Three dimensional assessment of the pharyngeal airway in individuals with non-syndromic cleft lip and palate

Introduction

Children with cleft lip and palate (CLP) are known to have airway problems. Previous studies have shown that individuals with CLP have a 30% reduction in nasal airway size compared to non-cleft controls. No reports have been found on cross-sectional area and volume of the pharyngeal airway in clefts. Introduction of Cone-Beam CT (CBCT) and imaging software has facilitated generation of 3D images for assessment of the cross-sectional area and volume of the airway.

Objective

To assess the pharyngeal airway in individuals with CLP using CBCT by measuring volume and smallest cross-sectional areas and compare with 19 age- and sex-matched non-cleft controls.

Methods

Retrospective study of CBCT data of pre-adolescent individuals (N = 19, Mean age = 10.6, 7 females, 12 males, UCLP = 6, BCLP = 3) from the Center for Craniofacial Anomalies. Volumetric analysis was performed using image segmentation features in CB Works 3.0. Volume and smallest cross-sectional were studied in both groups. Seven measurements were repeated to verify reliability using Pearson correlation coefficient. Volume and cross-sectional area differences were analyzed using paired t-tests.

Results

The method was found to be reliable. Individuals with CLP did not exhibit smaller total airway volume and cross sectional area than non-CLP controls.

Conclusion

3D imaging using CBCT and CB Works is reliable for assessing airway volume. Previous studies have shown that the nasal airway is restricted in individuals with CLP. In our study, we found that the pharyngeal airway is not compromised in these individuals.     

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

Background

The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium.

Objective

Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.

Methods

This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used.

Results

The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions.

Conclusions

Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Nicotinic acetylcholine receptor variants associated with susceptibility to chronic obstructive pulmonary disease: a meta-analysis

Background

Only 10-15% of smokers develop chronic obstructive pulmonary disease (COPD) which indicates genetic susceptibility to the disease. Recent studies suggested an association between COPD and polymorphisms in CHRNA coding subunits of nicotinic acetylcholine receptor. Herein, we performed a meta-analysis to clarify the impact of CHRNA variants on COPD.

Methods

We searched Web of Knowledge and Medline from 1990 through June 2011 for COPD gene studies reporting variants on CHRNA. Pooled odds ratios (ORs) were calculated using the major allele or genotype as reference group.

Results

Among seven reported variants in CHRNA, rs1051730 was finally analyzed with sufficient studies. Totally 3460 COPD and 11437 controls from 7 individual studies were pooled-analyzed. A-allele of rs1051730 was associated with an increased risk of COPD regardless of smoking exposure (pooled OR = 1.26, 95% CI 1.18-1.34, p < 10^-5). At the genotypic level, the ORs gradually increased per A-allele (OR = 1.27 and 1.50 for GA and AA respectively, p < 10^-5). Besides, AA genotype exhibited an association with reduced FEV1% predicted (mean difference 3.51%, 95%CI 0.87-6.16%, p = 0.009) and increased risk of emphysema (OR 1.93, 95%CI 1.29-2.90, p = 0.001).

Conclusions

Our findings suggest that rs1051730 in CHRNA is a susceptibility variant for COPD, in terms of both airway obstruction and parenchyma destruction.     

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

Early phase resolution of mucosal eosinophilic inflammation in allergic rhinitis

Background

It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue.

Objective

To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation.

Methods

Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days'' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period.

Results

Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment.

Conclusions

Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.     

Clara cell 10-kDa protein gene transfection inhibits NF-κB activity in airway epithelial cells

Background

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.

Method/Results

To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10''s effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10''s effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.

Conclusion

These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation.     

Respiratory Syncytial Virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice

Background

Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity.

Methods

Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood.

Results

RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes.

Conclusions

RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.     

Smoking accelerates aging of the small airway epithelium

Background

Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking.

Methods

Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function.

Results

Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers.

Conclusion

These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0094-1) contains supplementary material, which is available to authorized users.     

Functional characterization of the matrix metalloproteinase-1 cigarette smoke-responsive region and association with the lung health study

Background

Prior studies have demonstrated that the distal 1.5 kb of the MMP-1 promoter is fundamental in directing the induction of the MMP-1 gene by cigarette smoke.

Methods

To characterize the genetic variants in the MMP-1 cigarette smoke-responsive element, deep re-sequencing of this element was performed on DNA samples from participants in the Lung Health Study. Furthermore, evidence of Sp1 binding to the MMP-1 promoter was assessed using chromatin immunoprecipitation assays and the influence of cigarette smoke exposure on this interaction was evaluated in cultured human small airway epithelial cells.

Results

Ten polymorphisms (four novel) were detected in the cigarette smoke-responsive element. Chromatin immunoprecipitation assays to assess the protein-DNA interactions at Sp1 sites in the MMP-1 promoter showed increased binding to the Sp1 sites in the cigarette smoke-responsive element in small airway epithelial cells treated with cigarette smoke extract. In contrast, a Sp1 site outside of the element exhibited the opposite effect. None of the polymorphisms were more prevalent in the fast decliners versus the slow decliners (fast decliners = mean −4.14% decline in FEV1% predicted per year vs. decline in FEV1% predicted per year).

Conclusions

Sequencing analyses identified four novel polymorphisms within the cigarette smoke-responsive element of the MMP-1 promoter. This study identifies functional activity within the cigarette smoke-responsive element that is influenced by cigarette smoke and examines this region of the promoter within a small patient population.     

Does unrestrained single-chamber plethysmography provide a valid assessment of airway responsiveness in allergic BALB/c mice?

Background

Unrestrained plethysmography has been used to monitor bronchoconstriction because of its ease of use and ability to measure airway responsiveness in conscious animals. However, its reliability remains controversial.

Objective

To investigate if unrestrained plethysmography could provide a valid interpretation of airway responsiveness in allergic BALB/c mice.

Methods

Ovalbumin sensitized BALB/c mice were randomized to receive either a single-dose Ovalbumin challenge (OVA-1D group) or a three-dose Ovalbumin challenge (OVA-3D group). The OVA-1D group was further divided into OVA-1D-I (measured invasively, using lung resistance as the index of responsiveness) and OVA-1D-N group (measured non-invasively, using Penh as the index of responsiveness). Similarly the OVA-3D group was divided into OVA-3D-I and OVA-3D-N groups based on the above methods. The control groups were sensitized and challenged with normal saline. Bronchial alveolar lavage fluid was taken and airway histopathology was evaluated for airway inflammation. Nasal responsiveness was tested with histamine challenge.

Results

Compared with controls, a significant increase in airway responsiveness was shown in the OVA-1D-N group (P < 0.05) but not in the OVA-1D-I group. Both OVA-3D-I and OVA-3D-N groups showed higher responsiveness than their controls (P < 0.05). The nasal mucosa was infiltrated by eosinophic cells in all Ovalbumin immunized groups. Sneezing or nasal rubbing in allergic groups appeared more frequent than that in the control groups.

Conclusion

Penh can not be used as a surrogate for airway resistance. The invasive measurement is specific to lower airway. Penh measurement (done as a screening procedure), must be confirmed by a direct invasive measurement specific to lower airway in evaluating lower airway responsiveness.     

PLUNC Is a Novel Airway Surfactant Protein with Anti-Biofilm Activity

Background

The PLUNC (“Palate, lung, nasal epithelium clone”) protein is an abundant secretory product of epithelia present throughout the conducting airways of humans and other mammals, which is evolutionarily related to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family. Two members of this family - the bactericidal/permeability increasing protein (BPI) and the lipopolysaccharide binding protein (LBP) - are innate immune molecules with recognized roles in sensing and responding to Gram negative bacteria, leading many to propose that PLUNC may play a host defense role in the human airways.

Methodology/Principal Findings

Based on its marked hydrophobicity, we hypothesized that PLUNC may be an airway surfactant. We found that purified recombinant human PLUNC greatly enhanced the ability of aqueous solutions to spread on a hydrophobic surface. Furthermore, we discovered that PLUNC significantly reduced surface tension at the air-liquid interface in aqueous solutions, indicating novel and biologically relevant surfactant properties. Of note, surface tensions achieved by adding PLUNC to solutions are very similar to measurements of the surface tension in tracheobronchial secretions from humans and animal models. Because surfactants of microbial origin can disperse matrix-encased bacterial clusters known as biofilms [1], we hypothesized that PLUNC may also have anti-biofilm activity. We found that, at a physiologically relevant concentration, PLUNC inhibited biofilm formation by the airway pathogen Pseudomonas aeruginosa in an in vitro model.

Conclusions/Significance

Our data suggest that the PLUNC protein contributes to the surfactant properties of airway secretions, and that this activity may interfere with biofilm formation by an airway pathogen.     

Optimization of Multimodal Imaging of Mesenchymal Stem Cells Using the Human Sodium Iodide Symporter for PET and Cerenkov Luminescence Imaging

Purpose

The use of stably integrated reporter gene imaging provides a manner to monitor the in vivo fate of engrafted cells over time in a non-invasive manner. Here, we optimized multimodal imaging (small-animal PET, Cerenkov luminescence imaging (CLI) and bioluminescence imaging (BLI)) of mesenchymal stem cells (MSCs), by means of the human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) as reporters.

Methods

First, two multicistronic lentiviral vectors (LV) were generated for multimodal imaging: BLI, ¹²⁴I PET/SPECT and CLI. Expression of the imaging reporter genes was validated in vitro using ^99mTcO₄ ⁻ radioligand uptake experiments and BLI. Uptake kinetics, specificity and tracer elution were determined as well as the effect of the transduction process on the cell''s differentiation capacity. MSCs expressing the LV were injected intravenously or subcutaneously and imaged using small-animal PET, CLI and BLI.

Results

The expression of both imaging reporter genes was functional and specific. An elution of ^99mTcO₄ ⁻ from the cells was observed, with 31% retention after 3 h. After labeling cells with ¹²⁴I in vitro, a significantly higher CLI signal was noted in hNIS expressing murine MSCs. Furthermore, it was possible to visualize cells injected intravenously using BLI or subcutaneously in mice, using ¹²⁴I small-animal PET, CLI and BLI.

Conclusions

This study identifies hNIS as a suitable reporter gene for molecular imaging with PET and CLI, as confirmed with BLI through the expression of Fluc. It supports the potential for a wider application of hNIS reporter gene imaging and future clinical applications.     

Understanding the contribution of native tracheobronchial structure to lung function: CT assessment of airway morphology in never smokers

Background

Computed tomographic (CT) airway lumen narrowing is associated with lower lung function. Although volumetric CT measures of airways (wall volume [WV] and lumen volume [LV]) compared to cross sectional measures can more accurately reflect bronchial morphology, data of their use in never smokers is scarce. We hypothesize that native tracheobronchial tree morphology as assessed by volumetric CT metrics play a significant role in determining lung function in normal subjects. We aimed to assess the relationships between airway size, the projected branching generation number (BGN) to reach airways of <2mm lumen diameter –the site for airflow obstruction in smokers- and measures of lung function including forced expiratory volume in 1 second (FEV₁) and forced expiratory flow between 25% and 75% of vital capacity (FEF 25–75).

Methods

We assessed WV and LV of segmental and subsegmental airways from six bronchial paths as well as lung volume on CT scans from 106 never smokers. We calculated the lumen area ratio of the subsegmental to segmental airways and estimated the projected BGN to reach a <2mm-lumen-diameter airway assuming a dichotomized tracheobronchial tree model. Regression analysis was used to assess the relationships between airway size, BGN, FEF 25–75, and FEV₁.

Results

We found that in models adjusted for demographics, LV and WV of segmental and subsegmental airways were directly related to FEV₁ (P <0.05 for all the models). In adjusted models for age, sex, race, LV and lung volume or height, the projected BGN was directly associated with FEF 25–75 and FEV₁ (P = 0.001) where subjects with lower FEV₁ had fewer calculated branch generations between the subsegmental bronchus and small airways. There was no association between airway lumen area ratio and lung volume.

Conclusion

We conclude that in never smokers, those with smaller central airways had lower airflow and those with lower airflow had less parallel airway pathways independent of lung size. These findings suggest that variability in the structure of the tracheobronchial tree may influence the risk of developing clinically relevant smoking related airway obstruction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0181-y) contains supplementary material, which is available to authorized users.     

Sinupret Activates CFTR and TMEM16A-Dependent Transepithelial Chloride Transport and Improves Indicators of Mucociliary Clearance

Introduction

We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl⁻) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.

Methods

Primary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR^−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl⁻ transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca²⁺]_i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.

Results

Sinupret-mediated Cl⁻ secretion [ΔI_SC(µA/cm²)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR^−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca²⁺ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl⁻ secretion. Sinupret also enhanced CBF and ASL depth.

Conclusion

Sinupret stimulates CBF, promotes transepithelial Cl⁻ secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca²⁺-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl⁻ secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.     

Lipid Alterations in Experimental Murine Colitis: Role of Ceramide and Imipramine for Matrix Metalloproteinase-1 Expression

Background

Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction.

Methodology/Principal Findings

Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4⁺CD62L⁺ cells into RAG1^−/−-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS–induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1β in Caco-2-IEC and human intestinal fibroblasts.

Conclusions/Significance

Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1β is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.     

Airway Symptoms and Biological Markers in Nasal Lavage Fluid in Subjects Exposed to Metalworking Fluids

Backgrounds

Occurrence of airway irritation among industrial metal workers was investigated. The aims were to study the association between exposures from water-based metal working fluids (MWF) and the health outcome among the personnel, to assess potential effects on the proteome in nasal mucous membranes, and evaluate preventive actions.

Methods

The prevalence of airway symptoms related to work were examined among 271 metalworkers exposed to MWF and 24 metal workers not exposed to MWF at the same factory. At the same time, air levels of potentially harmful substances (oil mist, morpholine, monoethanolamine, formaldehyde) generated from MWF was measured. Nasal lavage fluid was collected from 13 workers and 15 controls and protein profiles were determined by a proteomic approach.

Results

Airway symptoms were reported in 39% of the workers exposed to MWF although the measured levels of MWF substances in the work place air were low. Highest prevalence was found among workers handling the MWF machines but also those working in the same hall were affected. Improvement of the ventilation to reduce MWF exposure lowered the prevalence of airway problems. Protein profiling showed significantly higher levels of S100-A9 and lower levels of SPLUNC1, cystatin SN, Ig J and β2-microglobulin among workers with airway symptoms.

Conclusions

This study confirms that upper airway symptoms among metal workers are a common problem and despite low levels of MWF-generated substances, effects on airway immune proteins are found. Further studies to clarify the role of specific MWF components in connection to airway inflammation and the identified biological markers are warranted.     

Association of LIN28B with Adult Adiposity-Related Traits in Females

Context

Pubertal timing is under strong genetic control and its early onset associates with several adverse health outcomes in adulthood, including obesity, type 2 diabetes and cardiovascular disease. Recent data indicate strong association between pubertal timing and genetic variants near LIN28B, but it is currently unknown whether the gene contributes to the association between puberty and adult disease.

Objective

To elucidate the putative genetic link between early puberty and adult disease risk, we examined the association of two genetic variants near LIN28B with adult body size and metabolic profiles in randomly ascertained adult Finnish males and females.

Methods

Two single nucleotide polymorphisms (SNPs), rs7759938, the lead SNP previously associated with pubertal timing and height, and rs314279, previously also associated with menarcheal age but only partially correlated with rs7759938 (r² = 0.30), were genotyped in 26,636 study subjects participating in the Finnish population survey FINRISK. Marker associations with adult height, weight, body mass index (BMI), hip and waist circumference, blood glucose, serum insulin and lipid/lipoprotein levels were determined by linear regression analyses.

Results

Both rs7759938 and rs314279 associated with adult height in both sexes (p = 2×10⁻⁶ and p = 0.001). Furthermore, rs314279 associated with increased weight in females (p = 0.001). Conditioned analyses including both SNPs in the regression model verified that rs314279 independently associates with adult female weight, BMI and hip circumference (p<0.005). Neither SNP associated with glucose, lipid, or lipoprotein levels.

Conclusion

Genetic variants near the puberty-associated gene LIN28B associate with adult weight and body shape in females, suggesting that the gene may tag molecular pathways influencing adult adiposity-related traits.