Rituximab in the Management of Autoimmune Bullous Diseases: A Treatment-Resistant Case Series from a Single Central European Referral Center.

Background and Objectives: Rituximab (RTX) has been the predominant treatment for autoimmune bullous diseases (AIBDs). The objective of this research was to assess the advantages and safety characteristics of RTX treatment in individuals with AIBD. This assessment focused on clinical remission and a reduction in glucocorticosteroid usage, its effect on the titers of autoantibodies targeting desmoglein-1 (DSG-1) and desmoglein-3 (DSG-3), and adverse occurrences during a 12-month follow-up period in a dermatology department within a Central European university context. Materials and Methods: Our case series involved eleven patients, including eight patients with pemphigus vulgaris, two with pemphigus foliaceus, and one with epidermolysis bullosa acquisita. They received a 1 g dose of rituximab, repeated over a two-week interval. Results: The reduction in a prednisone-equivalent dosage after 2, 6, and 12 months following the second RTX infusion was 65.05%, 73.99%, and 76.93%, in that order. The titers of antibodies against DSG-1 exhibited reductions of 43.29%, 75.86%, and 54.02% at 2, 6, and 12 months, respectively. By contrast, the antibody concentrations targeting DSG-3 displayed a decrease of 27.88%, 14.48%, and 5.09% at the corresponding time points. Over the course of the 12-month monitoring period, 18.18% of patients experienced disease relapse, while the remaining individuals achieved either complete or partial remission with minimal or no therapy. Adverse effects were noted in 36.36% of the patient population; they were mild, and no serious adverse effects were reported. Conclusions: RTX represents an efficacious and well-tolerated therapeutic option for the management of AIBD and merits consideration in cases of refractory AIBD. However, further research is imperative to delineate the most optimal dosage, dosing frequency, and total quantity of maintenance infusions required. Additionally, there is a compelling need for studies that explore the impact of RTX on individuals with AIBD who do not exhibit a significant reduction in anti-desmoglein autoantibody levels.

Novel nanoparticle AuNCs conjugated with Desmoglein-3 antibody for FL/CT dual-mode targeted imaging and precise treatment of lung squamous cell carcinoma.

Due to lack of effective, early and non-invasive diagnostic as well as treatment tools, the surgical treatment opportunities for lung squamous cell carcinoma (SCC) are limited, resulting in high mortality rates. Therefore, the combination of targeted recognition and precise treatment of lung SCC is of great significance. In this study, a multifunctional nanoparticle is designed and synthesized, which specifically identifies lung SCC cells for target imaging and therapy. Desmoglein-3 (Dsg-3), a transmembrane glycoprotein found in desmosomes, is highly expressed in lung SCC cells. Gold nanoclusters (AuNCs) conjugated with Dsg-3 antibodies to form Au-Dsg-3 through coupling reaction. The results showed that the fluorescence imaging (FI) intensity and computed tomography (CT) signal of Au-Dsg-3 significantly increased within 6 h in vitro and in vivo, achieving dual-modal imaging to detect lung SCC effectively. Besides, Au-Dsg-3 even integrates targeted photothermal therapy (PTT) characteristics in a single nanoparticle. When exposed to near-infrared radiation (NIR), the temperature of the tumor site increased rapidly and reached a high temperature of 53.3 °C after 600 s, causing tumor ablation and growth inhibition. In summary, Au-Dsg-3 provides a key platform for targeted biological imaging and collaborative PTT, which demonstrates good performance on lung SCC.

Genome-wide association study identifying variants related to performance and injury in high-performance athletes.

A growing body of evidence exists supporting the role that genetic variation plays in athletic performance and injury. This study sought to identify genetic variants associated with performance and lower limb musculoskeletal injury in a high-level athletic cohort. A total of 126 Estonian National Team members (Olympic athletes and participants of International Championships) (104 males, 82.5%) underwent a genome-wide association analysis between 2017 and 2018, to identify single-nucleotide polymorphisms (SNPs) associated with performance and/or injury. The athletic cohort was stratified within each sport based on performance and whether they were a medalist (n = 29) or not (n = 97), whether they sustained an injury (n = 47) or not (n = 79), and the type of injury (patella tendinopathy n = 22, Achilles tendinopathy n = 17, hamstring injury n = 3, anterior cruciate ligament rupture n = 6). Three SNPs demonstrated strong genome-wide association with athletic performance (podium/medalist versus not), including DSG1 (rs10502567, OR 14.3) and DSG4 (rs73410248, OR 17.4), while 76 SNPs demonstrated suggestive significance. Overall, 37 SNPs gave genome-wide suggestive association with any type of injury, including PAPPA2 (rs11580456, OR 13.8) and MAS1 (rs220735, rs170219, OR 3.1) which demonstrated positive signal with multiple SNPs. Several genes demonstrated positive association for the specific injury types, including COL22A1 (rs3924862) and PLXNA2 (rs11799530), as well as PAPPA2 (rs11580456), DOK5 (rs73142922), GNG12 (rs28435277), and DAP (rs267959, rs2930047, rs1080440, rs267939). The current study identified genetic variants associated with high-level athletic performance and musculoskeletal injury. Further work is required to permit integration of this and future knowledge into individualized training practices, as well as injury mitigation and rehabilitation programs.

Desmosomal Junctions and Connexin-43 Remodeling in High-Pacing-Induced Heart Failure Dogs.

BACKGROUND: While desmosomal junctions and gap junction remodeling are among the arrhythmogenic substrates, the fate of desmosomal and gap junctions in high-pacing-induced heart failure remains unclear. This aim of this study was to determine the fate of desmosomal junctions in high-pacing-induced heart failure. METHODS: Dogs were randomly divided into 2 equal groups, a high-pacing-induced heart failure model group (heart failure group, n = 6) and a sham operation group (control group, n = 6). Echocardiography and cardiac electrophysiological examination were performed. Cardiac tissue was analyzed by immunofluorescence and transmission electron microscopy. The expression of desmoplakin and desmoglein-2 proteins was detected by western blot. RESULTS: A significant decrease in ejection fraction, significant cardiac dilatation, diastolic and systolic dysfunction, and ventricular thinning occurred after 4 weeks in high-pacing-induced dog model of heart failure. Effective refractory period action potential duration at 90% repolarization was prolonged in the heart failure group. Immunofluorescence analysis and transmission electron microscopy demonstrated connexin-43 lateralization accompanies desmoglein-2 and desmoplakin remodeling in the heart failure group. Western blotting showed that the expression of desmoplakin and desmoglein-2 proteins was higher in heart failure than in normal tissue. CONCLUSION: Desmosome (desmoglein-2 and desmoplakin) redistribution and desmosome (desmoglein-2) overexpression accompanying connexin-43 lateralization were parts of a complex remodeling in high-pacing-induced heart failure.

Modulation of pulmonary desmosomes by inhaler therapy in preterm-born children with bronchopulmonary dysplasia.

Despite evidence demonstrating persistent lung function deficits in preterm-born children, especially in those who had bronchopulmonary dysplasia (BPD) in infancy, the underlying biological mechanisms explaining these lung function deficits remain poorly understood. We characterised the exhaled breath condensate (EBC) proteome in preterm-born children, with and without BPD; and before and after inhaler treatment. EBC from children aged 7-12 years, from the Respiratory Health Outcomes in Neonates (RHiNO) study, were analysed by Nano-LC Mass Spectrometry with Tandem Mass Tag labelling. Children with percent predicted forced expiratory volume in 1 second ≤ 85% were enrolled to a 12-week blinded randomised trial of inhaled corticosteroids alone (ICS) or with long-acting ß2-agonist (ICS/LABA) or placebo. EBC was analysed from 218 children at baseline, and 46 children received randomised inhaled therapy. 210 proteins were detected in total. For the 19 proteins present in every sample, the desmosome proteins: desmoglein-1, desmocollin-1 and plakoglobin were significantly decreased, and cytokeratin-6A was increased in preterm-born children with BPD when compared to preterm- and term-born controls. ICS/LABA treatment significantly increased abundance of desmoglein-1, desmocollin-1 and plakoglobin in the BPD group with low lung function, and significantly increased plakoglobin in those without BPD. No differences were noted after ICS treatment. Exploratory analyses of proteins not detected in all samples suggested decreased abundance of several antiproteases. This study provides proteomic evidence of ongoing pulmonary structural changes with decreased desmosomes in school-aged preterm-born children with BPD and low lung function, which was reversed with combined inhaled corticosteroids and long-acting ß2-agonists therapy.

Evaluation of clinical factors and outcome of systemic glucocorticoid therapy discontinuation in patients with pemphigus achieving complete remission.

Therapy discontinuation of systemic glucocorticoid treatment for pemphigus remains uncertain at the clinical end point of complete remission. The objective of this study was to identify the factors associated with achieving complete remission off therapy (CROT) and analyze the occurrence of relapse after therapy discontinuation. A retrospective cohort study was conducted at the Department of Dermatology of Peking University First Hospital. A total of 447 patients with pemphigus treated from 2005 to 2020 were identified. Univariate and multivariate analyses were conducted to analyze the associated factors of CROT and to evaluate the outcomes. The mean age was 48 years (±13.4 years), and 54.6% of the patients were women. During a median follow-up of 59 months (43-87.5 months), 160 of 447 (35.8%) patients achieved CROT after a median treatment duration of 51 months (38-66.2 months). Patients with a shorter therapy duration to complete remission on minimal therapy and negative desmoglein antibodies tested in remission were more likely to achieve early CROT. Thirty-five of 160 (21.9%) patients experienced relapse after CROT. Patients who discontinued therapy without guidance experienced significantly faster and higher occurrences of relapse than those withdrawing under guidance (log-rank p = 0.01). Minimal therapy maintenance ≤8 months from complete remission on minimal therapy and positive desmoglein antibodies tested at withdrawal increased the risk of early relapse after CROT.

Deregulated phenotype of autoreactive Th17 and Treg clone cells in pemphigus vulgaris after in-vitro treatment with desmoglein antigen (Dsg-3).

The loss of balance between regulatory T (Treg) and T helper 17 (Th17) causes loss of tolerance against desmoglein (Dsg)-3 leading to pemphigus vulgaris (PV), an autoimmune bullous skin disorder associated with autoantibodies against Dsg-3. We aimed to elucidate the complex relationship of Th17 and Treg cells, their molecules, and the underlying mechanism in the development of PV disease. Using cytokine secretion assays, Th17 and Treg cells were sorted by FACS Aria-III within Dsg-3-responsive PBMC population and homogeneous T cell clones were generated in-vitro. Different cell surface molecules like CD25, GITR, CD122, CD152, CD45RO, IL-23R, STAT3, STAT5, CD127, HLA-DR, CCR4, CCR5, CCR6 and CCR7 were studied. The functional response of Th17 and Treg cells were elucidated by measuring the levels of various cytokines released by IL-10 and IL-17 T cells. The mRNA expression of transcription factors (FoxP3 and RORγt) was also analyzed. IL-17 secreting (Th17) cells with phenotype CD4+IL-17+ were greatly increased and IL-10 secreting (Treg) cells with phenotype CD4+IL-10+ were reduced in PV cases than healthy controls. The qPCR analysis showing high expression of retinoic acid receptor-related orphan receptor gamma (RORγt) mRNA in comparison to forkhead box P3 (FoxP3) mRNA confirmed the development of pro-inflammatory Th17 response in PV. Further, the cytokine profile of pro-inflammatory and anti-inflammatory cytokines suggested defective suppressive functions in Treg cells with high inflammatory response. Our findings indicate that autoantigen Dsg-3 specifically allows the proliferation of IL-17 secreting T cells though has a negative effect on IL-10 secreting T cells leading to dysregulation of immunity in PV patients. This antagonistic relationship between Dsg-3-specific Th17 and Treg cells may be critical for the onset and persistence of inflammation in PV cases.

SARS-CoV-2 Infection in Pemphigus Vulgaris Two Weeks after Rituximab Therapy with Total Recovery: A Case Report.

The mortality risk factors for Corona Virus Disease-19 (COVID-19) infection (caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)) include advanced age, male sex, certain comorbidities, and immunosuppression (1). Pemphigus vulgaris is a rare mucocutaneous autoimmune disease with autoantibodies against desmosomal desmoglein-1 and desmoglein-3, resulting in acantholysis and blister formation. This epithelial barrier defect increases susceptibility to infections, which may lead to relapses (2). Additionally, therapy-associated immunosuppression can lead to severe infections. Corticosteroids are the mainstay therapy. For moderate and severe pemphigus, rituximab is recommended in first-line treatment along with other immunosuppressants, and it may also be added in refractory cases. It is a monoclonal antibody against CD20 with long-lasting B-cell depletion potency. Recovery of B-cell function may last from one to seven years. Consequently, patients receiving rituximab cannot produce enough COVID-19 specific plasma cells, leading to a severe course of COVID-19 (2). Shashidi-Dadras et al. reported five mild COVID-19 cases among 167 patients with pemphigus who had received rituximab one to five years earlier. The authors presumed rituximab use within five years increases COVID-19 susceptibility regardless the number of courses received (3). Among 48 patients with pemphigus treated with rituximab within five years, Uzuncakmak et al. reported one mild case of COVID-19 (in a patient who had received a single course seven months earlier) (4). In another study, high titers of SARS-CoV-2 antibodies and high counts of antibody-secreting cells were associated with severe COVID-19 (5), which may be the consequence of antibody-dependent enhancement (6). Mahmoudi et al. concluded that B-cells may not be necessary for recovery in COVID-19, but they may protect from reinfection (7). Considering these data, rituximab should be postponed during the pandemic (8). In exceptional cases, it may be applied with careful consideration of the risk-benefit ratio (2,4). Patients should be monitored for signs of COVID-19 before and during treatment. A 63-year-old woman with pemphigus vulgaris presented at our department with widespread skin lesions. Comorbidities included hypertension, hypothyroidism, and glaucoma. Diagnosis was established based on histology and direct and indirect immunofluorescent microscopy results. Both desmoglein-1 and desmoglein-3 autoantibodies were detectable by ELISA. The patient was initially treated with low-dose systemic methylprednisolone (8 mg/day), because glaucoma contraindicated a higher dose. Azathioprine was subsequently started (gradually increased from 0.6 to 2.5 mg/kg/day). Continuous mucocutaneous progression 4 weeks later led to the decision to add rituximab therapy. The patient was confirmed as SARS-CoV-2 negative and received 1000 mg 12 weeks after starting glucocorticoid treatment. Two weeks later, she developed fever and became SARS-CoV-2 positive, and therefore the second rituximab treatment had to be cancelled. The patient had fever for six weeks without any other complaints, hospitalization was not required, and immunosuppression was continued with 8 mg methylprednisolone and 2.5 mg/kg azathioprine. Two weeks after recovery, she was diagnosed with pulmonary embolism, but recovered completely. Pulmonary embolism is a relatively common complication of COVID-19 which may be triggered by inactivity, loss of body fluids due to fever, a hypercoagulable state, and direct toxic venous endothelial damage caused by the virus (9). At a follow-up 4 months later, minimal skin lesions and significantly decreased desmoglein-1 and desmoglein-3 titers were observed. Azathioprine and methylprednisolone therapy were continued, and a second dosage of rituximab was given 7 months from the first one without any side-effects. We conclude that rituximab is a highly effective therapy in pemphigus, but the risk-benefit ratio should be carefully considered during the COVID-19 pandemic. We have not observed irreversible or permanent consequences of its administration, but our patient had a potentially lethal complication, pulmonary embolism, which may be associated with a more severe COVID-19 course due to immunosuppression. Total recovery was observed despite COVID-19 shortly after the initiation of rituximab.

Pemphigus vulgaris: a case report.

Pemphigus vulgaris (PV) is an autoimmune mucocutaneous disorder of the oral cavity and is the most common subtype of pemphigus. The etiology remains obscure, although the disease is characterized by autoantibodies directed against the desmoglein component of the keratinocytes. It manifests clinically as vesicle, bullae or erosions of skin and mucous membrane and histopathologically shows the presence of acantholysis. The presence of exclusive oral lesions initially increases the chances of misdiagnosing the disease as another condition, posing diagnostic, therapeutic and prognostic difficulties, consequently prompt diagnosis and treatment can prevent untoward consequences. Demonstration of IgG antibodies against desmoglein in Immunofluroscence confirms the diagnosis. In here we report a case of a 55-year-old female patient suffering from PV emphasizing the significance of clinical examination, pertinent investigations, treatment rendered and its outcome.

Desmoglein-2 is important for islet function and ß-cell survival.

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic ß-cells. Although ß-cell targeted autoimmune processes and ß-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports ß-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing ß-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced ß-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine ß-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of ß-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.

Myocarditis-like Episodes in Patients with Arrhythmogenic Cardiomyopathy: A Systematic Review on the So-Called Hot-Phase of the Disease.

Arrhythmogenic cardiomyopathy (ACM) is a genetically determined myocardial disease, characterized by myocytes necrosis with fibrofatty substitution and ventricular arrhythmias that can even lead to sudden cardiac death. The presence of inflammatory cell infiltrates in endomyocardial biopsies or in autoptic specimens of ACM patients has been reported, suggesting a possible role of inflammation in the pathophysiology of the disease. Furthermore, chest pain episodes accompanied by electrocardiographic changes and troponin release have been observed and defined as the "hot-phase" phenomenon. The aim of this critical systematic review was to assess the clinical features of ACM patients presenting with "hot-phase" episodes. According to PRISMA guidelines, a search was run in the PubMed, Scopus and Web of Science electronic databases using the following keywords: "arrhythmogenic cardiomyopathy"; "myocarditis" or "arrhythmogenic cardiomyopathy"; "troponin" or "arrhythmogenic cardiomyopathy"; and "hot-phase". A total of 1433 titles were retrieved, of which 65 studies were potentially relevant to the topic. Through the application of inclusion and exclusion criteria, 9 papers reporting 103 ACM patients who had experienced hot-phase episodes were selected for this review. Age at time of episodes was available in 76% of cases, with the mean age reported being 26 years ± 14 years (min 2-max 71 years). Overall, 86% of patients showed left ventricular epicardial LGE. At the time of hot-phase episodes, 49% received a diagnosis of ACM (Arrhythmogenic left ventricular cardiomyopathy in the majority of cases), 19% of dilated cardiomyopathy and 26% of acute myocarditis. At the genetic study, Desmoplakin (DSP) was the more represented disease-gene (69%), followed by Plakophillin-2 (9%) and Desmoglein-2 (6%). In conclusion, ACM patients showing hot-phase episodes are usually young, and DSP is the most common disease gene, accounting for 69% of cases. Currently, the role of "hot-phase" episodes in disease progression and arrhythmic risk stratification remains to be clarified.

Expansion of mouse castration-resistant intermediate prostate stem cells in vitro.

BACKGROUND: Most castration-resistant prostate cancers (CRPCs) have a luminal phenotype with high androgen receptor (AR) and prostate-specific antigen (PSA) expression. Currently, it is difficult to culture castration-resistant luminal cells with AR and PSA expression. METHODS: We formulated a custom-made medium and isolated primary cells from the prostate of adult wild-type (WT) and TRAMP mice. The cells were characterized by immunofluorescence staining, transcriptomic analysis, and qRT-PCR verification. Their self-renewal and differentiation potential in vitro and in vivo were examined. We treated the cells with androgen deprivation and enzalutamide and performed immunofluorescence staining and western blotting to analyze their expression of AR and PSA. RESULTS: We isolated a novel type of castration-resistant intermediate prostate stem cells (CRIPSCs) from adult WT and TRAMP mice. The mouse CRIPSCs proliferated rapidly in two-dimensional (2D) culture dishes and can be cultured for more than six months. The mouse CRIPSCs expressed luminal markers (AR, PSA, and Dsg4), basal markers (CK5 and p63), Psca, and the intermediate cell marker (Ivl). Transcriptomic analysis showed that the mouse CRIPSCs had upregulated signaling pathways related to cancer development and drug resistance. In the long-term culture, TRAMP CRIPSCs had higher expression of the genes related to stem cells and cancers than WT mice. Both WT and TRAMP CRIPSCs formed organoids in Matrigel. WT CRIPSCs did not form prostate tissues when transplanted in vivo without urogenital sinus mesenchyme (UGM) cells. In contrast, TRAMP CRIPSCs formed prostate ducts in NOG mice without UGM  cells and differentiated into luminal, basal, and neuroendocrine cells. Androgens regulated AR translocation between the nucleus and cytoplasm in the mouse CRIPSCs. Treatment of androgen deprivation  (ADT) and enzalutamide reduced AR expression in WT and TRAMP CRIPSCs; however, this treatment promoted PSA expression in TRAMP, while not WT CRIPSCs, similar to the clinical observations of CRPC. CONCLUSIONS: Our study established a method for isolating and expanding mouse CRIPSCs in 2D culture dishes. Mouse CRIPSCs had markers of basal and luminal cells, including AR and PSA, and can differentiate into prostate organoids and tissues. TRAMP CRIPSCs had elevated PSA expression upon ADT and enzalutamide treatment. Our method can be translated into clinical settings for CRPC precision medicine.

FcRn Antagonism Leads to a Decrease of Desmoglein-Specific B Cells: Secondary Analysis of a Phase 2 Study of Efgartigimod in Pemphigus Vulgaris and Pemphigus Foliaceus.

Background: Immunoglobulin G (IgG) levels are maintained by the IgG-recycling neonatal Fc-receptor (FcRn). Pemphigus vulgaris and pemphigus foliaceus are debilitating autoimmune disorders triggered by IgG autoantibodies against mucosal and epidermal desmogleins. Recently, a phase 2 clinical trial (NCT03334058; https://clinicaltrials.gov/NCT03334058) was completed in participants with pemphigus using efgartigimod, an FcRn inhibitor, in combination with prednisone. Efgartigimod demonstrated an early effect on diease activity and was well tolerated. In addition to the safety and efficacy assessment, clinical trials present an opportunity to gain more insights into the mechanism of disease, the mode of action of treatment, and potential for corticosteroid-sparing activity. Objective: The aim of our study was to assess the impact of FcRn antagonism by efgartigimod on immunological parameters known to be directly involved in pemphigus pathology, such as cellular and serological responses. Methods: We investigated total and antigen-specific IgG subclass level kinetics during and after treatment, assessed antigen-specific B-cell responses, followed T- and B-cell immunophenotypes, and analyzed how different immunophenotypes link to clinical response. Results: Treatment resulted in reduction of total IgG as well as autoreactive IgG antibody levels. Surprisingly, unlike total IgG and vaccine- or natural-infection-elicited IgG, which returned to baseline levels after stopping efgartigimod treatment, autoreactive antibody levels remained low in several study participants. Efgartigimod showed no effect on total leukocytes, neutrophils, monocytes, or lymphocytes in patients treated with extended efgartigimod therapy. Intriguingly, antigen-specific analyses revealed a loss of desmoglein-specific B cells in several participants responding to efgartigimod, in line with prolonged reduction of pathogenic IgG levels. Conclusions: Efgartigimod treatment of participants with pemphigus improved their conditions and exerted an immunomodulatory effect beyond the blockade of IgG recycling. Further studies in larger populations with an appropriate placebo control are needed to confirm these potentially important observations to establish long-term clinical responses in autoimmune diseases.

Bidirectional regulation of desmosome hyperadhesion by keratin isotypes and desmosomal components.

Desmosomes are intercellular junctions which mediate cohesion and communication in tissues exposed to mechanical strain by tethering the intermediate filament cytoskeleton to the plasma membrane. While mature desmosomes are characterized by a hyperadhesive, Ca2+-independent state, they transiently loose this state during wound healing, pathogenesis and tissue regeneration. The mechanisms controlling the hyperadhesive state remain incompletely understood. Here, we show that upon Ca2+-induced keratinocyte differentiation, expression of keratin 17 (K17) prevents the formation of stable and hyperadhesive desmosomes, accompanied by a significant reduction of desmoplakin (DP), plakophilin-1 (PKP1), desmoglein-1 (Dsg1) and -3 (Dsg3) at intercellular cell borders. Atomic force microscopy revealed that both increased binding strength of desmoglein-3 molecules and amount of desmoglein-3 oligomers, known hallmarks of hyperadhesion, were reduced in K17- compared to K14-expressing cells. Importantly, overexpression of Dsg3 or DPII enhanced their localization at intercellular cell borders and increased the formation of Dsg3 oligomers, resulting in stable, hyperadhesive desmosomes despite the presence of K17. Notably, PKP1 was enriched in these desmosomes. Quantitative image analysis revealed that DPII overexpression contributed to desmosome hyperadhesion by increasing the abundance of K5/K17-positive keratin filaments in the proximity of desmosomes enriched in desmoglein-3. Thus, our data show that hyperadhesion can result from recruitment of keratin isotypes K5/K17 to desmosomes or from enhanced expression of DP and Dsg3 irrespective of keratin composition. The notion that hyperadhesive desmosomes failed to form in the absence of keratins underscores the essential role of keratins and suggest bidirectional control mechanisms at several levels.

Arrhythmogenic Right Ventricular Cardiomyopathy Accompanied by Chronic Myocarditis.

Patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) classically present with ventricular arrhythmias and less commonly heart failure. ARVC is an inherited cardiomyopathy and generally based on a variant of desmosomal genes. Recently, the association between myocardial inflammation and ARVC has been a matter of great concern. We encountered a patient with ARVC who had a desmoglein-2 mutation with advanced right ventricular failure accompanying a preserved left ventricular function. Concomitant right ventricular myocarditis was detected four years after the diagnosis of ARVC. ARVC and myocarditis might have a deep pathophysiological association, at least in some cases.

Autosomal recessive monilethrix: Novel variants of the DSG4 gene in three Chinese families.

BACKGROUND: Monilethrix is a rare hereditary hair loss disorder characterized by hair fragility and beaded hair shaft alterations. Monilethrix is classically inherited in an autosomal dominant (AD) fashion caused by variants in the hair keratin genes KRT81, KRT83, or KRT86. Interestingly, an autosomal recessive (AR) form of monilethrix with variants in DSG4 gene has also been reported in recent years. OBJECTIVE: To identify causative variants in Chinese patients with autosomal recessive (AR) form of monilethrix. METHODS: Three families with AR form of monilethrix were observed and sequence variant analysis of DSG4 was performed by polymerase chain reaction (PCR), quantitative real-time PCR, and DNA sequencing. RESULTS: All the patients had sparse, fragile hair involving the scalp, eyebrows, and eyelashes with keratotic follicular papules and pruritus since birth. Atypical-beaded hairs and broken hair shaft fragments were identified in all the patients under dermoscopy. Heterozygous variants c.837del and c. 2389C > T, a homozygous splice site variant c.2355 + 1G > A, and a homozygous 48,644 bp large deletion variant g.31381440_31430084del in the DSG4 gene were identified and verified in the families. CONCLUSION: This report provided further evidence for the phenotypic spectrum and clinical features of, and the expanded variant database of AR form of monilethrix.

Desmoglein-3 induces YAP phosphorylation and inactivation during collective migration of oral carcinoma cells.

Alterations of the Hippo-YAP pathway are potential targets for oral squamous cell carcinoma (OSCC) therapy, but heterogeneity in this pathway could be responsible for therapeutic resistance. We analysed the Hippo-YAP signatures in a cohort of characterised keratinocyte cell lines derived from the mouth floor and buccal mucosa from different stages of OSCC tumour progression and focused on the specific role of YAP on invasive and metastatic potential. We confirmed heterogeneity in the Hippo-YAP pathway in OSCC lines, including overexpression of YAP1, WWTR1 (often referred to as TAZ) and the major Hippo signalling components, as well as the variations in the genes encoding the intercellular anchoring junctional proteins, which could potentially regulate the Hippo pathway. Specifically, desmoglein-3 (DSG3) exhibited a unique and mutually exclusive regulation of YAP via YAP phosphorylation during the collective migration of OSCC cells. Mechanistically, such regulation was associated with inhibition of phosphorylation of epidermal growth factor receptor (EGFR) (S695/Y1086) and its downstream effectors heat shock protein beta-1 (Hsp27) (S78/S82) and transcription factor AP-1 (c-Jun) (S63), leading to YAP phosphorylation coupled with its cytoplasmic translocation and inactivation. Additionally, OSCC lines displayed distinct phenotypes of YAP dependency or a mixed YAP and TAZ dependency for cell migration and present distinct patterns in YAP abundance and activity, with the latter being associated with YAP nuclear localisation. In conclusion, this study provides evidence for a newly identified paradigm in the Hippo-YAP pathway and suggests a new regulation mechanism involved in the control of collective migration in OSCC cells.

Isolation and Genomic Analysis of Single Circulating Tumor Cell Using Human Telomerase Reverse Transcriptase and Desmoglein-2.

As epithelial cells in the circulation are considered to originate from the tumor, the epithelial cell adhesion molecule has been commonly used as a standard marker for circulating tumor cells (CTCs) isolation. However, it seems to disappear after the epithelial-mesenchymal transition that most cancer cells undergo for intravasation. Thus, more advanced techniques for CTC detection are needed to better understand the clinical significance of CTCs. A cancer cell-specifically-infecting or replicating virus that codes a fluorescent monitor gene can be a solution to efficiently detect CTCs. Thus, the authors designed an adenovirus to bind to desmoglein-2, which is highly expressed in most cancer cells. A cancer-specific human telomerase reverse transcriptase promoter is inserted to control a viral E1 region. The adenovirus is utilized to compare the number of CTCs from renal cell carcinoma and prostate cancer patients before and after surgery. The isolated two or three CTCs are eligible for whole genome sequencing. The genomic analysis proves the difference of variants between primary tumors and CTCs. Taken together, it is a fast and exact serial method for CTC isolation and the enriched genome sequencing may be used to determine the prognosis and as a point-of-care system for patients with cancer.

Independent DSG4 frameshift variants in cats with hair shaft dystrophy.

Investigations of hereditary phenotypes in spontaneous mutants may help to better understand the physiological functions of the altered genes. We investigated two unrelated domestic shorthair cats with bulbous swellings of the hair shafts. The clinical, histopathological, and ultrastructural features were similar to those in mice with lanceolate hair phenotype caused by loss-of-function variants in Dsg4 encoding desmoglein 4. We sequenced the genomes from both affected cats and compared the data of each affected cat to 61 control genomes. A search for private homozygous variants in the DSG4 candidate gene revealed independent frameshift variants in each case, c.76del or p.Ile26fsLeu*4 in case no. 1 and c.1777del or p.His593Thrfs*23 in case no. 2. DSG4 is a transmembrane glycoprotein located primarily in the extracellular part of desmosomes, a complex of adhesion molecules responsible for connecting the keratin intermediate filaments of neighbouring epithelial cells. Desmosomes are essential for normal hair shaft formation. Both identified DSG4 variants in the affected cats lead to premature stop codons and truncate major parts of the open-reading frame. We assume that this leads to a complete loss of DSG4 function, resulting in an incorrect formation of the desmosomes and causing the development of defective hair shafts. Together with the knowledge on the effects of DSG4 variants in other species, our data suggest that the identified DSG4 variants cause the hair shaft dystrophy. To the best of our knowledge, this study represents the first report of pathogenic DSG4 variants in domestic animals.

An unusual presentation of painless penile erosions of pemphigus vulgaris: A case report

Introduction@#Pemphigus vulgaris is a life-threatening, autoimmune bullous disease caused by desmogleins (Dsg) 1 and 3 au- toantibodies. It is a rare disease with an incidence rate of 0.5 to 3.2 per 100,000 per year. It typically presents as painful, flaccid blisters and erosions on both the skin and mucous membranes.@*Case report@#We present a 43-year-old male with painless penile erosions of 1-month duration. He was evaluated for sexually transmitted infections, but laboratory tests yielded negative results. Subsequently, vesicles and bullae on the back and hyper- keratotic lesions on the malar area appeared, leading to the differential diagnoses of bullous diseases. Skin biopsy was done revealing intraepidermal suprabasal blisters with acantholytic cells. Direct Immunofluorescence demonstrated positive inter- cellular deposits of IgG and C3. ELISA Dsg 1 and Dsg 3 were positive (ratio of 1.857 and 4.580, respectively). A final diagnosis of pem- phigus vulgaris (PV) was made. The patient has remained in remission after a 3-month course of prednisone and azathioprine.@*Conclusion@#This is a unique case of PV presenting with an unusual manifestation of painless penile erosions. There have been limited reports of PV with penile skin involvement and all cases presented with painful lesions. Because painless penile lesions as presenting feature is rare, the diagnosis may be easily missed. This case demonstrates that thorough dermatologic examination and early diagnosis despite atypical findings are crucial to provide timely and appropriate treatment as this determines the clinical outcome of the disease.