% of patients) are the major autoantigens in PV, additional structural and metabolic autoantigens have been identified including desmo- collins (Dsc) 1 and 3, muscarinic and nicotinic acetylcholine re- ceptors, mitochondrial antigens, thyroid peroxidase, hSPCA1, plakophilin 3, plakoglobin, and E-cadherin [18]. Studies have shown that autoantibodies against these additional targets may complement the effects of anti-Dsg autoantibodies and explain individual variations in pemphigus disease severity [18]. In paraneoplastic pemphigus (PNP), autoantibodies are directed against desmosomes including Dsg1 and Dsg3, α2-macroglobulin-like 1, and the plakins envoplakin, desmo- plakin I and II, plectin, periplakin and the hemidesmosome BP230. The presence of desmoplakin autoantibodies is also common to PV, PF, and BP. However, autoantibodies for envo- plakin and periplakin on immunoblot, as well as autoanti- bodies for desmoplakin (on indirect immunofluorescence and rat bladder epithelium), appear to be sensitive and specific for PNP diagnosis [19]. This has led to the development of an enzyme-linked immunosorbent assay (ELISA) that detects envoplakin in consideration for a diagnostic tool for PNP. PNP is associated in a majority of cases with non-Hod- gkin lymphoma, chronic lymphocytic leukemia and Castle- man disease [20]. A common clinical feature is stomatitis which presents with painful erosions and ulcerations of the oropharynx. Anti-envoplakin antibodies are highly specific for PNP and are used for the differentiation of PNP from other AIBDs [21]. In IgA pemphigus, a rare form of pemphigus with unclear etiology, serum IgA autoantibodies are associated with reac- tivity against the desmosomal cadherins Dsc1, Dsc2, Dsc3, Dsg1, and Dsg3 [17]. These circulating IgA antibodies lead to formation of pruritic and painful eruptions that present as vesicles and pustules on the skin [22]. As IgA pemphigus is so rare, there is currently no reported sex, age, or race dis- tribution of this disease. However, IgA pemphigus has been observed in all age demographics [23]. Pemphigoid diseases Pemphigoid diseases are characterized by subepidermal blister formation in the skin and mucous membranes [24]. Pemphigoid diseases occur when the immune system produces autoanti- bodies against proteins involved in the linkage between the epidermis and dermis. As a result of this autoimmune reac- tion, the epidermal layer separates from the dermis. Several different types of pemphigoid diseases exist including bullous pemphigoid (BP), pemphigoid gestationis, mucous membrane pemphigoid, linear IgA dermatosis and p200 pemphigoid [10]. The hemidesmosomal proteins, BP180 and BP230, which tether the 2 skin layers together, are the common autoanti- body targets in BP. BP is the most common AIBD in the gen- eral population, with an annual incidence ranging between 2.3 to 23 cases per million. BP disproportionately affects elderly people, with an incidence of 190-312 cases per million among those 80 years and older [14]. This disease manifests with bulging skin blisters and minimal mucous membrane involve- ment [25]. Unlike pemphigus, BP shows a negative Nikolsky sign (ie no splitting of skin upon applying pressure) [26]. Autoantibodies against BP180 represent the most sig- nificant biomarker in BP due to their high prevalence [27]. Additional screening for anti-BP230 antibodies is important as they occur in 40% of patients who are seronegative for anti-BP180 antibodies. The parallel detection of both an- ti-BP230 antibodies and anti-BP180 antibodies increases the sensitivity of BP detection significantly, to a combined 97.1% [28]. Pemphigoid gestationis is the manifestation of BP in pregnant women and is characterized by autoantibod- ies predominately against epitopes in the immunodominant NC16A domain of BP180 (BP180-NC16A) [29]. In chil- dren, linear IgA dermatosis occurs from the autoantibody recognition of the ectodomain fragment of BP180 [30]. In addition to BP180, laminin 332 is a major target in mucous membrane pemphigoid (MMP) [31]. Additionally, patients with mucous membrane pemphigoid may show antibodies against BP230, integrin α6/β4, and collagen VII [31,32]. The identification of anti-laminin 332 is important for determin- ing a patients prognosis as anti-laminin 332 positive patients seem to be at an increased risk of malignancies [33]. Epidermolysis bullosa acquisita EBA is a severe blistering dermatosis characterized by autoanti- bodies against type VII collagen [34]. EBA manifests as subepi- dermal blisters and erosions in response to the minor irritation of skin and affects both the skin and mucous membranes. The level of the cleavage in the basal membrane contributes to the various phenotypes of EBA, including the most common inflam- matory and mechanobullous (noninflammatory) variants [35]. Dermatitis herpetiformis DH is an itchy dermatosis affecting 10% of celiac patients. It manifests as blisters in the subepidermis of areas such as the Review | Dermatol Pract Concept. 2022;12(2):e2022116 5 elbows, knees, and buttocks. There is also minimal blistering of the mucous membranes. DH is one of many manifestations of gluten-sensitivity and is characterized by IgA autoantibod- ies against endomysium tissue/epidermal transglutaminase (anti-tTG/-eTG) and/or deamidated gliadin (IgA/IgG) [36]. In contrast to the increase in diagnosis of celiac disease, DH incidence appears to be decreasing (Table 1) [37]. Diagnostic approach The diagnosis of AIBDs requires the detection of both cir- culating and tissue-bound antibodies, and histopathology, in conjunction with clinical symptoms [38]. The pathway to AIBD diagnosis can be broken down into 4 pillars. First, the clinical manifestations of the disease must be assessed. Second, histopathology can be performed to provide infor- mation on the location of skin involvement (sub- or intraepi- dermal separation). Third, the detection of tissue bound autoantibodies by direct immunofluorescence (DIF) is done. DIF is the current diagnostic gold standard for AIBDs but gives limited information on the target antigens. DIF has a sensitivity of 82% - 91% and a specificity of 98% [17]. The fourth pillar is the identification of autoantibodies by sero- logical testing such as indirect immunofluorescence (IIF) mi- croscopy, monospecific ELISA or immunoblot techniques.17 Serological testing for the detection of circulating an- tibodies in AIBDs has the advantage of being minimally invasive and may be suitable for diagnostic purposes in conjunction with the clinical manifestations, and for aiding therapy decisions and disease prognosis [17]. Conventional serological detection of AIBD-specific antibodies involves an initial IIF screen using tissue substrates, followed by an anti- gen-specific assay such as ELISA. In 2016, the International Bullous Diseases Consensus Group met to standardize the diagnosis and management of pemphigus [39]. The diagnosis of pemphigus was agreed to be based on the clinical presentation and histopathology consistent with pemphigus and either a positive DIF micros- copy or serologic detection of autoantibodies against epithe- lial cell surface antigens [39]. The determination of serum autoantibodies was recommended for therapeutic decision making as serum levels of IgG against Dsg1 and Dsg3 cor- relate with the clinical activity of pemphigus. AIBD autoantibody screening using tissue IIF Due to their high sensitivity, tissue substrates are ideal for screening for AIBDs autoantibodies (esophagus, salt-split skin, bladder mucosa) [40]. The esophagus substrate yields characteristic honeycomb-like immunofluorescence pat- terns which can be differentiated when screening for anti- bodies in suspected cases of PV or PF. IIF using esophagus as a substrate has proven to be useful for the detection of autoantibodies against Dsg1 and Dsg3, with a sensitivity of 81% - 100% and a specificity of 89% - 100% [17,41]. For the differentiation of autoantibodies in subepidermal AIBDs, tissue sections of salt-split skin are used [42]. Salt-split skin substrate has a sensitivity of 73% - 96% and a specificity of 97% for such subepidermal antibodies. Additionally, as antibodies have varying antigenic binding properties on salt-split skin, this allows for the differentiation between the subepidermal AIBDs BP, pemphigoid gestationis, linear IgA dermatosis and other subepidermal AIBDs such as EBA, and anti-laminin-332-type MMP. Where BP180 and BP230 are located on the epidermal side of salt-split skin, collagen type VII and laminin 332 remain on the dermal side. Urinary bladder is an ideal substrate for distinguishing between PNP and other pemphigus diseases as plakins like envoplakin are highly expressed in the bladder while Dsg1 and Dgs3 are not [43]. Urinary bladder is therefore a highly specific substrate for PNP (99% - 100%) and having a sensi- tivity of 74% [17]. Finally, liver tissue is useful for the detec- tion of IgA autoantibodies against endomysium in DH [44]. The International Bullous Diseases Consensus Group recommends using IIF microscopy on monkey esophagus or human skin to detect autoantibodies against surface pro- teins of epidermal keratinocytes [39]. In cases of atypical presentation or the suspicion of another AIBD, the use of IIF microscopy on rat bladder and immunoblot/immuno- precipitation is discussed. They also describe the use of re- combinantly expressed Dsg1, Dsg3, or envoplakin substrates (EUROIMMUN) when Dsg- or envoplakin-specific ELISA cannot be used [39]. Antigen-specific detection of AIBDs The detection of antigen-specific autoantibodies in AIBDs can be achieved using monospecific IIF and ELISAs [17]. Monospecific IIF can be accomplished using transfected cells as a substrate in which the target antigen has been re- combinantly expressed. Additionally, designer antigens have been created to enhance diagnostic sensitivity and specificity of IIF. Such purified recombinant designer antigens are uti- lized as monospecific IIF substrates. BIOCHIPS, which are coated with an IIF substrate and arranged onto microscope slides, allow for autoantibody screening and confirmatory discrimination in a single incubation. In this way, various types of AIBDs can be screened for in one test. IIF BIO- CHIP mosaics contain combinations of different substrates (esophagus, salt-split skin, bladder mucosa, transfected cells, purified designer antigens). A study which compared the performance of the “Dermatology Mosaic 7” with a multi- step serum analysis using single antibody tests, found a 94% diagnostic agreement between both methods. Therefore, multiparametric BIOCHIP mosaics offer a cost and time ef- fective IIF method. 6 Review | Dermatol Pract Concept. 2022;12(2):e2022116 Autoantibodies can be mono-specifically identified us- ing ELISAs which utilize purified recombinant proteins [17]. Commercial assays which utilize the recombinant ectodomains of Dsg1 and Dsg3 have a high sensitivity and specificity for the detection of pemphigus foliaceus and pemphigus vulgaris (96% - 100%, 96% - 100% and 85% - 100%, 96 - 100%, respectively) [17]. In addition to their use as monospecific substrates in IIF, designer antigens have been developed for ELISAs (for the detection of antibodies against BP180 and BP230, deamidated gliadin peptides) to improve immunoreactivity. ELISA techniques provide quantitative measurement which is useful for the application of therapy monitoring. Profile ELISAs containing a combination of an- tigens enables the simultaneous detection of multiple AIBDs subtypes in patients with suspected AIBDs. Experts recommend determining anti-Dsg1 and/or an- ti-Dsg3 IgG antibodies by ELISA for the detection of PF, and mucosal/mucocutaneous PV (Mannose-Binding Lectin, EUROIMMUN) [39]. Serum concentrations of antibodies against Dsg1 and Dsg3 are associated with pemphigus dis- ease activity in and high levels of anti-Dsg1 by ELISA has a positive predictive value for skin relapses. Therefore, the determination of serum autoantibodies against skin struc- tural proteins by ELISA has a prognostic value for guiding pemphigus treatment. Conclusions A definitive diagnosis of AIBD is based on a combination of clinical signs and symptoms and the analysis of autoantibod- ies using IIF and ELISA. IIF, using various tissue substrates, is a useful application for antibody screening while transfected cells and purified antigen substrates are suitable for anti- gen-specific IIF. ELISA allows for the quantitative measure- ment of antibody levels to support the detection of different AIBD subtypes. Serological antibody testing is important for distinguishing between the various AIBD subtypes due to differences in their prognosis and treatment. Immunologic testing has also a key role in providing an accurate diagnosis as blistering skin diseases are easily misdi- agnosed. Oral blisters are often misdiagnosed as an infection such as candidiasis or herpes. Without a proper diagnosis, a patient is at risk of being mistreated, potentially with a chronic overexposure to steroids which may reduce some symptoms without fully addressing the underlying problem. Additionally, serological testing allows for the monitor- ing of AIBD disease. Serum levels of anti-BP180 antibodies correlate with disease activity of BP while anti-BP230 levels correlate with the disease duration [45]. Moreover, levels of anti-Dsg1 and Dsg3 are associated with severity of pemphi- gus diseases and response to therapy while anti-envoplakin titers correlate with the degree of PNP symptoms as well as differential diagnostic clarification [46]. The detection of an- ti-collagen type VII antibodies aids in the detection of EBA and allows for the differentiation of EBA from other AIBDs [47,48]. In addition to disease monitoring, correlations between lowered levels of AIBDs specific autoantibodies in response to therapy point to the use of serological testing for therapy monitoring purposes [49,50]. 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