The clinical efficacy and safety of anti-IgE therapy in recessive dystrophic epidermolysis bullosa.

The treatment of recessive dystrophic epidermolysis bullosa (RDEB) remains challenging. Elevated IgE levels have previously been reported in several RDEB patients. In this prospective, single-centre, open intervention study, elevated IgE levels were seen in 11 out of 12 patients with intense pruritus, and the patients with elevated IgE levels received anti-IgE therapy every 4 weeks for at least three cycles. Compared with the baseline, 10 patients with RDEB had good clinical outcomes with enhanced wound healing, a reduction in Birmingham (epidermolysis bullosa) EB severity score by 15%, a reduction in affected body surface area by 23.3%, amelioration of skin inflammation, and an increase in type VII collagen deposition by 13.1-fold. All the patients had a good tolerance to anti-IgE therapy. Furthermore, patients with higher IgE levels tended to have higher disease severity and more favorable clinical outcomes. Our report also suggested the potential role of IgE in the pathogenesis of inflammatory conditions associated with RDEB. (ChiCTR1900021437).

Visualization of autoantibodies and neutrophils in vivo identifies novel checkpoints in autoantibody-induced tissue injury.

In several autoimmune diseases, e.g., pemphigoid disease (PD), autoantibodies are the direct cause of pathology. Albeit key requirements for antibody-mediated diseases were identified, their interactions and exact temporal and spatial interactions remained elusive. The skin is easily accessible for imaging. Thus, we selected epidermolysis bullosa acquisita (EBA), a PD with autoantibodies to type VII collagen (COL7), to visualize interactions of autoantibodies, target tissue and effector cells (neutrophils). Following injection into mice, anti-COL7 IgG bound to the dermal-epidermal junction (DEJ) within minutes. We unexpectedly observed an inhomogeneous distribution of autoantibodies along the DEJ. Thus, we hypothesized that specific external triggers may affect autoantibody distribution. Indeed, mechanical irritation led to an increased autoantibody binding along the DEJ. Subsequently, anti-COL7 IgG was injected into mice expressing green fluorescent protein under the LysM promoter (LysM-eGFP) mice. This allows to visualize myeloid cells in vivo in these animals. Using multiphoton imaging, we observed a limited extravasation of LysM-eGFP+ cells into skin was observed within 24 hours. Intriguingly, LysM-eGFP+ cells did not immediately co-localize with autoantibodies, which was only noted at later time points. Of note, interactions of LysM-eGFP+ with the autoantibodies at the DEJ were short-lived. Collectively, our results define the following checkpoints for autoantibody-induced tissue injury: (i) autoantibody egress to target tissue influenced by mechanical trigger factors, (ii) neutrophil recruitment into the vicinity of autoantibody deposits and (iii) short-term neutrophil localization to these deposits, as well as (iv) delayed recruitment of neutrophils with subsequent autoantibody-induced inflammation.

Efficacy of Human Placental-Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal-epidermal junction (DEJ). We recently demonstrated that human cord blood-derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1-/- ) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental-derived stem cells (HPDSCs), currently being used as human leukocyte antigen-independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full-term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1-/- mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short-term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1-/- mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti-C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018;7:530-542.

Vancomycin Mediates IgA Autoreactivity in Drug-Induced Linear IgA Bullous Dermatosis.

Vancomycin (VCM) is known to induce linear IgA bullous dermatosis (LAD). However, in contrast to conventional LAD, in which circulating IgA autoantibodies against basement membrane proteins are commonly detected, patient sera from VCM-induced LAD yields negative results in indirect immunofluorescence microscopy, and the targeted autoantigen remains undetermined. By using sera from a typical patient with VCM-induced LAD, we identified that co-incubation of sera with VCM resulted in linear IgA deposition at the basement membrane zone by indirect immunofluorescence. Patient sera reacted with the dermal side of 1 mol/L NaCl-split skin and with the recombinant noncollagenous (i.e., NC1) domain of type VII collagen by both immunoblot and ELISA in the presence of VCM. The investigation of an additional 13 patients with VCM-induced LAD showed that 10 out of the 14 sera (71.4%) reacted with the NC1 domain of type VII collagen by ELISA when spiked with VCM, whereas only 4 (28.6%) tested positive without it. The enhancement of reactivity to NC1 by VCM, as determined by optical density via ELISA, was observed in 10 out of the 14 sera (71.4%). These findings indicate that type VII collagen is a target autoantigen in VCM-induced LAD and that VCM mediates IgA autoreactivity against type VII collagen, providing an insight into mechanisms involved in drug-induced autoimmune disease.

Epidermal aspects of type VII collagen: Implications for dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita.

Type VII collagen (COL7), a major component of anchoring fibrils in the epidermal basement membrane zone, has been characterized as a defective protein in dystrophic epidermolysis bullosa and as an autoantigen in epidermolysis bullosa acquisita. Although COL7 is produced and secreted by both epidermal keratinocytes and dermal fibroblasts, the role of COL7 with regard to the epidermis is rarely discussed. This review focuses on COL7 physiology and pathology as it pertains to epidermal keratinocytes. We summarize the current knowledge of COL7 production and trafficking, its involvement in keratinocyte dynamics, and epidermal carcinogenesis in COL7 deficiency and propose possible solutions to unsolved issues in this field.

Epidermolysis Bullosa (EB) Acquisita in an Adult Patient with Previously Unrecognized Mild Dystrophic EB and Biallelic COL7A1 Mutations.

Circulating anti-type VII collagen autoantibodies are frequently detected in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, evidence supporting their pathogenic role in inducing epidermolysis bullosa acquisita (EBA) has been provided for only one individual with dominant dystrophic epidermolysis bullosa (DDEB). We describe here a patient who presented with dystrophic toenails since early childhood and developed trauma-induced skin blisters and oral erosions at age 26 years. Direct immunofluorescence showed IgG deposits with a u-serrated pattern along the cutaneous basement membrane zone, while no change in the expression of collagen VII could be detected by antigen mapping. High-titre anti-collagen VII antibodies were detected by enzyme-linked immunoassay (ELISA). In parallel, sequencing of epidermolysis bullosa (EB) genes identified compound heterozygous COL7A1 missense c.410G>A (p.Arg137Gln) and splicing c.3674C>T (p.Ala1225_Gln1241del) mutations, previously unrecognized in dystrophic epidermolysis bullosa (DEB). Thus, our patient had RDEB "nails-only" and developed mechanobullous EBA in adulthood. These data support a pathogenic role of circulating autoantibodies to collagen VII in inducing EBA in selected patients with DEB. Unforeseen worsening of skin symptoms in DEB should prompt laboratory investigations for EBA.

Case of epidermolysis bullosa acquisita with concomitant anti-laminin-332 antibodies.

Subepidermal autoimmune blistering disease including bullous pemphigoid, pemphigoid gestationis, mucous membrane pemphigoid, anti-laminin-γ1 pemphigoid, linear immunoglobulin A bullous disease and epidermolysis bullosa acquisita (EBA), are all characterized by direct immunofluorescence microscopy or immunoglobulin deposition on the basement membrane zone. Among them, EBA is a rare acquired subepidermal autoimmune blistering disease of the skin and mucous membranes reactive with type VII collagen, a major component of the epidermal basement membrane zone. Anti-laminin-332-type mucous membrane pemphigoid has pathogenic autoantibodies against laminin-332, which is a basement membrane heterotrimeric protein composed of α3, ß3 and γ2 laminin chains. We describe a 73-year-old Japanese man presenting with multiple, annular, tense blisters on the lower legs and oral lesions. Despite the severe clinical manifestations, the disease was successfully controlled by combination therapy of oral prednisolone and mizoribine. This case was confirmed to have autoantibodies to both type VII collagen and laminin-332 α3 chain by indirect immunofluorescence of 1 mol NaCl-split normal human skin, various immunoblot analyses and enzyme-linked immunosorbent assays. This case was a rare case of EBA with concomitant anti-laminin-332 antibodies.

Structural proteins of the dermal-epidermal junction targeted by autoantibodies in pemphigoid diseases.

The dermal-epidermal junction consists of a network of several interacting structural proteins that strengthen adhesion and mediate signalling events. This structural network consists of hemidesmosomal-anchoring filament complexes connecting the basal keratinocytes to the basement membrane. The anchoring filaments in turn interact with the anchoring fibrils to attach the basement membrane to the underlying dermis. Several of these structural proteins are recognized by autoantibodies in pemphigoid diseases, a heterogeneous group of clinically and immunopathologically diverse entities. Targeted proteins include the two intracellular plakins, plectin isoform 1a and BP230 (also called bullous pemphigoid antigen (BPAG) 1 isoform e (BPAG1e)). Plectin 1a and BP230 are connected to the intermediate filaments and to the cell surface receptor α6ß4 integrin, which in turn is connected to laminin 332, a component of the anchoring filaments. Further essential adhesion proteins are BP180, a transmembrane protein, laminin γ1 and type VII collagen. Latter protein is the major constituent of the anchoring fibrils. Mutations in the corresponding genes of these adhesion molecules lead to inherited epidermolysis bullosa emphasizing the importance of these proteins for the integrity of the dermal-epidermal junction. This review will provide an overview on the structure and function of the proteins situated in the dermal-epidermal junction targeted by autoantibodies.

Unusual presentation of systemic lupus erythematosus.

Bullous systemic lupus erythematosus is a rare distinctive subepidermal blistering disorder that can occur in patients with systemic lupus erythematosus (SLE). It is histologically characterized by a neutrophil-predominant infiltrate in the subepidermal region with deposition of immunoglobulins (IgG, IgA, IgM), C3, and auto-antibodies against collagen VII. Herein, we report a case of a 13-year-old girl who presented with bullous SLE and Class III lupus nephritis, which is extremely rare at this age.

Clinical features and diagnosis of epidermolysis bullosa acquisita.

INTRODUCTION: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune blistering disease of skin and mucous membranes. EBA is caused by autoantibodies against type VII collagen, which is a major component of anchoring fibrils, attaching epidermis to dermis. Binding of autoantibodies to type VII collagen leads to skin fragility and, finally, blister formation. The clinical picture of EBA is polymorphic, with several distinct phenotypes being described. Despite recent progress in understanding the pathophysiology of EBA, its diagnosis is still challenging. Areas covered: This review provides an update on the clinical manifestations and diagnostic methods of EBA. We searched PubMed using the terms 'epidermolysis bullosa acquisita' covering articles in English between 1 January 2005 and 31 May 2016. Relevant older publications were retrieved form cited literature. Expert commentary: While the clinical picture is highly variable, diagnosis relies on direct immunofluorescence (IF) microscopy of a perilesional skin biopsy. Linear deposits of IgG, IgA and/or C3 along the dermal-epidermal junction with an u-serrated pattern are diagnostic for EBA alike the detection of serum autoantibodies against type VII collagen. Several test systems for the serological diagnosis of EBA have recently become widely available. In some patients, sophisticated diagnostic approaches only available in specialized centers are required.

Coexistence of acquired hemophilia A and epidermolysis bullosa acquisita: Two case reports and published work review.

Epidermolysis bullosa acquisita (EBA) is a rare chronic subepidermal bullous autoimmune disease. The occurrence of acquired hemophilia A (AHA) is low and so the coexistence of EBA and AHA is extremely rare. We herein described a case of EBA coexisting with AHA and a case of EBA coexisting with AHA and hepatitis B. These EBA may be related to the pathogenesis of AHA. In this study, we analyzed the clinical features in the two Chinese cases of EBA coexisting with AHA, and found esophageal hemorrhage and hematemesis were the main symptoms of both patients. Cyclosporin, prednisone and lamivudine effectively control EBA with AHA and hepatitis B. The dose of cyclosporin should be more than 4 mg/kg per day and the period of treatment should be longer than 5 months to reduce the risk of EBA co-occurring with AHA.

Sublamina densa-type linear IgA bullous dermatosis with IgA autoantibodies specific for type VII collagen: a case report and clinicopathological review of 32 cases.

Linear IgA bullous dermatosis (LABD) is a rare autoimmune bullous disorder characterized by linear deposits of IgA at the basement membrane zone(BMZ) and/or by circulating IgA anti-BMZ antibodies. Comparing with other immuno-bullous diseases, LABD represents a heterogeneous disease entitywith diversity of pathogenic IgA autoantibodies to different hemidesmosomal antigens and an association with malignancies and occasional drug use. We herein present an 82-year-old Japanese man with LABD, whose indirect immunofluorescence using 1M NaCl-split skin showed positive staining for IgA at the dermal side alone. Fluorescence overlay antigen mapping using laser scanning confocal microscopy (FOAM-LSCM) was employed to examine the in vivo bound patient's IgA, which was specific for type VII collagen (COL7), a prominent antigen of the sublamina densa. One year later, he developed malignant lymphoma, suggesting the diagnosis of paraneoplastic LABD. We reviewed 32 cases of sublamina-densa type LABD with anti-COL7 IgA antibodies thus far reported in the literature to compare the clinicopathological characteristics of this rare disease variant and emphasize that COL7 is the main autoantigen in sublamina densa disease.

Autoimmunity and Cytokine Imbalance in Inherited Epidermolysis Bullosa.

In order to evaluate the serum anti-skin autoantibodies and cytokine concentrations in patients with different epidermolysis bullosa (EB) types and severity, 42 EB patients and 38 controls were enrolled. Serum anti-skin antibodies were significantly higher in the patients than in the controls (p = 0.008, p < 0.001, p < 0.001, p < 0.001 and p < 0.001 for desmoglein 1 (DSG1) desmoglein 3 (DSG3), bullous pemphigoid 180 (BP180), BP230 and type VII collagen (COL7), respectively). The same trend was observed for interleukin (IL)-1ß, IL-2, IL-6, IL-10, tumor necrosis factor-ß, and interferon-γ (p < 0.001, p < 0.001, p < 0.001, p = 0.008, p < 0.001 and p = 0.002, respectively). Increases in anti-skin antibodies and cytokine concentrations were higher in patients with recessive dystrophic EB than in those with different types of EB, in generalized cases than in localized ones, and in patients with higher Birmingham Epidermolysis Bullosa Severity (BEBS) scores than in those with a lower score. The BEBS score was directly correlated with BP180, BP230, COL7 (p = 0.015, p = 0.008 and p < 0.001, respectively) and IL-6 (p = 0.03), whereas IL-6 appeared significantly associated with DSG1, DSG3, BP180, BP230 and COL7 (p = 0.015, p = 0.023, p = 0.023, p = 0.015 and p = 0.005, respectively). This study showed that autoimmunity and inflammatory responses are frequently activated in EB, mainly in severe forms, suggesting the use of immunosuppressive drugs or biologicals that are active against pro-inflammatory cytokines to reduce clinical signs and symptoms of disease.

Radiosensitive Hematopoietic Cells Determine the Extent of Skin Inflammation in Experimental Epidermolysis Bullosa Acquisita.

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation. Identifying the factors that influence this disease variability could provide unrecognized insights into pathogenesis. We established a novel Ab transfer-induced model of epidermolysis bullosa acquisita (EBA), an autoimmune disease characterized by (muco)-cutaneous blistering caused by anti-type VII collagen (COL7) autoantibodies. Blistering after anti-COL7 IgG (directed against the von Willebrand factor A-like domain 2) transfer showed clear variability among inbred mouse strains, that is, severe cutaneous blistering and inflammation in C57BL/6J and absence of skin lesions in MRL/MpJ mice. The transfer of anti-COL7 IgG into irradiated, EBA-resistant MRL/MpJ mice, rescued by transplantation with bone marrow from EBA-susceptible B6.AK-H2k mice, induced blistering. To the contrary, irradiated EBA-susceptible B6.AK-H2k mice that were rescued using MRL/MpJ bone marrow were devoid of blistering. In vitro, immune complex activation of neutrophils from C57BL/6J or MRL/MpJ mice showed an impaired reactive oxygen species release from the latter, whereas no differences were observed after PMA activation. This finding was paralleled by divergent expression profiles of immune complex-activated neutrophils from either C57BL/6J or MRL/MpJ mice. Collectively, we demonstrate that radiosensitive cells determine the varying extent of skin inflammation and blistering in the end-stage effector phase of EBA.

Caspase-1-independent IL-1 release mediates blister formation in autoantibody-induced tissue injury through modulation of endothelial adhesion molecules.

Although reports documented aberrant cytokine expression in autoimmune bullous dermatoses (AIBDs), cytokine-targeting therapies have not been established in these disorders. We showed previously that IL-6 treatment protected against tissue destruction in experimental epidermolysis bullosa acquisita (EBA), an AIBD caused by autoantibodies to type VII collagen (COL7). The anti-inflammatory effects of IL-6 were mediated by induction of IL-1ra, and prophylactic IL-1ra administration prevented blistering. In this article, we demonstrate elevated serum concentrations of IL-1ß in both mice with experimental EBA induced by injection of anti-COL7 IgG and in EBA patients. Increased IL-1α and IL-1ß expression also was observed in the skin of anti-COL7 IgG-injected wild-type mice compared with the significantly less diseased IL-1R-deficient or wild-type mice treated with the IL-1R antagonist anakinra or anti-IL-1ß. These findings suggested that IL-1 contributed to recruitment of inflammatory cells into the skin. Accordingly, the expression of ICAM-1 was decreased in IL-1R-deficient and anakinra-treated mice injected with anti-COL7. This effect appeared to be specifically attributable to IL-1 because anakinra blocked the upregulation of different endothelial adhesion molecules on IL-1-stimulated, but not on TNF-α-stimulated, cultured endothelial cells. Interestingly, injection of caspase-1/11-deficient mice with anti-COL7 IgG led to the same extent of skin lesions as in wild-type mice. Collectively, our data suggest that IL-1, independently of caspase-1, contributes to the pathogenesis of EBA. Because anti-IL-1ß in a prophylactic setting and anakinra in a quasi-therapeutic setting (i.e., when skin lesions had already developed) improved experimental EBA, IL-1 appears to be a potential therapeutic target for EBA and related AIBDs.

Conditional depletion of mast cells has no impact on the severity of experimental epidermolysis bullosa acquisita.

The role of mast cells (MCs) in autoimmunity is the matter of an intensive scientific debate. Based on observations in different MC-deficient mouse strains, MCs are considered as fundamental players in autoimmune diseases. However, most recent data suggest that the outcome of such diseases is strongly affected by the individual mouse strain used. By the use of two c-Kit mutant MC-deficient mouse strains and one c-Kit-independent strain, we here investigated the role of MCs in a systemic Ab transfer model of epidermolysis bullosa acquisita, a subepidermal autoimmune blistering skin disease characterized by autoantibodies against type VII collagen. While C57BL/6J-Kit(W-sh/W-sh) mice developed an unexpected increased blistering phenotype, no significant differences to WT controls were seen in WBB6F1 -Kit(W/W-v) or the novel Mcpt5-Cre iDTR animals. Interestingly, in a local Ab transfer model, which induces a localized disease, we showed that application of high concentrations of anti-COL7 (where COL7 is type VII collagen) Abs induced MC activation and MC-dependent edema formation that did, however, not contribute to blister induction. Our results indicate that in the autoimmune disorder epidermolysis bullosa acquisita MCs do not contribute to the immune-mediated tissue injury. Modern c-Kit mutant-independent MC-deficient mouse strains will help to further redefine the role of MCs in autoimmunity.

Detection of Type VII Collagen Autoantibodies Before the Onset of Bullous Systemic Lupus Erythematosus.

IMPORTANCE: Anti-type VII collagen autoantibodies are often detectable in patients with bullous systemic lupus erythematosus (BSLE). However, the timing of their appearance preceding the onset of disease is unknown to date. OBSERVATIONS: We report the case of a 50-year-old woman with a history of SLE who was seen with vesicles and bullae around her lips, trunk, axillae, arms, and thighs. Histologic analysis and immunofluorescence and immunoblot studies confirmed the diagnosis of BSLE. Immunoblotting and enzyme-linked immunosorbent assay studies of the patient's serum obtained 3 months before the onset of BSLE showed the presence of anti-type VII collagen autoantibodies. Levels of anti-type VII collagen IgG increased after bullous lesions appeared. Within 1 month after initiating dapsone therapy and increasing the dosage of prednisone, skin lesions promptly resolved. One year after the onset of BSLE, the anti-type VII collagen IgG decreased below levels observed before the inception of the bullous lesions. CONCLUSIONS AND RELEVANCE: Anti-type VII collagen autoantibodies can precede the clinical appearance of BSLE. The subsequent increase and decrease in levels of circulating anti-type VII collagen autoantibodies, which mirrored skin disease activity, support a potential role in their initiation of disease.

GM-CSF modulates autoantibody production and skin blistering in experimental epidermolysis bullosa acquisita.

GM-CSF activates hematopoietic cells and recruits neutrophils and macrophages to sites of inflammation. Inhibition of GM-CSF attenuates disease activity in models of chronic inflammatory disease. Effects of GM-CSF blockade were linked to modulation of the effector phase, whereas effects on early pathogenic events, for example, Ab production, have not been identified. To evaluate yet uncharacterized effects of GM-CSF on early pathogenic events in chronic inflammation, we employed immunization-induced epidermolysis bullosa acquisita (EBA), an autoimmune bullous disease caused by autoantibodies to type VII collagen. Compared to wild-type mice, upon immunization, GM-CSF(-/-) mice produced lower serum autoantibody titers, which were associated with reduced neutrophil numbers in draining lymph nodes. The same effect was observed in neutrophil-depleted wild-type mice. Neutrophil depletion in GM-CSF(-/-) mice led to a stronger inhibition, indicating that GM-CSF and neutrophils have additive functions. To characterize the contribution of GM-CSF specifically in the effector phase of EBA, disease was induced by transfer of anti-type VII collagen IgG into mice. We observed an increased GM-CSF expression, and GM-CSF blockade reduced skin blistering. Additionally, GM-CSF enhanced reactive oxygen species release and neutrophil migration in vitro. In immunization-induced murine EBA, treatment with anti-GM-CSF had a beneficial effect on established disease. We demonstrate that GM-CSF modulates both autoantibody production and skin blistering in a prototypical organ-specific autoimmune disease.