A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1ß upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1ß in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex.

Quantitative proteomics reveals an altered cystic fibrosis in vitro bronchial epithelial secretome.

Alterations in epithelial secretions and mucociliary clearance contribute to chronic bacterial infection in cystic fibrosis (CF) lung disease, but whether CF lungs are unchanged in the absence of infection remains controversial. A proteomic comparison of airway secretions from subjects with CF and control subjects shows alterations in key biological processes, including immune response and proteolytic activity, but it is unclear if these are due to mutant CF transmembrane conductance regulator (CFTR) and/or chronic infection. We hypothesized that the CF lung apical secretome is altered under constitutive conditions in the absence of inflammatory cells and pathogens. To test this, we performed quantitative proteomics of in vitro apical secretions from air-liquid interface cultures of three life-extended CF (ΔF508/ΔF508) and three non-CF human bronchial epithelial cells after labeling of CF cells by stable isotope labeling with amino acids in cell culture. Mass spectrometry analysis identified and quantitated 666 proteins across samples, of which 70 exhibited differential enrichment or depletion in CF secretions (±1.5-fold change; P < 0.05). The key molecular functions were innate immunity (24%), cytoskeleton/extracellular matrix organization (24%), and protease/antiprotease activity (17%). Oxidative proteins and classical complement pathway proteins that are altered in CF secretions in vivo were not altered in vitro. Specific differentially increased proteins-MUC5AC and MUC5B mucins, fibronectin, and matrix metalloproteinase-9-were validated by antibody-based assays. Overall, the in vitro CF secretome data are indicative of a constitutive airway epithelium with altered innate immunity, suggesting that downstream consequences of mutant CFTR set the stage for chronic inflammation and infection in CF airways.

Development of glandular models from human nasal progenitor cells.

Hyperplasia/hypertrophy of submucosal glands contributes to mucus overproduction in chronic diseases of the upper and lower respiratory tracts, especially in adult and pediatric chronic rhinosinusitis. Mechanisms that lead to glandular hyperplasia/hypertrophy are markedly understudied, reflecting a lack of in vitro model systems wherein airway epithelial progenitor cells differentiate into glandular cells. In this study, we developed and compared several in vitro three-dimensional systems using human nasal epithelial basal cells (HNEBCs) cultured by different methods on two types of extracellular matrices. We demonstrate that HNEBCs cultured on Matrigel (Corning, Tewksbury, MA) form glandular acini-like structures, whereas HNEBCs embedded in a collagen type I matrix form a network of tubules. Fibroblast-conditioned medium increases tubule formation in collagen type I. In contrast, HNEBCs cocultured with fibroblasts self-aggregate into organotypic structures with tubules and acini. These observations provide morphological evidence that HNEBCs are pluripotent and retain the capacity to differentiate into structures resembling specific structural components of submucosal glands depending on the extracellular matrices and culture conditions. The resultant models should prove useful in targeting cross-talk between epithelial cells and fibroblasts to decipher molecular mechanisms and specific signals responsible for the development of glandular hyperplasia/hypertrophy, which in turn may lead to new therapeutic strategies for chronic rhinosinusitis and other inflammatory respiratory diseases characterized by glandular hyperplasia/hypertrophy.

Proteomic analysis of pediatric sinonasal secretions shows increased MUC5B mucin in CRS.

BACKGROUND: Chronic rhinosinusitis (CRS) is characterized by mucous overproduction and submucosal gland hyperplasia. The global protein profile of sinonasal secretions in pediatric CRS has not been studied. We hypothesized that MUC5B, a glandular mucin, would be relatively increased in CRS secretions compared to other mucins. METHODS: Secretions were collected at Children's National Health System (Children's National) from CRS patients undergoing sinus surgery and from control patients without CRS undergoing craniofacial procedures. Proteins were extracted, digested to peptides, and analyzed by mass spectometry. Fold change significance was calculated using the QSpec algorithm. Western blot analysis was performed to validate proteomic findings. RESULTS: In total, 294 proteins were identified. Although both MUC5B and MUC5AC were identified in a majority of samples, the relative abundance of MUC5B was found to be significantly higher (P < 0.05). Western blot data validated these findings. Other proteins with the highest significant positive-fold change in CRS samples were BP1 fold-containing family A member 1, chitinase-3-like protein 1, plastin-2, serpin 10, and BP1 fold-containing family B member 1. CONCLUSION: Overall, our data demonstrate an increase of MUC5B abundance in the sinus secretions of pediatric patients with CRS.

Premature infants have impaired airway antiviral IFNγ responses to human metapneumovirus compared to respiratory syncytial virus.

BACKGROUND: It is unknown why human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) cause severe respiratory infection in children, particularly in premature infants. Our aim was to investigate if there are defective airway antiviral responses to these viruses in young children with history of prematurity. METHODS: Nasal airway secretions were collected from 140 children ≤ 3 y old without detectable virus (n = 80) or with PCR-confirmed HMPV or RSV infection (n = 60). Nasal protein levels of IFNγ, CCL5/RANTES, IL-10, IL-4, and IL-17 were determined using a multiplex magnetic bead immunoassay. RESULTS: Full-term children with HMPV and RSV infection had increased levels of nasal airway IFNγ, CCL5, and IL-10 along with an elevation in Th1 (IFNγ)/Th2 (IL-4) ratios, which is expected during antiviral responses. In contrast, HMPV-infected premature children (< 32 wk gestation) did not exhibit increased Th1/Th2 ratios or elevated nasal airway secretion of IFNγ, CCL5, and IL-10 relative to uninfected controls. CONCLUSION: Our study is the first to demonstrate that premature infants have defective IFNγ, CCL5/RANTES, and IL-10 airway responses during HMPV infection and provides novel insights about the potential reason why HMPV causes severe respiratory disease in children with history of prematurity.

Rhinovirus-induced airway cytokines and respiratory morbidity in severely premature children.

BACKGROUND: Rhinovirus (RV) has been linked to the pathogenesis of asthma. Prematurity is a risk factor for severe RV infection in early life, but is unknown if RV elicits enhanced pro-asthmatic airway cytokine responses in premature infants. This study investigated whether young children born severely premature (<32 wks gestation) exhibit airway secretion of Th2 and Th17 cytokines during natural RV infections and whether RV-induced Th2-Th17 responses are linked to more respiratory morbidity in premature children during the first 2 yrs of life. METHODS: We measured Th2 and Th17 nasal airway cytokines in a retrospective cohort of young children aged 0-2 yrs with PCR-confirmed RV infection or non-detectable virus. Protein levels of IL-4, IL-13, TSLP, and IL-17 were determined with multiplex immunoassays. Demographic and clinical variables were obtained by electronic medical record (EMR) review. RESULTS: The study comprised 214 children born full term (n = 108), preterm (n = 44) or severely premature (n = 62). Natural RV infection in severely premature children was associated with elevated airway secretion of Th2 (IL-4 and IL-13) and Th17 (IL-17) cytokines, particularly in subjects with history of bronchopulmonary dysplasia. Severely premature children with high RV-induced airway IL-4 had recurrent respiratory hospitalizations (median 3.65 hosp/yr; IQR 2.8-4.8) and were more likely to have at least one pediatric intensive care unit admission during the first 2 yrs of life (OR 8.72; 95% CI 1.3-58.7; p = 0.02). CONCLUSIONS: Severely premature children have increased airway secretion of Th2 and Th17 cytokines during RV infections, which is associated with more respiratory morbidity in the first 2 yrs of life.

Directional secretomes reflect polarity-specific functions in an in vitro model of human bronchial epithelium.

The polarity of the conducting airway epithelium is responsible for its directional secretion. This is an essential characteristic of lung integrity and function that dictates interactions between the external environment (apical) and subepithelial structures (basolateral). Defining the directional secretomes in the in vitro human bronchial epithelial (HBE) differentiated model could bring valuable insights into lung biology and pulmonary diseases. Normal primary HBE cells (n = 3) were differentiated into respiratory tract epithelium. Apical and basolateral secretions (24 h) were processed for proteome profiling and pathway analysis. A total of 243 proteins were identified in secretions from all HBE cultures combined. Of these, 51% were classified as secreted proteins, including true secreted proteins (36%) and exosomal proteins (15%). Close examination revealed consistent secretion of 69 apical proteins and 13 basolateral proteins and differential secretion of 25 proteins across all donors. Expression of Annexin A4 in apical secretions and Desmoglein-2 in basolateral secretions was validated using Western blot or ELISA in triplicate independent experiments. To the best of our knowledge, this is the first study defining apical and basolateral secretomes in the in vitro differentiated HBE model. The data demonstrate that epithelial polarity directs protein secretion with different patterns of biological processes to the apical and basolateral surfaces that are consistent with normal bronchial epithelium homeostatic functions. Applying this in vitro directional secretome model to lung diseases may elucidate their molecular pathophysiology and help define potential therapeutic targets.

Mitotic asynchrony induces transforming growth factor-ß1 secretion from airway epithelium.

We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-ß1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-ß1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-ß1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-ß1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony.

The human secretome atlas initiative: implications in health and disease conditions.

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts toward secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study. This article is part of a Special Issue entitled: An Updated Secretome.

IL-1ß induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone.

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 ß (IL-1ß) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1ß-induced upregulation of MUC5AC. Data show that ligand binding of IL-1ß to the IL-1ß receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1ß exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1ß activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1ß-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.

Glucocorticoid receptor and histone deacetylase-2 mediate dexamethasone-induced repression of MUC5AC gene expression.

Airway occlusion in obstructive airway diseases is caused in part by the overproduction of secretory mucin glycoproteins through the up-regulation of mucin (MUC) genes by inflammatory mediators. Some pharmacological agents, including the glucocorticoid dexamethasone (Dex), repress mucin concentrations in lung epithelial cancer cells. Here, we show that Dex reduces the expression of MUC5AC, a major airway mucin gene, in primary differentiated normal human bronchial epithelial (NHBE) cells in a dose-dependent and time-dependent manner, and that the Dex-induced repression is mediated by the glucocorticoid receptor (GR) and two glucocorticoid response elements (GREs) in the MUC5AC promoter. The pre-exposure of cells to RU486, a GR antagonist, and mutations in either the GRE3 or GRE5 cis-sites abolished the Dex-induced repression. Chromatin immunoprecipitation (ChIP) assays showed a rapid temporal recruitment of GR to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in NHBE and in A549 cells. Immunofluorescence showed nuclear colocalization of GR and histone deacetylase-2 (HDAC2) in MUC5AC-expressing NHBE cells. ChIP also showed a rapid temporal recruitment of HDAC2 to the GRE3 and GRE5 cis-elements in the MUC5AC promoter in both cell types. The knockdown of HDAC2 by HDAC2-specific short interfering RNA prevented the Dex-induced repression of MUC5AC in NHBE and A549 cells. These data demonstrate that GR and HDAC2 are recruited to the GRE3 and GRE5 cis-sites in the MUC5AC promoter and mediate the Dex-induced cis repression of MUC5AC gene expression. A better understanding of the mechanisms whereby glucocorticoids repress MUC5AC gene expression may be useful in formulating therapeutic interventions in chronic lung diseases.

Δ-9,11 modification of glucocorticoids dissociates nuclear factor-κB inhibitory efficacy from glucocorticoid response element-associated side effects.

Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids--drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17ß-hydroxy-11ß-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.

Spiritual and Emotional Experience With a Diagnosis of Breast Cancer: A Scoping Review.

BACKGROUND: The breast cancer diagnosis causes a high level of suffering and distress in patients who experience difficulties in coping. There is a need to improve knowledge of emotional and spiritual coping in response to the stressful situation of women who must face this diagnosis. OBJECTIVES: The aims of this study were to map women's spiritual and emotional coping experiences reported after a breast cancer diagnosis and examine the proposed interventions and suggestions for clinical practice. METHODS: A scoping review was performed by searching the Scientific Electronic Library Online, Scopus, Cumulative Index to Nursing and Allied Health Literature, Latin American & Caribbean Health Sciences Literature, Medical Literature Analyses and Retrieval System Online, Spanish Bibliographic Index of Health Sciences, PSYCINFO, and Google Scholar databases using Medical Subject Headings terms. Additional pertinent studies were identified by reviewing the bibliographies of the included studies. Twenty articles were included according to the recommendations for scoping reviews. RESULTS: Study findings regarding emotional and spiritual coping with the diagnosis and proposed interventions were synthesized. A thematic list of interventions and recommendations for clinical practice is also provided. CONCLUSIONS: The studies demonstrated that women with breast cancer are challenged by their emotions and experiences. The review highlights the importance of spiritual coping for redefining women's meaning in life. In clinical practice, caring for women's inherent needs when they are coping with a diagnosis is important to establish integral care. IMPLICATIONS FOR PRACTICE: Nurses can evaluate coping strategies, offer support for adaptation to the disease, provide qualified listening, help women in their search for significance while coping with cancer, and help them identify ways to overcome this stressful situation. Similarly, they can encourage patients to find spiritual comfort and emotional support.

Human bronchial epithelial cells differentiate to 3D glandular acini on basement membrane matrix.

To create a model system that investigates mechanisms resulting in hyperplasia and hypertrophy of respiratory tract submucosal glands, we developed an in vitro three-dimensional (3D) system wherein normal human bronchial epithelial (HBE) cells differentiated into glandular acini when grown on a basement membrane matrix. The differentiation of primary HBE cells into glandular acini was monitored temporally by light microscopy. Apoptosis-induced lumen formation was observed by immunofluorescence analysis. The acinar cells expressed and secreted MUC5B mucin (marker for glandular mucous cells) and lysozyme, lactoferrin, and zinc-α2-glycoprotein (markers for glandular serous cells) at Day 22. ß-Tubulin IV, a marker for ciliated cells, was not detected. Expression of mucous and serous cell markers in HBE glandular acini demonstrated that HBE cells grown on a basement membrane matrix differentiated into acini that exhibit molecular characteristics of respiratory tract glandular acinar cells. Inhibition studies with neutralizing antibodies resulted in a marked decrease in size of the spheroids at Day 7, demonstrating that laminin (a major component of the basement membrane matrix), the cell surface receptor integrin α6, and the cell junction marker E-cadherin have functional roles in HBE acinar morphogenesis. No significant variability was detected in the average size of glandular acini formed by HBE cells from two normal individuals. These results demonstrated that this in vitro model system is reproducible, stable, and potentially useful for studies of glandular differentiation and hyperplasia.

Glandular gene expression of sinus mucosa in chronic rhinosinusitis with and without cystic fibrosis.

Secretory cells in submucosal glands (SMGs) secrete antibacterial proteins and mucin glycoproteins into the apical lumen of the respiratory tract, and these are critical for innate immune mucosal integrity. Glandular hyperplasia is manifested in diseases with obstructive respiratory pathologies associated with mucous hypersecretion, and is predominant in the sinus mucosa of patients with chronic rhinosinusitis (CRS), cystic fibrosis (CF), and clinical symptoms of CRS. To gain insights into the molecular basis of SMG hyperplasia in CRS, gene expression microarray analyses were performed to identify the differences in global and specific gene expression in the sinus mucosa of control, CRS, and CRS/CF patients. A marked up-regulation of 11 glandular-associated genes in CRS and CRS/CF sinus mucosa was evident. The RNA and protein expressions of the four most highly up-regulated genes (DSG3, KRT14, PTHLH, and OTX2) were evaluated. An increased expression of DSG3, KRT14, and PTHLH was demonstrated at the mRNA and protein levels in both CRS and CRS/CF sinus mucosa, whereas the increased expression of OTX2 was evident only for CRS/CF sinus mucosa, implicating OTX2 as a CF-specific gene. Immunofluorescence analysis localized DSG3, PTHLH, and OTX2 to serous cells, and KRT14 to myoepithelial cells, in SMGs. Because glandular hyperplasia is a central histologic feature of CRS, the identification of overexpressed glandular genes in the sinus mucosa lays the groundwork for future studies of glandular hyperplasia, and may ultimately lead to the development of novel treatments for mucous hypersecretion in patients with CRS.

Asthmatic airway epithelium is intrinsically inflammatory and mitotically dyssynchronous.

Asthma is an inflammatory condition for which anti-inflammatory glucocorticoids are the standard of care. However, similar efficacy has not been shown for agents targeting inflammatory cells and pathways. This suggests a noninflammatory cell contributor (e.g., epithelium) to asthmatic inflammation. Herein, we sought to define the intrinsic and glucocorticoid-affected properties of asthmatic airway epithelium compared with normal epithelium. Human primary differentiated normal and asthmatic airway epithelia were cultured in glucocorticoid-free medium beginning at -48 hours. They were pulsed with dexamethasone (20 nM) or vehicle for 2 hours at -26, -2, +22, and +46 hours. Cultures were mechanically scrape-wounded at 0 hours and exposed continuously to bromodeoxyuridine (BrdU). Cytokine secretions were analyzed using cytometric bead assays. Wound regeneration/mitosis was analyzed by microscopy and flow cytometry. Quiescent normal (n = 3) and asthmatic (n = 6) epithelia showed similar minimal inflammatory cytokine secretion and mitotic indices. After wounding, asthmatic epithelia secreted more basolateral TGF-ß1, IL-10, IL-13, and IL-1ß (P < 0.05) and regenerated less efficiently than normal epithelia (+48 h wound area reduction = [mean ± SEM] 50.2 ± 7.5% versus 78.6 ± 7.7%; P = 0.02). Asthmatic epithelia showed 40% fewer BrdU(+) cells at +48 hours (0.32 ± 0.05% versus 0.56 ± 0.07% of total cells; P = 0.03), and those cells were more dyssynchronously distributed along the cell cycle (52 ± 10, 25 ± 4, 23 ± 7% for G1/G0, S, and G2/M, respectively) than normal epithelia (71 ± 1, 12 ± 2, and 17 ± 2% for G1/G0, S, and G2/M, respectively). Dexamethasone pulses improved asthmatic epithelial inflammation and regeneration/mitosis. In summary, we show that inflammatory/fibrogenic cytokine secretions are correlated with dyssynchronous mitosis upon injury. Intermittent glucocorticoids simultaneously decreased epithelial cytokine secretions and resynchronized mitosis. These data, generated in an airway model lacking inflammatory cells, support the concept that epithelium contributes to asthmatic inflammation.

IL-8 regulates mucin gene expression at the posttranscriptional level in lung epithelial cells.

Airway inflammation and mucus hypersecretion/overproduction/obstruction are pathophysiological characteristics of cystic fibrosis, asthma, and chronic obstructive pulmonary disease. Up-regulation of airway mucin genes by inflammatory/immune response mediators is one of the major contributors to mucin overproduction. IL-8, a potent proinflammatory mediator and neutrophil chemoattractant, is present at high levels in the airway secretions of such patients. In this study, the effects of IL-8 on expression of two major airway mucin genes, MUC5AC and MUC5B, were evaluated. IL-8 increased the mRNA abundance of both mucin genes in two human respiratory tract-derived cell lines (A549 and NCI-H292) in a time- and concentration-dependent manner. IL-8 also increased MUC5AC and MUC5B mRNA levels in primary normal differentiated human bronchial epithelial cells, with a high concentration of IL-8 required to increase MUC5B mRNA levels. IL-8 did not transcriptionally up-regulate MUC5AC gene expression, but rather increased the stability of the MUC5AC transcript, suggesting regulation at the posttranscriptional level. In addition, IL-8 altered the levels of RNA-binding proteins to specific domains in the 3'-untranslated region of the MUC5AC transcript. Taken together, these data indicate that the IL-8-induced binding of RNA-binding proteins to the 3'-untranslated region of MUC5AC is a potential mechanism for regulating MUC5AC gene expression at the posttranscriptional level, thus suggesting a new role whereby IL-8 sustains mucin gene expression in inflamed airways.

MUC5B Is the predominant mucin glycoprotein in chronic otitis media fluid.

Chronic otitis media (COM), e.g. "glue" ear is characterized by middle ear effusion and conductive hearing loss. Although mucous glycoproteins (mucins), which contribute to increased effusion viscosity, have been analyzed in ear tissue specimens, no studies have been reported that characterize the molecular identity of secreted mucin proteins present in actual middle ear fluid. For this study, effusions from children with COM undergoing myringotomy at Children's National Medical Center, Washington, DC were collected. These were solubilized and gel fractionated, and the protein content was identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics approach. Western blot analyses with mucin specific antibodies and densitometry were performed to validate the mass spectrometry findings. LC-MS/MS results identified mucin MUC5B by >26 unique peptides in six of six middle ear effusion samples, whereas mucin MUC5AC was only identified in one of six middle ear effusions. These findings were validated by Western blot performed on the same six and on an additional 11 separate samples where densitometry revealed on average a 6.4-fold increased signal in MUC5B when compared with MUC5AC (p = 0.0009). In summary, although both MUC5AC and MUC5B mucins are detected in middle ear effusions, MUC5B seems to be predominant mucin present in COM secretions.

Increased poly(ADP-ribose) polymerase (PARP)-1 expression and activity are associated with inflammation but not goblet cell metaplasia in murine models of allergen-induced airway inflammation.

Inflammation plays a key role in lung injury and in the pathogenesis of asthma. Two murine models of allergic airway inflammation-sensitization and challenge to ovalbumin (OVA) and intratracheal exposure to interleukin-13 (IL13)-were used to evaluate the expression of poly(ADP-ribose) polymerase-1 (PARP-1) in allergic airway inflammation. Inflammation is prominent in OVA-induced allergic asthma, but this inflammation is greatly reduced by a PARP-1 inhibitor and almost eliminated when PARP-1 knockout mice are subjected to the OVA model. The present study temporally evaluated PARP-1 protein expression, localization, and activity, as well as inflammation and goblet cell metaplasia (GCM), in murine lungs following a single OVA challenge or IL13 exposure. Following OVA challenge PARP-1 protein expression and activity were greatly increased, being maximal at 3 to 5 days following OVA exposure and beginning to decrease by day 8. These changes correlated with the timing and degree of inflammation and GCM. In contrast, PARP-1 protein or activity did not change following single IL13 exposure, though GCM was manifested without inflammation. This study demonstrates that both PARP-1 protein and activity are increased by allergen-activated inflammatory mediators, excluding IL13, and that PARP-1 increase does not appear necessary for GCM, one of the characteristic markers of allergic airway inflammation in murine models.