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BackgroundStaphylococcus aureus and Staphylococcus epidermidis are the most abundant bacteria found on the skin of patients with atopic dermatitis (AD). S aureus is known to exacerbate AD, whereas S epidermidis has been considered a beneficial commensal organism.ObjectiveIn this study, we hypothesized that S epidermidis could promote skin damage in AD by the production of a protease that damages the epidermal barrier.MethodsThe protease activity of S epidermidis isolates was compared with that of other staphylococcal species. The capacity of S epidermidis to degrade the barrier and induce inflammation was examined by using human keratinocyte tissue culture and mouse models. Skin swabs from atopic and healthy adult subjects were analyzed for the presence of S epidermidis genomic DNA and mRNA.ResultsS epidermidis strains were observed to produce strong cysteine protease activity when grown at high density. The enzyme responsible for this activity was identified as EcpA, a cysteine protease under quorum sensing control. EcpA was shown to degrade desmoglein-1 and LL-37 in vitro, disrupt the physical barrier, and induce skin inflammation in mice. The abundance of S epidermidis and expression of ecpA mRNA were increased on the skin of some patients with AD, and this correlated with disease severity. Another commensal skin bacterial species, Staphylococcus hominis, can inhibit EcpA production by S epidermidis.ConclusionS epidermidis has commonly been regarded as a beneficial skin microbe, whereas S aureus has been considered deleterious. This study suggests that the overabundance of S epidermidis found on some atopic patients can act similarly to S aureus and damage the skin by expression of a cysteine protease.

BACKGROUND: Brutons tyrosine kinase (BTK) is important in B-cell signalling. Efficacy has been reported for BTK inhibitors (BTKi) in human autoimmune diseases. Canine pemphigus foliaceus (cPF) is one of the most common canine autoimmune skin diseases. OBJECTIVES: To determine the safety and efficacy of the BTKi PRN1008 in the treatment of cPF. ANIMALS: Four privately owned dogs. METHODS AND MATERIALS: Four dogs diagnosed with PF were administered BTKi PRN1008. Initial dosages approximated to 15 mg/kg once daily, increased to twice daily if inadequate response was seen. Treatment continued for 20 weeks, attempting to decrease to every other day. Dogs were monitored with complete blood counts, serum biochemistry panels and urinalyses, and evaluated with a modified version of a validated human Pemphigus Disease Activity Index (cPDAI). Serum anti-desmocollin-1 (DSC-1) and desmoglein-1 (DSG-1) immunoglobulin (Ig)G titres were performed before and after the treatment period. Drug bound to target was measured in peripheral blood mononuclear cells (PBMC). RESULTS: All four dogs showed reduction in lesions and cPDAI score during the first two weeks of treatment. Three dogs continued to improve and sustained near complete remission by 20 weeks, at which point three responses were considered good and one fair. Final daily dosages were in the range 17-33 mg/kg. Anti-DSC-1 IgG titre decreased dramatically in one dog, was undetectable in two and was uninterpretable in one dog. No dogs had detectable IgG to DSG1. A possible adverse event occurred in one dog. CONCLUSIONS AND CLINICAL IMPORTANCE: BTKi PRN1008 monotherapy may have some beneficial effects in some cases of cPF.

There is concern that using depot-medroxyprogesterone acetate (DMPA) for pregnancy prevention heightens HIV susceptibility. While no clinical data establishes causal link between HIV acquisition and use of this injectable progestin, prior work from our laboratory showed that DMPA comparably lowers genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and weakens genital epithelial barrier function in female mice and women. We likewise saw DMPA increase mouse susceptibility to multiple genital pathogens including HIV. Herein, we sought to confirm and extend these findings by comparing genital epithelial barrier function in untreated rhesus macaques (RM) vs. RM treated with DMPA or DMPA and estrogen (E). Compared to controls, genital tissue from RM with pharmacologically relevant serum levels of medroxyprogesterone acetate displayed significantly lower DSG1 levels and greater permeability to low molecular mass molecules. Conversely, DMPA-mediated effects on genital epithelial integrity and function were obviated in RM administered DMPA and E. These data corroborate the diminished genital epithelial barrier function observed in women initiating DMPA and identify RM as a useful preclinical model for defining effects of exogenous sex steroids on genital pathogen susceptibility. As treatment with E averted DMPA-mediated loss of genital epithelial barrier function, our results also imply that contraceptives releasing progestin and E may be less likely to promote transmission of HIV and other sexually transmitted pathogens than progestin-only compounds.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing this devastating disease. Here, we observed that non-desmoglein (Dsg) AuAbs in the sera of patients with Dsg1/3 AuAb-negative acute PV are pathogenic, because IgGs from these individuals induced skin blistering in neonatal mice caused by suprabasal acantholysis. Serum levels of AuAbs to desmocollin 3 (Dsc3), M3 muscarinic acetylcholine receptor (M3AR), and secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1) correlated with the disease stage of PV. Moreover, AuAb absorption on recombinant Dsc3, M3AR, or SPCA1 both prevented skin blistering in the passive transfer of AuAbs model of PV in BALB/c mice and significantly decreased the extent of acantholysis in a neonatal mouse skin explant model. Although acantholytic activities of each of these immunoaffinity-purified AuAbs could not induce a PV-like phenotype, their mixture produced a synergistic effect manifested by a positive Nikolskiy sign in the skin of neonatal mice. The downstream signaling of all pathogenic non-Dsg AuAbs involved p38 mitogen-activated protein kinase (MAPK)-mediated phosphorylation and elevation of cytochrome c release and caspase 9 activity. Anti-Dsc3 and anti-SPCA1 AuAbs also activated SRC proto-oncogene, nonreceptor tyrosine kinase (SRC). Of note, although a constellation of non-Dsg AuAbs apparently disrupted epidermal integrity, elimination of a single pathogenic AuAb could prevent keratinocyte detachment and blistering. Therefore, anti-Dsg1/3 AuAb-free PV can be a model for elucidating the roles of non-Dsg antigen-specific AuAbs in the physiological regulation of keratinocyte cell-cell adhesion and blister development.

Bacteria that reside on the skin can influence the behavior of the cutaneous immune system, but the mechanisms responsible for these effects are incompletely understood. Colonization of the skin by Staphylococcus aureus (S. aureus) is increased in atopic dermatitis and can result in increased severity of the disease. In this study, we show that S. aureus stimulates human keratinocytes to increase their endogenous protease activity, including specific increases in trypsin activity. This increased protease activity coincided with increased expression of mRNA for kallikreins (KLKs), with KLK6, 13, and 14 showing the greatest induction after exposure to S. aureus. Suppression of mRNA for these KLKs in keratinocytes by targeted small interfering RNA silencing before S. aureus exposure blocked the increase in protease activity. Keratinocytes exposed to S. aureus showed enhanced degradation of desmoglein-1 and filaggrin, whereas small interfering RNA for KLK6, KLK13, and KLK14 partially blocked this degradation. These data illustrate how S. aureus directly influences the skin barrier integrity by stimulating endogenous proteolytic activity and defines a previously unknown mechanism by which S. aureus may influence skin diseases.

BackgroundSevere dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported.ObjectiveWe studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis.MethodsHistopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents.ResultsNo mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide.ConclusionsSAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

BackgroundSevere dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported.ObjectiveWe studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis.MethodsHistopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents.ResultsNo mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide.ConclusionsSAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

Corneocyte desquamation has been ascribed to the following: 1) proteolytic degradation of corneodesmosomes (CDs); 2) disorganization of extracellular lamellar bilayers; and/or 3) "swell-shrinkage-slough" from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in D-Squame strips from the first versus fifth stripping ("outer" vs. "mid"-stratum corneum (SC), respectively) from nine normal adult forearms. Strippings were either processed for standard electron microscopy (EM) or for ruthenium-, or osmium-tetroxide vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol, which, to our knowledge, is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded desmoglein 1/desmocollin 1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging, and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae, further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.

Corneocyte desquamation has been ascribed to the following: 1) proteolytic degradation of corneodesmosomes (CDs); 2) disorganization of extracellular lamellar bilayers; and/or 3) "swell-shrinkage-slough" from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in D-Squame strips from the first versus fifth stripping ("outer" vs. "mid"-stratum corneum (SC), respectively) from nine normal adult forearms. Strippings were either processed for standard electron microscopy (EM) or for ruthenium-, or osmium-tetroxide vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol, which, to our knowledge, is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded desmoglein 1/desmocollin 1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging, and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae, further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.