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Figure 3: Demonstration of the pathogenicity of anti-type VII collagen antibodies. (a) Immune complexes (IC) of anti-COL7 IgG (Ab) and COL7 (AG) are able to activate neutrophils isolated from either healthy humans or mice. Endpoint measurement is the release of reactive oxygen species (ROS) by chemiluminescence. Detection of elastase by ELISA is an additional measurement for neutrophil activation in this assay. For AIBD, this assay was first described for type XVII collagen and anti-type XVII collagen antibodies obtained from patients with bullous pemphigoid [73]. Later, the same method was adopted for EBA [61]. The figure shows a schematic result (ROS release) from neutrophils activated with either antigen or antibody alone, or with immune complexes. (b) Incubation of cryosections of normal human or mouse skin with antibodies targeting COL7, followed by incubation with neutrophils from healthy donors induces dermal-epidermal separation. This so-called “cryosection assay” also allows to investigate neutrophil activation. This assay was initially developed using sera from BP patients [74, 75] and was later adopted for EBA [60]. Shown are H&E stained sections of cryosections of normal human skin incubated with either normal human serum (NHS) or IgG from an EBA patient. After the washing of antibodies, neutrophils from healthy human donors were added to the sections. While no pathology is observed in the section incubated with NHS, a clear separation of the epidermis from the underlying dermis is present in the section incubated with anti-COL7 IgG. * indicates split formation. (c) Injection of rabbit or human anti-COL7 IgG into mice leads to IgG and complement (C3) deposition along the dermal-epidermal junction (arrow heads). Subsequently, subepidermal blistering (*) accompanied with a dermal leukocyte infiltration (arrow heads) develops. Clinically, mice present with erythema, crusts, and alopecia. Fresh blisters are rarely observed, most likely due to the thin epidermis in mice. This antibody transfer (passive) EBA mouse model was first described independently in 2005 by two groups [44, 63]. The histology and images are from C57Bl/6 mice injected with rabbit anti-COL7 IgG, 12 days after the first antibody injection. (d) Immunization of susceptible mouse strains with recombinant proteins located within the NC1 domain leads to autoantibody production, which can be detected by direct IF microscopy from skin biopsies. Subsequent to autoantibody deposition at the dermal-epidermal junction, complement activation (evidenced by C3 deposition at the dermal-epidermal junction) and histological and clinical findings duplicating the human disease are observed.