Background The characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan (HA), a major extracellular matrix component, accumulates during inflammatory lung diseases including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that HA and its receptors would play a role in human asthma. Objective To investigate the role of HA and HA binding proteins in human asthma. Methods Twenty-one subjects with asthma and 25 normal control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage (BAL). Fibroblasts were cultured, HA and HA synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of HA binding proteins, CD44, TLR2 and TLR4 on BAL macrophages was determined by flow cytometry. IL-8 production by macrophages in response to HA fragment stimulation was compared. Results Airway fibroblasts from asthma patients produced significantly increased concentrations of lower molecular weight HA compared to those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from asthmatic subjects showed an increase in responsiveness to low molecular weight HA stimulation, as demonstrated by increased IL-8 production. Conclusions HA homeostasis is deranged in asthma with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to HA fragments suggests a novel pro-inflammatory mechanism by which persistence of HA fragments could contribute to chronic inflammation and airway remodeling in asthma.

BACKGROUND AND PURPOSEInhalation of a b-adrenoceptor agonist (b-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of b-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this b2-adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by b2-adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional b-agonist-induced b2-adrenoceptor desensitization in the context of allergic inflammatory airway disease. EXPERIMENTAL APPROACHWe combined chronic allergen exposure with repeated b-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of b2-adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of b2-adrenoceptors (KO). Associated inflammatory changes -cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and b2-adrenoceptor density (radioligand binding) -were also measured. KEY RESULTSb2-Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype. CONCLUSIONS AND IMPLICATIONSThis animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic bagonist treatment on b-adrenoceptor function in asthma. Such information may guide the clinical use of b-agonists and provide insight into development of novel b-adrenoceptor ligands for the treatment of asthma.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry-based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma. These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. .

We performed epi-mode pump-probe imaging of melanin in excised human pigmented lesions and both hemoglobin and melanin in live xenograft mouse melanoma models to depths greater than 100 µm. Eumelanin and pheomelanin images, which have been previously demonstrated to differentiate melanoma from benign lesions, were acquired at the dermal-epidermal junction with cellular resolution and modest optical powers (down to 15 mW). We imaged dermal microvasculature with the same wavelengths, allowing simultaneous acquisition of melanin, hemoglobin and multiphoton autofluorescence images. Molecular pump-probe imaging of melanocytes, skin structure and microvessels allows comprehensive, non-invasive characterization of pigmented lesions.