Each microarray chip consisted of proteins representing 701 unique human genes and their transcript variants, and peptides of the Dsg 1 and Dsg 3 extracellular domains 1C5 (Table S1; Fig. molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the GNF351 disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, GNF351 CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients. Introduction Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against stratified squamous epithelium. PV antibodies demonstrate epithelial cell-surface staining by indirect immunofluorescence (IIF), and, because this staining appears between cells, initially the antibodies were described as intercellular antibodies [1], [2]. Although the incidence of PV is only 1 to 16 per million population per year [3], [4], this disease represents a significant burden to health care professionals, and the health care system. Systemic administration of glucocorticosteroid hormones is essential to establish control of disease during the acute stage [5]. While glucocorticosteroid treatment is life saving, it may cause severe side effects, including death [6], [7]. The development of nonsteroidal treatment has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte detachment in PV. During the last decade, the studies of autoimmune responses in PV have been supplemented and, to some extent, replaced by analyzing the levels of antibodies to desmoglein (Dsg) 3 by enzyme linked immunosorbent assay (ELISA) representing a hallmark and a diagnostic criterion of PV [8]. However, Dsg 3 antibody levels do not always correspond to the presence of cell-surface antibodies by IIF or correlate with disease activity [9], [10], [11] or predict relapse of the disease [12]. Furthermore, anti-Dsg antibodies can be absent in the active stage of disease but present in PV patients during remission [13], [14], [15], [16], [17], GNF351 [18], patients with unrelated medical conditions, and healthy subjects, including relatives of PV patients [17], [19], [20], [21], [22], [23], [24], [25], [26]. For example, 16 PV patients positive for cell-surface antibodies by IIF had normal Dsg 3 antibody levels [27]. Identification of proteins targeted by autoantibodies in PV is a subject of intense research. The first evidence that keratinocyte antigens other than Dsg 1 and Dsg 3 are pathophysiologically relevant was provided by experiments showing the ability to induce suprabasal acantholysis and gross skin blisters in neonatal mice by passive transfer of PV antibodies [28]. In this model, murine epidermis lacks Dsg 3 and the passively transferred PV IgG lacks Dsg 1 antibody. Hence, the injected PV antibodies cause blisters by targeting non-Dsg 1 and Dsg 3 keratinocyte antigens. Current understanding, however, does not adequately explain the mechanism of acantholysis in patients lacking Dsg 1 and 3 antibodies. Furthermore, results of a recent study Rabbit Polyclonal to Neuro D indicate that autoreactivity in PV relies on somatic mutations generated in response to an antigen unrelated to Dsg 3 [29]. Taken together, these facts justify a search for novel targets of pemphigus autoimmunity. In general, autoimmune diseases are characterized by the presence of multiple types of autoantibodies mediating a coordinated immunological attack against a fraction of the tissue proteome. For example, 116 autoantibodies were described in patients with systemic lupus erythematous [30]. The number of targeted self- antigens varies dramatically from patient to patient..