Laura Y. McGirt

Tight junction defects in patients with atopic dermatitis.

BACKGROUND Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. OBJECTIVE We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). METHODS Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illuminas BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. RESULTS We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. CONCLUSION Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides

The pathogenesis of mycosis fungoides (MF), the most common cutaneous T-cell lymphoma (CTCL), is unknown. Although genetic alterations have been identified, none are considered consistently causative in MF. To identify potential drivers of MF, we performed whole-genome sequencing of MF tumors and matched normal skin. Targeted ultra-deep sequencing of MF samples and exome sequencing of CTCL cell lines were also performed. Multiple mutations were identified that affected the same pathways, including epigenetic, cell-fate regulation, and cytokine signaling, in MF tumors and CTCL cell lines. Specifically, interleukin-2 signaling pathway mutations, including activating Janus kinase 3 (JAK3) mutations, were detected. Treatment with a JAK3 inhibitor significantly reduced CTCL cell survival. Additionally, the mutation data identified 2 other potential contributing factors to MF, ultraviolet light, and a polymorphism in the tumor suppressor p53 (TP53). Therefore, genetic alterations in specific pathways in MF were identified that may be viable, effective new targets for treatment.

Activation of Epidermal Toll-Like Receptor 2 Enhances Tight Junction Function: Implications for Atopic Dermatitis and Skin Barrier Repair

Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus (S. aureus) skin infections. S. aureus is sensed by many pattern recognition receptors including toll-like receptor (TLR) 2. We hypothesized that an effective innate immune response will include skin barrier repair and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, CLDN1, CLDN23, occludin and ZO-1 in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape-stripping. Tlr2−/− mice had a delayed and incomplete barrier recovery following tape-stripping. AD subjects had reduced epidermal TLR2 expression as compared to nonatopic (NA) subjects, which inversely correlated (r= 0.654, P= 0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may play a role in their incompetent skin barrier.

miR-223 regulates cell growth and targets proto-oncogenes in mycosis fungoides/cutaneous T-cell lymphoma.

The pathogenesis of the cutaneous T-cell lymphoma (CTCL), mycosis fungoides (MF) is unclear. MicroRNA (miRNA) are small non-coding RNA that target mRNA leading to reduced mRNA translation. Recently, specific miRNA were shown to be altered in CTCL. We identified significantly reduced expression of miR-223 in early stage MF skin, and the levels of miR-223 diminished further in advanced stage disease. CTCL peripheral blood mononuclear cells and cell lines also had reduced miR-223 as compared to controls. Elevated expression of miR-223 in these cell lines reduced cell growth and clonogenic potential, whereas inhibition of miR-223 increased cell numbers. Investigations into putative miR-223 targets with oncogenic function, including E2F1 and MEF2C, and the predicted miR-223 target, TOX, revealed all three are targeted by miR-223 in CTCL. E2F1, MEF2C, and TOX proteins were decreased with miR-223 overexpression, while miR-223 inhibition led to increased protein levels in CTCL. In addition, we showed the 3′-UTR of TOX mRNA was a genuine target of miR-223. Therefore, reduced levels of miR-223 in MF/CTCL lead to increased expression of E2F1, MEF2C, and TOX, which likely contribute to the development and/or progression of CTCL. Thus, miR-223 and its targets may be useful for the development of new therapeutics for MF/CTCL.

Inhibition of Histone Deacetylase 3 Causes Replication Stress in Cutaneous T Cell Lymphoma

Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death.

Biomarkers of Th2 polarity are predictive of staphylococcal colonization in subjects with atopic dermatitis

Background  Staphylococcal colonization of the skin is commonly observed in subjects with atopic dermatitis (AD) and correlates with disease severity. Little is known about whether the degree of T‐helper 2 (Th2) polarity in these subjects can also affect the frequency of bacterial colonization in this disease.

The use of blind skin biopsy in the diagnosis of intravascular B-cell lymphoma

Intravascular B-cell lymphoma is a rare type of non-Hodgkins lymphoma that is characterized by a clonal proliferation of lymphoblasts within small blood vessels. Patients present with nonspecific symptoms and are often only given a diagnosis at autopsy. We report a case of intravascular B-cell lymphoma, characterized by pyrexia, anemia, thrombocytopenia, and mental status decline, without obvious cutaneous manifestations, that was diagnosed with blind skin biopsy.

Sodium thiosulfate in the treatment of non-uremic calciphylaxis

Calciphylaxis is a metastatic calcification‐induced vasculopathy that results in the occlusion of small blood vessels. Although calciphylaxis is normally associated with end‐stage renal disease, calciphylaxis from non‐uremic origin occurs as well. While the number of reports continues to increase, a standard treatment for non‐uremic calciphylaxis has yet to be established. Sodium thiosulfate (STS), which has been proven to be effective in the treatment of uremic calciphylaxis, shows promise; however, reports of its use in non‐uremic cases are limited. We describe a case of non‐uremic calciphylaxis in a patient with normal renal and parathyroid function who had complete resolution of disease after treatment with STS, and we review similar cases in the published work. Based on the successful outcomes detailed in this case series, STS appears to be an effective therapy for non‐uremic calciphylaxis.

Predictors of response to extracorporeal photopheresis in advanced mycosis fungoides and Sézary syndrome

Background: Extracorporeal photopheresis (ECP) has been utilized for more than 20 years to treat cutaneous T‐cell lymphoma (CTCL), but a clinical response can take up to 9 months to manifest. This study was undertaken to determine whether clinical features, laboratory values, cytokine levels, or gene expression levels of tumor markers are useful to predict the subsequent response to ECP in CTCL patients with blood involvement.

Genetic markers associated with progression in early mycosis fungoides

Mycosis fungoides (MF) is a rare, but potentially devastating malignancy. It classically presents with cutaneous patches and plaques and can progress to tumours on the skin with lymph node, blood and visceral involvement. While most patients with MF have a relatively benign disease course, a subset of patients will develop progressive disease that is often fatal.