Pemphigus Vulgaris and Bullous Pemphigoid: Update on Diagnosis and Treatment

Autoimmune bullous disorders are a heterogeneous spectrum of skin disorders characterized by the production of autoantibodies against adhesion molecules of the skin. The 2 major groups of diseases are “pemphigus diseases” and “autoimmune bullous diseases of the pemphigoid type.” Pemphigus diseases are a group of autoimmune blistering diseases of the skin and mucous membranes characterized by intraepithelial cleft and acantholysis. The main subtypes of pemphigus include pemphigus vulgaris, pemphigus foliaceus, and paraneoplastic pemphigus. Diagnosis is based on clinical manifestations and confirmed with histological, immunofluorescence, and serological testing. Recently multivariant enzyme-linked immunosorbent assay systems have been developed as practical screening tools for patients with suspected autoimmune bullous dermatoses. The current first-line treatment of pemphigus is based on systemic corticosteroids that are often combined with immunosuppressive adjuvants, such as azathioprine, mycophenolate mofetil, and the anti-CD20 monoclonal antibody rituximab, usually at initiation of treatment. Rituximab efficacy is higher when it is administered early in the course of the disease. Therefore, it should be used as first-line treatment to improve efficacy and reduce cumulative doses of corticosteroids and their side effects. Treatment of bullous pemphigoid is based on disease extension. Localized and mild forms can be treated with superpotent topical corticosteroids or with nonimmunosuppressive agents. In patients with generalized disease or whose disease is resistant to the treatments described above, systemic corticosteroids are preferred and effective. Adjuvant immunosuppressants are often combined with steroids for their steroid-sparing effect.

ABDs of the pemphigoid type or autoimmune subepidermal blistering diseases of the skin and mucosae constitute a large group of diseases characterized by the production of circulating autoantibodies against several structural proteins of the basement membrane zone, leading histologically to subepidermal blistering. The main disorders include BP, pemphigoid gestationis, mucous membrane pemphigoid, epidermolysis bullosa acquisita, and anti-p200 pemphigoid [2] (Table 3). BP is characterized by the generation of autoantibodies directed in particular against BP180/collagen XVII and BP230/dystonin.

Epidemiology
Prospective studies suggest the incidence rates of ABDs are in the range of 14.5-20.4/million [3][4][5]. Most of the available epidemiological data derive from PV, the most frequently reported disorder among the PDs, and BP [3].
PV incidence appears to be highly variable according to geographic regions and ethnic groups. The incidence rates reported in European prospective studies range between 0.5 and 4.0/million [5,6]. Higher rates, up to 16.1/million/year, have been observed in subjects from Israel and Iran [7,8]. In fact, the disease is more common among individuals of Ashkenazi origin, but also in ethnic groups from Iraq and Iran.
Therefore, ethnic differences should be taken into account when comparing incidence rates in countries with populations of Jewish heritage [3,9]. Pemphigus is most frequently diagnosed between ages 50 and 60 in European countries, but it can be seen earlier outside of Europe.
are a major cause of severe morbidity and considerable mortality [1].
Based on the available literature data, this paper aims to provide an up-to-date overview on diagnosis and therapy of pemphigus vulgaris (PV) and bullous pemphigoid (BP), which represent the 2 major diseases in the heterogeneous clinical spectrum of ABDs.

Classification
The classification of an ABD relies on the level of blistering and considers 2 major groups of diseases, namely "pemphigus diseases"(PDs) and "ABDs of the pemphigoid type." PDs are characterized by the production of pathogenic autoantibodies directed against different proteins of the desmosome, leading histologically to intraepithelial blistering.
There are several variants of pemphigus, but the 3 major forms include PV, pemphigus foliaceus (PF), and paraneoplas-   [17]. Environmental factors, in particular pathogens, have been hypothesized as causes of reverting immunological tolerance with resulting autoantibody production [18]. A possible link with rotavirus infection has been suggested because of cross-reactive VH1-46 antibodies, which are able both to disrupt keratinocyte adhesion and inhibit rotavirus replication [19]. An additional relationship between the exposure to a noninfectious environmental antigen and the development of an autoimmune response to self-antigen has been documented in fogo selvagem, the endemic form of PF. In this condition, cross-reactive epitopes on Dsg1 and LJM11 sand fly salivary gland antigen could drive the production of pathogenic IgG4 autoantibodies anti-Dsg1 [20].
Although antibody-mediated disease mechanisms in PV are widely characterized, the T cell implication has to be better clarified. Autoreactive CD4+ T lymphocytes have been implicated in the regulation of the production of pathogenic anti-Dsg3 autoantibodies by B cells [21]. There is a strong association between PV and distinct major histocompatibility complex class II haplotypes, which are considered essential for the presentation of specific Dsg3 peptides to autoreactive CD4+ helper T cells [18,22].
The causes of loss of tolerance leading to the production of antibodies anti-BP180/collagen XVII and BP230/dystonin, and less frequently also against other antigens in BP, are not known. BP has been linked to environmental factors as physical agents, such as radiation therapy, ultraviolet radiation [23] trauma, neurodegeneration or neuroinflammation [24] and drugs, in particular gliptins [25]. Muramatsu et al demonstrated that Tregs are essential in preventing the spontaneous production of BP autoantibodies as well [26].
An imbalance between autoreactive helper T cells and Tregs and a T cell-independent activation of a toll-like receptor The yearly incidence of BP reported in European prospective studies varies between 2.5 and 13/million [5,6]. The largest series of patients (N = 869) collected in a retrospective historical cohort from the UK showed higher incidence rates of 42.8/million [10]. Rising incidence rates of BP in Europe have been linked to risk factors, including population aging; drugs, in particular antidiabetics; diuretics; phenothiazines; major cognitive impairment; and disabling neurological disorders [3,11]. BP affects mostly the elderly: it is generally seen in individuals older than 70 years. Overall incidence is higher in females [3].

Genetics
There is evidence that genetics plays a critical role in PV development, severity, and prognosis. An increased frequency of HLA-DRB1*04:02 haplotypes in Ashkenazi Jewish patients [12] and DQB1*05:03 in non-Jewish Caucasian populations [13] has been observed. The coexistence of alleles of DRB1*14/DQB1*05 and A*11/DQB1*05 would be strongly influential for the predisposition to the disease, while that of HLA-B*50/DQB1*02 could play a protective role [14]. In addition, multiple single nucleotide polymorphisms within different genes may confer susceptibility to the diseases, probably in a population-specific way [15].
Serological typing for HLA class II antigens in patients with ABD of the pemphigoid type revealed a highly significant association with HLA-DQβ1*0301 [16].

Pathophysiology
PV and BP are characterized by the loss of tolerance to autoantigens expressed primarily in the skin, in particular desmosomal proteins in PV and components of the hemidesmosomes in BP.
In PDs, the production of pathogenic immunoglobulin G (IgG) autoantibodies, mainly IgG4, against the desmosomal  ognized as robust tools to correctly assess disease activity [27].

Clinical Presentation
Classic BP affects elderly individuals, usually above age 70 years. The spectrum of clinical presentation is highly variable. In a consistent proportion of patients, the blistering eruption is preceded by a prodromal nonbullous phase, usually lasting weeks to months and even, in rare cases, remaining the only manifestation of the disease [28]. During the nonbullous phase, pruritic, erythematous, or urticarial patches and plaques occur. Also eczematous, polycyclic, targetoid, nodular, or lichenoid lesions may be observed ( Figure 3). The bullous phase is characterized by tense bullae on an erythematous, urticarial base, localized or widespread   This test does not replace histological examination, since acantholytic keratinocytes may also be observed in other dermatoses as a secondary phenomenon to inflammation or ballooning degeneration.

Bullous Pemphigoid
The histopathological assessment of an early bulla shows a subepidermal blister containing a net of fibrin with a variable number of eosinophils and/or neutrophils accompanied by a dermal inflammatory infiltrate mainly consisting of eosinophils and neutrophils [3,33]. In the nonbullous phase, histopathological findings may be nonspecific, since only subepidermal clefts and eosinophilic spongiosis may be observed [34].

Direct Immunofluorescence
DIF is the most reliable and sensitive diagnostic test for ABDs for the most part. However, nonspecific staining may occasionally be seen in other cutaneous disorders with occasional false-positive findings [35,36]. DIF investigates the skin or children. Acral involvement is common, especially on the face (62%), palms and soles (79%); localized lesions on the genital area may be observed in 17% of cases [32].   Dsg1 and Dsg3 generally correlate with the extent and clinical activity of disease, and high levels of anti-Dsg1 by ELISA has a positive predictive value for skin relapses [37]. Therefore, ELISA may represent a good serological marker of disease activity, although evidence about its predictive value from large prospective cohort studies is lacking [37]. One must keep in mind that anti-Dsg antibodies have been occasionally discovered in sera of normal patients and those affected with other bullous diseases [41]. In addition, a small number of pemphigus patients may not show the PV phenotype expected by their Dsg autoantibody serum profile [42].

Bullous Pemphigoid
IIF may demonstrate circulating IgG antibodies binding to the basal membrane. The most specific test substrate for BP is salt-split skin, which is healthy human skin in which subepidermal splitting was induced by 1 mol/L sodium chloride solution [43]. Other substrates include monkey or rabbit esophagus, with possible lower sensitivity [38].

Treatment Pemphigus Vulgaris
The morbidity and mortality of PV have remained high owing to complications of therapy, such as pneumonia, septicemia, mucous membrane and shows antibody deposition on the keratinocyte cell surface (in PDs) or along the basement cell membrane (in BP).

Pemphigus Vulgaris
Biopsy of perilesional skin or mucosa shows deposits of IgG at the keratinocyte cell membrane [22,37]. IgG deposition is seen in up to 100% of patients with active disease. Complement (C3) deposition may not be observed, since IgG4, the dominant subclass of IgG involved in PV, does not fix complement.

Bullous Pemphigoid
It is best to biopsy perilesional skin from a recent blister. The

Serological Studies
The method of serological detection of the autoantibodies largely relies on indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) tests. Recently, novel diagnostic multivariant assays were developed as practical screening tools for patients with suspected ABD with the aim of processing the most common autoantibodies simultaneously [39,40].

Pemphigus Vulgaris
IIF allows for the detection of circulating autoantibodies against proteins or epithelial keratinocytes by incubating patient serum with appropriate commercially available substrates containing the target antigen. The testing study is conducted on monkey esophagus or epithelial substrates. PV sera produces a characteristic smooth and reticular pattern on most epithelial layer, referred as "fishnet-like," "chicken wire," or "honeycomb" pattern [37,38].
Cloning of the gene coding for the major pemphigus antigens, Dsg1 and Dsg3, has enabled the production of recombinant proteins, which are used to detect IgG autoantibodies by ELISA [41].  [47,48]. In particular, a meta-analysis including 578 patients with pemphigus showed a remission rate of 76% following a single cycle of rituximab, with a relapse rate of 20% (2 years) to 60% (5 years) [49]. More robust evidence showed a higher efficacy when it was administered early in the course of the disease [50].  The dosing protocol corresponds to that of the European Medicine Agency's "Summary of product characteristics" and US Food and Drug Administration's highlights of prescribing information. Different dosing protocols are reported in expert recommendations and therapeutic guidelines [42,50].

Bullous Pemphigoid
Although there is no generally accepted classification of disease severity, a classification of BP into mild (<10% affected body surface area), moderate, and severe forms has been suggested [57]. The effectiveness of topical therapy with superpotent topical corticosteroids in localized and moderate forms of BP is supported by a Cochrane review [38,58]. Be aware that the dose of 40 g every day or 10 to 30 g every day [59,60], which has proven equally effective as systemic prednisolone, is equivalent to about a tube per day. Practicability and limitations of this drug regimen are that for some patients with BP, a twice-daily topical application [57] on widespread areas or the daily purchase of an ointment tube may not be manageable. In the localized, nonsevere forms, immunomodulatory, nonimmunosuppressant drugs such as doxycycline may be considered a viable option [61]. Although its exact mechanism of action is not well understood, doxycycline proved to be not inferior to oral prednisolone for short-term blister control and is safer in the long term [61,62]. Systemic corticosteroid therapy (prednisone 0.5 mg/kg/day) represents the treatment of choice for severe forms [38].  [53,54]. Additional areas that deserve further studies are the combination protocols with corticosteroids, the possibility of using CD20 inhibitors alone, prevention of relapses, the role of anti-Dsg ELISA values as biomarkers to drive further infusions, and the benefit from combining other adjuvants in patient management. Consequently, the therapeutic algorithm of PV could be redefined in the future ( Figure 6).
Topical therapy of PV is essentially symptomatic and is practiced to alleviate inflammation and prevent secondary infections. Usually corticosteroids, calcineurin inhibitors or corticosteroids and antibiotics in combination are administered on the cutaneous and mucous lesions. Cutaneous erosive lesions should be covered using low-adhesive wound dressings. Supportive care for oral lesions includes gel-containing local anesthetics and proper dental care.
Limited experience proved rituximab useful in refractory PF as well [55,56]. immune suppression in PV by therapeutic immunoadsorption of pathogenic autoantibodies [68,69]. A promising approach is the use of reengineering chimeric autoantibody receptor T cells. Patient-derived T cells are modified ex vivo to express a chimeric antibody receptor, which allows selective recognition and consequent killing of anti-Dsg3 autoreactive B lymphocytes [70,71]. Also in BP, new therapeutic targets aim to provide new treatment strategies that may go beyond nonspecific immunosuppression. In addition to omalizumab, monoclonal antibodies to IL-5 such as mepolizumab and bertilimumab, an anti-eotaxin-1 antibody, are currently being investigated in BP [65]. In the context of immunomodulatory drugs, dimethyl fumarate, which is a prodrug utilized in psoriasis and multiple sclerosis, is also under investigation for BP because of its pleotropic anti-inflammatory effects [72].
at the dermal-epidermal basement membrane zone and inflammatory response might be not fully ruled by a B-cell downregulation and a decrease in autoantibody titers [65].
Recent studies support a pathogenic role of IgE in the development of BP. This hypothesis is endorsed by the finding of IgE deposition in the basement membrane zone in patients with BP. In addition, there is correlation between serum levels of IgE autoantibodies against BP180 and BP disease activity [62]. Use of IgE targeted therapies, such as omalizumab, has been shown as promising in recent studies [66], with 80% complete response rates and mean time to recurrence of 3.4 months [64].

Conclusions
Current treatment paradigms in ABD are traditionally based on the administration of immunosuppressive drugs, often in combination. The advent of rituximab, the monoclonal IgG antibody against CD20, has revolutionized the treatment of pemphigus, indicating that a complete remission off therapy is now possible. Therefore, additional studies are investigating other anti-CD20 monoclonal antibodies [67].
Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells without affecting protective immunity. Innovative approaches include antigen-specific