Mannan-Binding Lectin Levels and Activity Are Not Altered in Atopic Dermatitis Patients with a History of Eczema Herpeticum

Background. Eczema herpeticum (EH) is a potentially serious, systemic complication in subjects with atopic dermatitis (AD) caused by herpes simplex virus (HSV). The innate immune dysregulation that predisposes these subjects to cutaneous viral infections is not well understood. We tested the hypothesis that defects in mannan-binding lectin (MBL) may be associated with an increased risk of EH. Methods. We evaluated serum MBL levels and functional activity in 13 AD subjects with a history of EH (EH+) and 21 AD subjects with no history of EH (EH−). MBL levels were detected by enzyme immunoassay. MBL pathway functional activity was evaluated by determining MBL C4b deposition capacity. Results. We found no statistical difference in MBL serum levels or function between EH+ and EH− groups. Conclusion. Considering the limitations of this study (e.g., small samples size) our findings suggest that MBL defects do not play a role in EH

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 noncoding RNAs that target mRNA leading to reduced mRNA translation. Recently, specific miRNA were shown to be altered in CTCL. We detected significantly reduced expression of miR-223 in early-stage MF skin, and further decreased levels of miR-223 in advanced-stage disease. CTCL peripheral blood mononuclear cells and cell lines also had reduced miR-223 as compared with 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 that all three were targeted by miR-223 in CTCL. E2F1, MEF2C, and TOX proteins were decreased with miR-223 overexpression, whereas miR-223 inhibition led to increased protein levels in CTCL. In addition, we showed that 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 contributes 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

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 skin infections. S. aureus is sensed by many pattern recognition receptors, including Toll-like receptor 2 (TLR2). 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, claudin-1 (CLDN1), claudin-23 (CLDN23), occludin, and Zonulae occludens 1 (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 with nonatopic 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 have a role in their incompetent skin barrier

Clinical Study Mannan-Binding Lectin Levels and Activity Are Not Altered in Atopic Dermatitis Patients with a History of Eczema Herpeticum

Background. Eczema herpeticum (EH) is a potentially serious, systemic complication in subjects with atopic dermatitis (AD) caused by herpes simplex virus (HSV). The innate immune dysregulation that predisposes these subjects to cutaneous viral infections is not well understood. We tested the hypothesis that defects in mannan-binding lectin (MBL) may be associated with an increased risk of EH. Methods. We evaluated serum MBL levels and functional activity in 13 AD subjects with a history of EH (EH+) and 21 AD subjects with no history of EH (EH−). MBL levels were detected by enzyme immunoassay. MBL pathway functional activity was evaluated by determining MBL C4b deposition capacity. Results. We found no statistical difference in MBL serum levels or function between EH+ and EH− groups. Conclusion. Considering the limitations of this study (e.g., small samples size) our findings suggest that MBL defects do not play a role in EH

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

HDIs show selective inhibition of HDACs in CTCL cell lines.

<p>(A)Western blot analysis of whole cell lysates from Wild-type (WT) and <i>Hdac3</i>-null livers. Histones H3 and H4 served as loading controls. (B) Upper Panel: Western blot analysis of NIH 3T3 cells following treatment with various HDIs (indicated above each lane). Anti-histone H3 was used as a loading control. Lower panel: Western blot analysis of NIH 3T3 cells treated with either Trichostatin A (TSA) (1 µM), sodium butyrate (NaB) (5 mM), or increasing concentrations of nicotinamide (mM). (C) Western blot analysis of whole cell lysates prepared from cells that were transfected with either non-targeting siRNAs (NT) or siRNAs directed to the indicated Hdacs. (D) Western blot analysis of H3K56ac using whole cell lysates prepared from cells treated with the indicated amounts of RGFP966 for 24 hr. (E & F) Western blot analysis of (E) HH or (F) Hut78 cell lines treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, 10 µM 136, or 10 µM 966. Cells were treated for 24 hr and then harvested for protein isolation. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side-by-side comparison of DMSO and Depsipeptide. Histones H3 and H4 were used as loading controls.</p

CTCL cell lines are sensitive to pan and selective HDIs.

<p>(A) Growth curves of HDI treated HH cells (left) or Hut78 cells (right). Cells were treated once with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (B) Dose curves of 966 treated HH cells (left) and Hut78 cells (right). The experiment was performed in the same manner as (A) except that the cells treated were treated once with 2 µM, 5 µM, or 10 µM of 966 at hour 0. For both (A) and (B), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 233: p = 0.004, and 966: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 233: p = 0.006, and 966: p = 0.006. (B) HH cells (left), Depsi: p = 0.0008, 966 2 µM: p = 0.02, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 966 2 µM: p = 0.03, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006.</p

iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

<p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p