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Bullous pemphigoid (BP) varies in severity and stratified treatments are needed. However, there are no definitive standards for choosing appropriate treatments. To elucidate the factors involved in choosing treatments and the clinical outcomes of BP, we retrospectively reviewed the clinical records of 78 BP patients at a single center. Of the 78 patients, 49 (62.8%) were treated with oral prednisolone (PSL) and 29 (37.2%) were treated without PSL. The patients with older age, lower Bullous Pemphigoid Disease Area Index (BPDAI), and/or lower anti-BP180NC16a antibody titer at onset tended to be treated without oral PSL. Notably, only 9.1% patients without PSL experienced relapse, whereas 36.7% patients with oral PSL experienced relapse when the PSL was around 0.1 mg/kg. It suggests that the patients with mild disease severity could be well controlled without oral PSL. Receiver-operator curve analysis demonstrated that the cut-off value for the use of oral PSL was 31 for total BPDAI and was 7 for BPDAI skin urticaria/erythema, with a high (>0.9) area under the curve. Notably, none of the patients who were negative for the anti-BP180NC16a antibody at onset experienced relapse even though they were treated without PSL. In conclusion, in BP patients who were negative for anti-BP180NC16a antibody at onset, with a total BPDAI score of less than 31 or with an urticaria/erythema score of less than 7 can be treated without PSL. When the PSL is tapered to around 0.1 mg/kg, we should carefully monitor the patients to detect relapse.

Background Regulatory T (Treg) cells play an essential role in peripheral immune tolerance. Bullous pemphigoid (BP) is the most common blistering disease and is caused by autoantibodies to two BP antigens: type XVII collagen and BP230. Recently, we reported that Treg cell dysfunction may cause the production of autoantibodies to BP antigens. Several studies have suggested an association between Treg cells and BP pathogenesis. However, Treg cells are heterogeneous in humans, leading to inconsistent results in previous studies. Objective To assess functional Treg subsets in BP. Methods We examined three distinct Treg subsets in conventional BP (cBP) patients before versus after systemic corticosteroid treatment, dipeptidyl peptidase-4 inhibitor-associated BP (DPP-4i-BP) patients, younger controls and older controls. Results We found that total Treg cells and all Treg cell subsets were increased in cBP patients before treatment and decreased by systemic corticosteroid treatment. In contrast, neither total Treg cells nor all Treg subsets were increased in DPP-4i-BP. Notably, CD45RA− Foxp3hi effector Treg cells positively correlated with disease severity in cBP, whereas CD45RA+Foxp3lo naive Treg cells positively correlated with the disease severity in DPP-4i-BP. Conclusion These findings suggest that Treg cells are differently involved in the pathogeneses of cBP and DPP-4i-BP.

A long-term immunosuppressive treatment can provoke latent infections. Autoimmune blistering diseases (AIBD) are mostly treated with systemic immunosuppressive agents. To prevent the reactivation or exacerbation of existing latent infections, patients must be screened for infectious diseases before immunosuppressive treatments are initiated. However, the prevalence of infectious diseases in AIBD remains to be elucidated. To evaluate the necessity of screening infectious diseases in AIBD, we retrospectively reviewed the clinical records of 215 patients at a single center with AIBD for hepatitis B virus (HBV), hepatitis C virus (HCV), Mycobacterium tuberculosis, Treponema pallidum, human T-cell leukemia virus type 1 (HTLV-1) and HIV infections. Approximately 40% of patients were infected with HBV. During systemic corticosteroid treatment, HBV DNA became positive in 3.4% of cases. Antibodies to HCV, interferon-γ release assays for M. tuberculosis and the T. pallidum latex agglutination test were positive in 0.6%, 6.6% and 1.2% cases, respectively. Neither HTLV-1 nor HIV infections were detected. In conclusion, checks for HBV and M. tuberculosis infections should be made before immunosuppressive treatments are started, because of the high prevalence of these potentially life-threatening infections. Other infections should be tested for depending on the patient's risk factors.

Immunoglobulins (Igs) consist of two antigen-binding regions (Fab) and one constant region (Fc). Protein A and protein G are bacterial proteins used for the purification of IgG by virtue of their high affinities for the Fc fragment. Rheumatoid factors are autoantibodies against IgG Fc fragments, which are present in the body under physiological conditions. Little is known about the influence of Fc-binding proteins on the pathogenicity of antibody-induced autoimmune diseases. Pemphigoid diseases are a group of autoimmune subepidermal blistering disorders that includes bullous pemphigoid and mucous membrane pemphigoid. IgGs targeting the non-collagenous NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) are known to induce skin fragility in mice and the depletion of BP180 in keratinocytes. In this study, mAb against NC16A in combination with Fc-binding proteins was found to enhance BP180 depletion. Although mAb against the C-terminus of BP180 does not show pathogenicity in vivo or in vitro, mAb treatment with Fc-binding proteins clearly induced skin fragility in mice and BP180 depletion in keratinocytes. Anti-BP180 mAbs and Fc-binding proteins were colocalized in the cytoplasm and at the basement membrane zone. Cell adhesion strengths were decreased in parallel with BP180 amounts. Clinically, bullous pemphigoid patients had higher rheumatoid factor titers than controls. Anti-BP180 mAb in combination with high-titer rheumatoid factor serum was found to enhance BP180 depletion. Furthermore, saliva from mucous membrane pemphigoid patients contained larger quantities of bacteria and Fc-binding proteins than controls. Our results suggest that Fc-binding proteins (rheumatoid factor or protein G) may enhance the pathogenicity of autoantibodies in pemphigoid diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8+ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, which likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.

Background Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP. Methods Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients. Results There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/β4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17. Conclusion We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP.

Background Epidermolysis bullosa (EB) is a group of hereditary disorders caused by mutations in the genes encoding structural molecules of the dermal-epidermal junction (DEJ). Cell-based therapies such as allogeneic mesenchymal stem/stromal cell (MSC) transplantation have recently been explored for severe EB types, such as recessive dystrophic EB (RDEB). However, hurdles exist in current MSC-based therapies, such as limited proliferation from a single cell source and limited cell survival due to potential allogenic rejection. Objectives We aimed to develop MSCs from keratinocyte-derived induced pluripotent stem cells (iPSCs). Methods Keratinocyte-derived iPSCs (KC-iPSCs) of a healthy human and an RDEB patient were cultured with activin A, 6-bromoindirubin-3′-oxime and bone morphogenetic protein 4 to induce mesodermal lineage formation. These induced cells were subjected to immunohistochemical analysis, flow cytometric analysis and RNA microarray analysis in vitro, and were injected subcutaneously and intravenously to wounded immunodeficient mice to assess their wound-healing efficacy. Results After their induction, KC-iPSC-induced cells were found to be compatible with MSCs. Furthermore, with the subcutaneous and intravenous injection of the KC-iPSC-induced cells into wounded immunodeficient mice, human type VII collagen was detected at the DEJ of epithelized areas. Conclusions We successfully established iPSC-derived MSCs from keratinocytes (KC-iPSC-MSCs) of a normal human and an RDEB patient. KC-iPSC-MSCs may have potential in therapies for RDEB.

Pyoderma gangrenosum is a chronic non-infectious neutrophilic dermatosis that causes undermining ulcers. Topical therapies for the deep ulcers of pyoderma gangrenosum have not been established. To investigate whether negative-pressure wound therapy is effective for a pyoderma gangrenosum ulcer, we used the PICO single use negative-pressure wound therapy system (Smith & Nephew, London, UK) for two pyoderma gangrenosum patients. In these cases, the ulcers decreased in size and necrolytic tissue was removed notably. Moreover, there were no secondary infections nor was there Koebner phenomena. Our cases suggest that portable negative-pressure wound therapy can be a treatment option for deep, intractable ulcers caused by pyoderma gangrenosum. Because portable negative-pressure wound therapy devices afford increased mobility to patients, they can give the patient a better quality of life than standard negative-pressure wound therapy systems do.

Background Epidermolysis bullosa (EB) is a congenital, refractory skin disease and there are no fundamental treatments. Recently, allogenic cell therapies are beginning to be applied as potential treatments, that are based on the concept that the allogenic cells can migrate into the skin and reconstitute the skin components. Although the mechanisms of cell migration into skin are not fully understood, chemokines are regarded as key factors in recruiting bone marrow-derived cells. Objectives Our study aims to elucidate the expression of chemokines in the EB patients. Methods We determined the expression of wound-healing related chemokines in the sera, keratinocytes, and skin tissues of EB patients and compared them to those of healthy volunteers by enzyme-linked immunosorbent assays, quantitative reverse transcription PCR, and immunofluorescence staining. Results The serum levels of CXCL12 and HMGB1 were found to be significantly elevated in the EB patients. Conversely, the serum levels of CCL21 were found to be lower in the EB patients than in healthy controls. In addition, the serum levels of CXCL12 tended to increase and the serum levels of CCL27 tended to decrease with an increase in the affected body surface areas. To detect the origin of the circulating chemokines, we performed immunofluorescence staining. CCL21, CCL27, HMGB1 and CXCL12 were stained more broadly in the EB patient tissues than those in the control tissues. Conclusions These results suggest that fluctuations in chemokine levels may contribute in a coordinated way to the wound-healing process and lend clues toward efficient cell therapies for EB.

Background More than half of patients with pemphigus experience relapse during the disease course. The risk factors and clinical and immunological characteristics of relapse remain largely unclear. Objectives To elucidate the risk factors and clinical features of pemphigus relapse. Methods We carried out a retrospective review of the clinical records of 42 cases of pemphigus at a single centre. Results Sixty-two per cent of patients experienced relapse, usually when oral prednisolone was tapered to around 0 center dot 1 mg kg(-1). In mucocutaneous pemphigus vulgaris (mcPV), the initial doses (mean +/- SD) of prednisolone were significantly lower in patients with relapse (0 center dot 78 +/- 0 center dot 24 mg kg(-1)) than in those without relapse (1 center dot 01 +/- 0 center dot 01 mg kg(-1)). At relapse, mcPV shifted to mucosal dominant PV (mPV; 40%), pemphigus foliaceus (PF) (20%) or 'other' (20%). In contrast, relapsing mPV and PF had the same clinical phenotypes as the initial phenotypes. Patients with both anti-desmoglein (Dsg)1 and anti-Dsg3 antibodies at onset had recurrence with anti-Dsg3 antibodies alone (40%), with both anti-Dsg1 and anti-Dsg3 antibodies (30%), with anti-Dsg1 antibody alone (20%) or were subthreshold (10%). Conclusions mcPV shows transitions in clinical phenotype and autoantibody profile at relapse. At least 1 mg kg(-1) daily of prednisolone, especially for patients with mcPV, and prudent tapering around 0 center dot 1 mg kg(-1) may lead to better outcomes.

Background Bullous pemphigoid (BP) is the most common autoimmune blistering disease. BP180 is the primary autoantigen of BP, and in a portion of BP cases, BP230 is the only target of autoantibodies. Such BP is called BP230-type BP. BP230-type BP tends to show milder clinical phenotypes than conventional BP, but the reason is unclear. The pathogenic roles of autoantibodies and complement activation have been shown in conventional BP, but the distribution of IgG subclasses and the degree of complement deposition in BP230-type BP remain unclear. Objective To compare the distribution of IgG subclasses and the degree of complement deposition in BP230-type BP with those in conventional BP with autoantibodies to BP180 and BP230 (BP180-BP230-type BP). Methods The diagnosis of BP was confirmed by the histopathology of the lesions, the deposition of IgG and complement in the perilesional skin and the presence of circulating autoantibodies to BP180 and BP230. The disease severity was determined by bullous pemphigoid disease area index. The deposition of IgG subclasses and complement deposition were examined by direct immunofluorescence of the perilesional skin in 6 BP230-type BP cases and 11 BP180-BP230-type BP cases. Results Sixty seven percent of BP230-type BP cases show a mild clinical phenotype. All BP230-type BP cases and 82% of BP180-BP230-type BP cases were found to demonstrate the clear deposition of IgG4 at the basement membrane zone of skin specimens. Notably, the deposition of IgG1 and IgG3 was faint or negative in all of the BP230-type BP cases, whereas they were clearly detected in 91% and 64% of the BP180-BP230-type BP cases, respectively. The deposition of complement C3 tended to be weaker in BP230-type BP than in BP180-BP230-type BP. Conclusion The mild clinical phenotype of BP230-type BP may correlate with the weaker deposition of IgG1, IgG3 and complement in the skin lesions.