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Background: effective treatment for severe asthma is a significant unmet need. While eosinophilic inflammation caused by Type 2 cytokines is responsive to corticosteroid and biologic therapies, many severe asthmatics exhibit corticosteroid‐unresponsive mixed granulocytic inflammation. Objective: here, we tested the hypothesis that the pro‐allergic cytokine, IL‐13, can drive both corticosteroid‐sensitive and corticosteroid‐resistant responses. Results: by integration of in vivo and in vitro models of IL‐13 driven inflammation, we identify a role for the epidermal growth factor receptor (EGFR/ERBB1) as a mediator of corticosteroid‐unresponsive inflammation and bronchial hyperresponsiveness (BHR) driven by IL‐13. Topological data analysis using human epithelial transcriptomic data from the U‐BIOPRED cohort identified severe asthma groups with features consistent with the presence of IL‐13 and EGFR/ERBB activation, with involvement of distinct EGFR ligands. Our data suggest that IL‐13 may play a dual role in severe asthma: on the one hand driving pathologic corticosteroid‐refractory mixed granulocytic inflammation, but on the other hand underpinning beneficial epithelial repair responses, which may confound responses in clinical trials. Conclusion and clinical relevance: detailed dissection of those molecular pathways that are downstream of IL‐13 and utilize the ERBB receptor and ligand family to drive corticosteroid refractory inflammation should enhance the development of new treatments that target this sub‐phenotype(s) of severe asthma, where there is an unmet need.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection carries a substantial risk of severe and prolonged illness; treatment options are currently limited. We assessed the efficacy and safety of inhaled nebulised interferon beta-1a (SNG001) for the treatment of patients admitted to hospital with COVID-19.MethodsWe did a randomised, double-blind, placebo-controlled, phase 2 pilot trial at nine UK sites. Adults aged 18 years or older and admitted to hospital with COVID-19 symptoms, with a positive RT-PCR or point-of-care test, or both, were randomly assigned (1:1) to receive SNG001 (6 MIU) or placebo by inhalation via a mouthpiece daily for 14 days. The primary outcome was the change in clinical condition on the WHO Ordinal Scale for Clinical Improvement (OSCI) during the dosing period in the intention-to-treat population (all randomised patients who received at least one dose of the study drug). The OSCI is a 9-point scale, where 0 corresponds to no infection and 8 corresponds to death. Multiple analyses were done to identify the most suitable statistical method for future clinical trials. Safety was assessed by monitoring adverse events for 28 days. This trial is registered with Clinicaltrialsregister.eu (2020-001023-14) and ClinicalTrials.gov (NCT04385095); the pilot trial of inpatients with COVID-19 is now completed.FindingsBetween March 30 and May 30, 2020, 101 patients were randomly assigned to SNG001 (n=50) or placebo (n=51). 48 received SNG001 and 50 received placebo and were included in the intention-to-treat population. 66 (67%) patients required oxygen supplementation at baseline: 29 in the placebo group and 37 in the SNG001 group. Patients receiving SNG001 had greater odds of improvement on the OSCI scale (odds ratio 2·32 [95% CI 1·07-5·04]; p=0·033) on day 15 or 16 and were more likely than those receiving placebo to recover to an OSCI score of 1 (no limitation of activities) during treatment (hazard ratio 2·19 [95% CI 1·03-4·69]; p=0·043). SNG001 was well tolerated. The most frequently reported treatment-emergent adverse event was headache (seven [15%] patients in the SNG001 group and five [10%] in the placebo group). There were three deaths in the placebo group and none in the SNG001 group.InterpretationPatients who received SNG001 had greater odds of improvement and recovered more rapidly from SARS-CoV-2 infection than patients who received placebo, providing a strong rationale for further trials.FundingSynairgen Research.

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

Background: Altered collagen architecture with increased “bone-type” pyridinoline collagen cross-linking, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in Idiopathic Pulmonary Fibrosis (IPF). We recently identified that hypoxia inducible factor (HIF) pathway activation promotes induction of the bone-type collagen cross-linking enzymes lysyl hydroxylase 2 and lysyl oxidase-like 2, yet the consequences of this on collagen structure-function in lung fibrosis remain unknown. Methods: Using a long term 3D in vitro model of the fibroblastic focus we cultured primary lung fibroblasts from IPF donors without or with the HIF-stabilising compound IOX2. Hydroxyproline and mature pyridinium cross-links were measured by colorimetric assays, and collagen ultrastructure assessed by electron microscopy (EM). The biomechanical effects of HIF stabilisation were investigated by parallel plate compression testing. Results: IOX2 stabilised HIF within the 3D fibroblastic focus model, promoting HIF pathway activation to disproportionately induce collagen-modifying enzymes relative to collagen fibril synthesis. After 6 weeks of culture, mature pyridinium cross-links were significantly increased by IOX2 to levels we have previously observed in IPF tissue. Ultrastructural analysis of the collagen fibrils with EM identified that IOX2 significantly reduced fibril diameter to sizes comparable with collagen fibrils enzymatically extracted from IPF tissue. IOX2 induced a greater than 3-fold increase in tissue stiffness. Conclusions: HIF pathway activation is a core regulator of bone-type collagen fibrillogenesis and altered structure-function in lung fibrosis.

Background: Maternal asthma is a risk factor for asthma and airway hyperresponsiveness (AHR) in children. The asthma susceptibility gene, ADAM33, has been associated with AHR and impaired lung function in early life. Aims and Objectives: Our aim was to study how a maternal allergic environment in pregnancy interacts with offspring-ADAM33 and the effects on their lungs after birth. We hypothesised that effects of maternal allergic airway inflammation (AAI) will be different in Adam33 knock-out (KO) compared to wild-type (WT) offspring. Methods: Heterozygous (Adam33+/-) pregnant dams were challenged with allergen to induce AAI and control dams with saline. WT and KO offspring from the same litters were studied 4 weeks post partum for AHR. Bronchoalveolar lavage and lung tissue were studied by RTqPCR, Western blots, immunostainings and precision-cut lung slices (PCLS) in the presence of agonists and antagonists. Results: Allergen-naive 4-week old WT offspring of mothers with AAI showed AHR in vivo, whereas KO offspring were protected. The pulmonary muscarinic M1 receptor was increased in all offspring of AAI dams. Ex vivo PCLS studies with methacholine and muscarinic receptor antagonists confirmed vagal reflex bronchoconstriction in WT while KO offspring were protected by a decreased airway smooth muscle constriction, as shown with a thromboxane-receptor agonist. Conclusions: Gene-environment interactions between Adam33 and maternal AAI determine AHR in early life. While maternal AAI induces AHR in WT offspring via vagal responses, Adam33 KO alters the airway smooth muscle function and suppresses AHR, pointing to a new asthma AHR therapy.

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial–mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial–mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Super-resolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit, however, most of the techniques are hampered by the need for fluorescent labels. Non-linear label-free techniques such as Second Harmonic Generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve super-resolution SHG (SR-SHG). A resolution of ~λ/6 with respect to the fundamental wavelength, that is, a ~2.3-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarisation properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

Rationale: ADAMs (proteinases with a disintegrin and a metalloproteinase domain) have not been well studied in chronic obstructive pulmonary disease (COPD).Objectives: To investigate whether ADAM9 ...

Background Maternal allergic asthma is a strong risk factor for the development of asthma and airway hyperresponsiveness (AHR) in children. ADAM33, an asthma susceptibility gene, has been associated with AHR and impaired lung function in early life. Our aim was to investigate how the maternal allergic environment during pregnancy interacts with the ADAM33 status of their offspring, and the effects this has on the lungs of offspring after birth. We hypothesised that the effects of maternal allergy will be different in Adam33 knock-out (KO) compared to wild-type (WT) offspring Methods Allergic airway inflammation (AAI) during pregnancy was induced in heterozygous (Adam33±) mice through intranasal house dust mite (HDM) challenges. Control mice were challenged with saline. WT and KO (Adam33-/-) offspring from the same litters were studied 4 weeks post partum (pp). Lung function was measured in response to increasing doses of methacholine. Bronchoalveolar lavage fluid (BALF) and lung tissue were obtained for RTqPCR, Western Blots and immunostainings. Precision-cut lung slices (PCLS) from 4-weeks old offspring were investigated for airway contraction in response to different agonists and antagonists in vitro. Results Allergen-naive WT offspring of allergic mothers showed AHR 4 weeks pp compared to those of control mothers, whereas KO offspring from the same litter were protected. Expression of the muscarinic M1 receptor was elevated in both KO and WT offspring lungs of HDM-challenged dams. Experiments using muscarinic receptor antagonists and methacholine in PCLS confirmed that maternal AAI causes increased bronchoconstriction through vagal reflexes in WT offspring. KO offspring were protected from this effect due to decreased sensitivity of airway smooth muscle, suggested by a delayed response to a thromboxane-receptor agonist in PCLS. Conclusions Our studies show how gene-environment interactions between Adam33 and maternal AAI determine development of AHR in early life. While the AAI of the mother leads to an increased pulmonary muscarinic M1 receptor expression, the absence of Adam33 alters the airway smooth muscle function in the offspring. Together these changes manifest in AHR only in WT offspring, but not in KO offspring. Further studies are needed to determine how ADAM33 KO changes smooth muscle function in the lungs.

Sarcoidosis is a multisystem disease of unknown aetiology. Up to 90% of sarcoidosis patients have lung involvement including pulmonary fibrosis. This leads to impaired quality of life and sometimes mortality. To better understand the nature of the lung matrix we generated novel 3D multicellular foci from individual patient cells. Primary lung fibroblasts were isolated from explant tissue from patients with sarcoidosis. These were then grown in optimal conditions to promote 3D multicellular foci formation for a total of six weeks, treated +/- transforming growth factor-beta-1 (TGFβ1). Alpha smooth muscle actin (aSMA) and collagen mRNA expression were assess­ed by RTqPCR and gross mechanical stiffness of the resulting multicellular foci was assessed by parallel plate compression testing. Total collagen was assessed by hydroxyproline assay (ELISA), pyridinoline (PYD) concentrations were measured by ELISA and LC-MS to assess collagen cross-linking. Treatment of sarcoidosis fibroblasts with 2.5ng/mL TGFβ1 caused a 6-fold increase in collagen-1 (p TGFβ1 increases fibrotic markers in sarcoidosis fibroblasts cultured in this long-term cell culture model.

Mast cells (MCs) are immune cells classically associated with allergic diseases including asthma where they promote bronchoconstriction and Type 2 inflammation. However, they are also key immune cells against parasitic, bacterial and viral infections by releasing immune and inflammatory mediators. Rhinoviruses (RV) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of RV replication is the airway epithelium and MCs localise to the bronchial epithelium in asthma. The subsequent epithelial cell damage caused by RV leads to IL-33 release. This promotes Type 2 cytokine release by MCs contributing to airway inflammation. As IL-33 has been shown to increase MC immune responses against RSV, we hypothesised that IL-33 has a role in mediating innate immune responses in MCs against RV. The human MC cell line LAD2 was treated with IL-33 (1 - 10 ng/ml) plus RV (MOI 1) or UV-irradiated RV as control. Total RNA was extracted at 2, 4, 8 and 24 hours after treatment. Gene expression and viral copy number were quantified by RT-qPCR and protein release by MSD/ELISA. We found that IL-33 significantly induced a time and concentration dependent expression of Type 2 and pro-inflammatory cytokine genes. Furthermore, we found induction of interferon (IFN) and IFN-inducible genes suggesting activation of an innate anti-viral response. However, IL-33 was also found to induce ICAM1, the receptor for major group RV entry, and in the presence of RV, IL-33 facilitated MC infection. These results demonstrate that IL-33 is able to induce protective IFN responses in MCs, however this effect is counterbalanced by upregulation of ICAM1, rendering the MCs more susceptible to RV infection.

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.

Background: Airborne particulate matter (PM) is a leading cause of mortality and respiratory morbidity, associated with asthma, COPD, lung cancer, and IPF. Effects of inhaled PM may depend on PM chemistry, and thus source. Ships, dockside and freight activities are major sources of PM in port cities, but the chemistry and toxicology of such PM is poorly understood; shoreside sources receive little attention, especially the unregulated ultrafine (UF) fraction. Aim: To determine the chemical and toxicological properties of port-generated PM. Methods: Size-fractionated airborne PM (diameter 10-2.5µm (coarse[C]), 2.5-0.1µm (fine[F]), Results: PM concentrations were generally within WHO guidelines. Chemical analysis showed site-specific PM composition, with increased markers of ship emissions (V, Ni) in F and UF fractions at the cruise site, only during cruise season. Notably, cruise ship-associated UFPM elicited greater inflammatory mediator release than PM from other sites. Conclusions: Port PM has varying compositional signatures depending on its source and size. The increased inflammogenicity of cruise ship-associated UFPM merits further study to understand potential effects on exposed individuals.

Introduction: The lung epithelium is a functional interface between our body and the outside environment and it plays a key role in the respiratory physiology and pathology. Recurring alveolar epithelial injury is a hallmark of idiopathic pulmonary fibrosis (IPF) that lead to aberrant fibroblast activation resulting in the progressive destruction of the lung parenchymal architecture and impairment of gas exchange. The complex interactions between the persistent injured epithelium and the abnormal activated fibroblasts seems to be the main factors determining initiation and progression of the disease in association with aging, environmental exposure and genetic susceptibility. A better understanding of the epithelial-mesenchymal signaling in IPF is needed for the development of more efficient therapeutic strategies that promote restoration of normal epithelium integrity and limit the progression of fibrosis. Aim: Characterize the paracrine signaling between alveolar cells (AECs) and lung fibroblasts (FFs) and investigate its role in injury/repair of the epithelium. Methods: we established and characterize an ex-vivo co-culture models of AECs and FFs to mimic the epithelial-mesenchymal paracrine interaction in injured alveoli. Results and Conclusions: we detected abnormal epithelialization in co-culture of AECs and IPF FFs compared to co-culture of AECs and healthy FFs. Mass spectrometry analysis of fibroblasts secretome identified different extracellular matrix (ECM) proteins that are involved in tissue repair and fibrosis. Gene silencing of the ECM protein osteonectin in IPF FFs restored the alveolar epithelium homeostasis. These results suggest that IPF FFs secrete factors that alter the normal epithelial repair responses preventing the restoration of tissue homeostasis.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a poor prognosis and limited therapeutic options. Increasing evidence suggests that alveolar epithelial damage and resulting abnormal epithelial-mesenchymal crosstalk may contribute to the aberrant wound healing response observed in the lungs of IPF patients. The epithelial-mesenchymal trophic unit (EMTU) describes the functional interactions between epithelial cells, mesenchymal cells and the ECM which are necessary to regulate lung development, repair of damaged tissue and inflammatory responses. Thus characterization of EMTU in IPF will help to understand the pathofisiology of the disease. Aim: Characterize the paracrine crosstalk between alveolar cells (AECs) and lung fibroblasts (FFs) and investigate its role in injury and repair of the epithelium. Methods: we enstablished an ex-vivo co-culture models of AECs and FFs to mimic the EMTU of the respiratory epithelium. Epithelial cell layer intergrity was assed by immunofluorescence. Oxidative stress and injury/repair response were analysed using qPCR, western blot and time-laspe microscopy. Results and Conclusions: we detected abnormal epithelialization in co-culture of AECs and IPF FFs compared to co-culture of AECs and healthy FFs while western blot and qPCR data show increase in markers of cell motility. These results suggest that IPF FFs alter the normal epithelial repair responses preventing the restoration of lung tissue homeostasis. Therefore characterization of the EMTU in IPF will help the development of more efficient therapeutic strategies to limit the progression of fibrosis and promote restoration of the normal lung epithelium in IPF patients.

Background: In underground railways, airborne particulate matter (PM) concentrations are significantly elevated compared to outdoors. Underground PM is especially rich in iron and transition metals; in the ultrafine fraction (UFPM; diameter Aim: To analyse cellular responses to underground UFPM beyond the constrained range of markers commonly used. Methods: UFPM was collected from a European underground station. UFPM-induced antioxidant response element (ARE) activation by the transcription factor Nrf2 was measured in a reporter epithelial cell line transiently transfected with an Nrf2-binding ARE-luciferase construct. Healthy-donor primary bronchial epithelial cells (PBECs) were cultured at air-liquid interface (ALI) to form mucociliary cultures, and exposed to 25µg/ml UFPM for 6h/24h. RNAseq was performed to identify differentially expressed genes (DEGs) following UFPM exposure. Results: An antioxidant response to underground UFPM was seen with significant activation of ARE after 24h UFPM exposure. In PM-exposed ALI cultures, after 6h there were 52 DEGs (13 up/39 down), particularly associated with epithelial maintenance, whereas after 24h there were 23 DEGs (17 up/6 down), notably relating to redox homeostasis and metal binding. There was prolonged up-regulation of several members of the metal-binding antioxidant metallothionein (MT) family, partially responsive to iron chelation (desferrioxamine) and ROS scavenging (N-acetylcysteine). Conclusion: These data suggest time-dependent responses to metal-rich UFPM and highlight novel markers of exposure to PM constitutents, such as MTs.

Mast cells (MCs) are immune cells classically associated with allergic diseases including asthma where they promote bronchoconstriction and Type 2 inflammation. However, they are also key immune cells against parasitic, bacterial and viral infections by releasing immune and inflammatory mediators. Rhinoviruses (RV) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of RV replication is the airway epithelium and MCs localise to the bronchial epithelium in asthma. The subsequent epithelial cell damage caused by RV leads to IL-33 release. This promotes Type 2 cytokine release by MCs contributing to airway inflammation. As IL-33 has been shown to increase MC immune responses against RSV, we hypothesised that IL-33 has a role in mediating innate immune responses in MCs against RV. The human MC cell line LAD2 was treated with IL-33 (1 - 10 ng/ml) plus RV (MOI 1) or UV-irradiated RV as control. Total RNA was extracted at 2, 4, 8 and 24 hours after treatment. Gene expression and viral copy number were quantified by RT-qPCR and protein release by MSD/ELISA. We found that IL-33 significantly induced a time and concentration dependent expression of Type 2 and pro-inflammatory cytokine genes. Furthermore, we found induction of interferon (IFN) and IFN-inducible genes suggesting activation of an innate anti-viral response. However, IL-33 was also found to induce ICAM1, the receptor for major group RV entry, and in the presence of RV, IL-33 facilitated MC infection. These results demonstrate that IL-33 is able to induce protective IFN responses in MCs, however this effect is counterbalanced by upregulation of ICAM1, rendering the MCs more susceptible to RV infection.

Superresolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit; however, most of the techniques are hampered by the need for fluorescent labels. Nonlinear label-free techniques such as second-harmonic generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve SR-SHG. A resolution of ∼ λ / 6 with respect to the fundamental wavelength, that is, a ∼ 2.3 -fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarization properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarization-resolved SHG by observing anisotropy in signals. These new, to the best of our knowledge, findings allowed us to visualize biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to nondestructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

Background Maternal allergy is a strong risk factor for developing asthma and airway hyperresponsiveness (AHR). ADAM33 has been identified as an asthma susceptibility gene and is associated with AHR and impaired lung function in early life. Our aim was to investigate the effects of maternal murine allergic airway inflammation on the lungs of offspring before and after birth. We hypothesised that the effects of maternal allergy will be modified in Adam33 knock out (KO) compared to wild-type (WT) offspring. Methods Allergic airway inflammation in pregnant heterozygous (Adam33±) mice was induced by exposure to house dust mite (HDM) through intranasal challenges during pregnancy. Control mice were challenged with saline. WT (Adam33+/+) and KO (Adam33-/-) offspring from the same litters were studied on embryonic day (ED)17.5 and 2 or 4 weeks post partum (pp). Lung function was measured in response to increasing doses of methacholine and bronchoalveolar lavage fluid (BALF) was collected for differential cell counts. Lung tissue was obtained for RTqPCR, Western blot and immunohistochemistry. Results At 4 weeks pp, WT offspring of HDM challenged mothers showed significantly enhanced AHR compared to WT offspring of control mothers. KO of Adam33 protected against AHR in the offspring of allergic mothers. Adam33 mRNA expression was significantly enhanced in WT lungs of HDM challenged mothers at ED17.5, but unchanged pp. Differential cell counts in the BALF and mRNA expression of inflammatory mediators indicated an absence of allergic airway inflammation in all of the offspring. Remodelling genes were not affected at any time point studied. In contrast, Cholinergic Receptor Muscarinic 1 (Chrm1) mRNA was increased at 4 weeks in all offspring of HDM challenged mothers. Conclusions This study identifies an in utero gene-environment interaction involving Adam33. This interaction has implications for the subsequent development of AHR in early life. Further studies are needed to elucidate the precise mechanism(s) whereby ADAM33 mediates its effects. Our data suggest modulation of the contractility of the airways, possibly involving muscarinic receptors.

Introduction Most asthma has its origin in early life and probably involves gene-environment interactions. The asthma susceptibility gene ADAM33 is associated with bronchial hyperresponsiveness (BHR) and reduced lung function in young children. It encodes a membrane-anchored metalloprotease, which is shed as a soluble protein (sADAM33) whose levels are increased in asthma. We have previously shown that sADAM33, promotes airway remodelling and augments allergic airway inflammation in juvenile mice (Davies ER et al, JCI Insight 2016). This might be initiated by ADAM33 induced innate lymphocytes (Kelly JFC et al, Thorax 2017). The aim of this work was to evaluate the effect of sADAM33 on the allergic airway responses of neonates. Methods Human sADAM33 was induced in lungs of double transgenic (Ccsp/ADAM33) (dTg) mice from in utero up to 4 weeks post-partum. dTg mice or single transgenic (sTg) controls were challenged with house dust mite extract (HDM) 3 times a week for 2 weeks from 3 or 14 days post-partum. BHR and inflammation were quantified. Lung tissue was analysed by RT-qPCR and immunohistochemistry (IHC). Results After HDM challenge from day 3, Type 2-responsive genes Il-5, Ccl11/Eotaxin and Muc5ac were significantly increased. Whilst an increase in BHR was observed after HDM challenge, there was no significant difference between sADAM33-expressing and control mice. In contrast, when challenged from day 14, sADAM33-expressing mice had a more robust eosinophilic inflammatory response in the bronchoalveolar lavage fluid with increased Il-5 and Ccl11/Eotaxin mRNA expression compared to littermate controls. This was also associated with increased Acta2 mRNA expression and BHR. Conclusion These data indicate that sADAM33 does not augment allergic responses in neonatal mice as robustly as in older mice. This suggests that the immune microenvironment in neonates is not sufficiently mature to respond to the pro-allergic effects of sADAM33 that may induce innate lymphocytes to make the airways more susceptible to allergic airway inflammation.

Rationale ADAM33 is an asthma susceptibility gene that plays a role in both airway remodelling and susceptibility to allergic airways disease. To study the role of ADAM33 in asthma we have exposed an Adam33 Knockout (KO) mouse to a house dust mite (HDM) sensitisation and challenge protocol. We have found that these mice exhibit less remodelling, bronchial hyperresponsiveness (BHR) and eosinophilic inflammation than wild type (WT) mice (ER Davies et al., JCI-Insight 2016), however the mechanisms which contribute to this protective phenotype are not well understood. Methods To study how the response to HDM is altered by loss of Adam33 we challenged WT and KO mice with HDM or saline and took whole lung RNA samples for next generation RNA sequencing (RNAseq). Gene set enrichment analysis was used to identify pathways and gene ontology terms associated with the differential response to HDM between KO and WT mice. Results Control WT and KO mice were found to have very similar gene expression profiles at baseline (5 differentially expressed genes, including Adam33, FDR p Discussion The alteration in the pulmonary metabolic gene signature may underpin a shift in immune cell activation and/or modification of smooth muscle energy expenditure during airway contraction. These changes may explain why the KO mouse is protected from both allergic responses and BHR. Further work will aim to identify the source of the different metabolic behaviour at a cellular level and to assess oxidative stress in lungs of normal and Adam33 KO mice.

A disintegrin and metalloproteinase 33 (ADAM33) is an asthma susceptibility gene. A soluble, catalytically active form (sADAM33) has been identified in bronchoalveolar lavage fluid, levels of which correlate with disease severity. To model the role of sADAM33 in asthma, a transgenic mouse, expressing human sADAM33 was generated. sADAM33 mice were found to be more susceptible than controls to allergic airways disease. This led to the hypothesis that induction of sADAM33 expression changes the underlying immune microenvironment in the airway, promoting susceptiblity to allergic airways disease. To study this, RNA samples from whole lungs of sADAM33 mice and control mice were sequenced for differential expression analysis. Gene ontology identified predominantly immune-related biological processes associated with sADAM33 expression. Validation across a wider cohort of samples using RTqPCR confirmed the up-regulation of lymphocyte effector cell markers (Nkp46, Gzmb), as well as negative regulators of effector responses (Ido1, Pd-l1). Protein levels of GZMB measured by ELISA were significantly increased in sADAM33 mice. Analysis of lymphocyte populations by flow cytometry identified an increased population of CD3-, NKp46+, KLRG1+ positive lymphocytes, which may represent an altered phenotype of innate lymphocyte populations in the airway. The biology of innate lymphocytes is complex, but there is increasing support for their involvement in allergic disease. The altered phenotype of this cell population within the sADAM33 mice may play an important role in their increased susceptibility to allergic airways disease and warrants further investigation.

Maternal allergy is a strong risk factor for developing asthma and airway hyperresponsiveness (AHR). The asthma susceptibility gene ADAM33 is associated with AHR and impaired lung function in early life. Our aim was to investigate gene-environment interactions involving Adam33 by determining the effects of maternal murine allergic airway inflammation on the lungs of offspring. We hypothesised that this will be modified in Adam33 knock out (KO) compared to wild-type (WT) offspring. Allergic airway inflammation was induced by exposure to house dust mite (HDM) allergen in pregnant heterozygous Adam33 mice. WT and KO offspring from the same litters were studied on embryonic day (ED)17.5 and 2 and 4 weeks post partum. Lung function was performed in response to methacholine, bronchoalveolar lavage fluid (BALF) collected for differential cell counts and lung tissue for RTqPCR. Compared with saline challenged mothers, 4-week-old WT offspring of HDM challenged mothers showed significantly enhanced AHR; KO offspring of HDM challenged mothers were protected against development of AHR. No inflammatory genes were up-regulated in the lungs and no inflammatory cells were present in the BALF at 2 or 4 weeks. While typical remodelling genes were not up-regulated at the time points studied, Adam33 mRNA expression was significantly increased in WT lungs at ED17.5 and Cholinergic Receptor Muscarinic 1 (Chrm1) mRNA at 4 weeks in offspring of HDM challenged mothers. This study identifies an in utero gene-environment interaction involving ADAM33 that has implications for the development of AHR in early life. Further studies are needed to elucidate the mechanisms whereby ADAM33 mediates this.

Effective treatment for severe corticosteroid refractory asthma is a significant unmet clinical need. It affects only a small percent of the asthmatic population but accounts for a disproportionate use of healthcare resources. The aim was to study IL-13 transgenic mice to test the hypothesis that a subset of IL-13 mediated airway responses are corticosteroid-unresponsive and contribute to ongoing airways symptoms. IL-13 expression in the lungs was induced using Doxycycline in Ccsp-rtTA/Otet-Il-13 (Ccsp/Il-13) mice. Where indicated, mice received intra-peritoneal injections of 3mg/kg Dexamethasone for 3-7 days. Lungs were analysed for airway hyperreactivity and inflammation. Compared to controls, Ccsp/Il-13 mice showed significantly increased airway hyperresponsiveness (AHR) to methacholine and immunohistochemistry revealed increased bronchial smooth muscle and goblet cell metaplasia. The bronchoalveolar lavage fluid contained mixed eosinophilic and neutrophilic inflammation, but neutrophils predominated. Characteristic Th2-responsive genes as well as genes associated with Th17 responses were significantly elevated. Treatment with steroids did not abrogate AHR, even though eosinophilia and the ‘Th2’ gene signature were significantly reduced. Neutrophils and the ‘Th17’ signature remained elevated. Although IL-13 promotes eosinophilic airways disease, it can also drive corticosteroid refractory inflammation characterised by persistent neutrophilia, Th17 cytokines and maintenance of AHR. The Ccsp/Il-13 mouse may be a useful model for dissecting the molecular pathways and mechanisms associated with predominant neutrophilic, corticosteroid refractory airway disease.

Background: “Velcro-type” crackles on chest auscultation are considered a typical acoustic finding of Fibrotic Interstitial Lung Disease (FILD), however whether they may have a role in the early detection of these disorders has been unknown. This study investigated how “Velcro-type” crackles correlate with the presence of distinct patterns of FILD and individual radiologic features of pulmonary fibrosis on High Resolution Computed Tomography (HRCT).Methods: lung sounds were digitally recorded from subjects immediately prior to undergoing clinically indicated chest HRCT. Audio files were independently assessed by two chest physicians and both full volume and single HRCT sections corresponding to the recording sites were extracted. The relationships between audible “Velcro-type” crackles and radiologic HRCT patterns and individual features of pulmonary fibrosis were investigated using multivariate regression models.Results: 148 subjects were enrolled: bilateral “Velcro-type” crackles predicted the presence of FILD at HRCT (OR 13.46, 95% CI 5.85–30.96, p < 0.001) and most strongly the Usual Interstitial Pneumonia (UIP) pattern (OR 19.8, 95% CI 5.28–74. 25, p < 0.001). Extent of isolated reticulation (OR 2.04, 95% CI 1.62–2.57, p < 0.001), honeycombing (OR 1.88, 95% CI 1. 24–2.83, < 0.01), ground glass opacities (OR 1.74, 95% CI 1.29–2.32, p < 0.001) and traction bronchiectasis (OR 1.55, 95% CI 1.03–2.32, p < 0.05) were all independently associated with the presence of “Velcro-type” crackles.Conclusions: “Velcro-type” crackles predict the presence of FILD and directly correlate with the extent of distinct radiologic features of pulmonary fibrosis. Such evidence provides grounds for further investigation of lung sounds as an early identification tool in FILD.

SHARP (Severe Heterogeneous Asthma Research collaboration, Patient-centred) is set up to harmonise severe asthma management across Europe and unravel the heterogeneity of severe asthma in a patient-centred wayhttp://ow.ly/s1x730mwLpk

Rationale Severe corticosteroid refractory asthma is a significant unmet medical need. It accounts for 10% of the asthma population and 50% of the health economic burden. Recent understanding of asthma heterogeneity has evolved beyond clinical characteristics, allowing definition of distinct disease phenotypes such as those defined by levels of Type 2 inflammation (Type-2 high ‘eosinophilic’ disease and Type-2 low ‘neutrophilic’ disease). However, a recent study using dupilumab (an antibody that blocks the common IL-4 and IL-13 receptor chain, IL-4Rα) as an add-on therapy in adults with uncontrolled persistent asthma showed efficacy irrespective of baseline eosinophil count (Wenzel et al, Lancet 2016). The aim of this work was to use IL-13 transgenic mice to test the hypothesis that a subset of IL-13 mediated airway responses are corticosteroid-unresponsive and contribute to ongoing airways symptoms. Methods IL-13 expression in the lungs was induced using Doxycycline (DOX) in Ccsp-rtTA/Otet-Il-13 double-transgenic ( Ccsp/Il-13 ) mice. Littermate control single transgenic mice also received DOX. Where indicated, mice received daily intra-peritoneal injections of 3 mg/kg Dexamethasone (Dex) for 3–7 days and control mice received saline. Methacholine challenge and lung function measurements were performed and lungs harvested for mRNA analysis and immunohistochemistry (IHC). BALF was obtained for ELISA and differential cell counts. Results Compared to controls, Ccsp/Il-13 mice showed significantly increased airway hyperresponsiveness (AHR) to methacholine and IHC revealed increased bronchial smooth muscle and goblet cell metaplasia. The BALF of these mice contained mixed eosinophilic and neutrophilic inflammation, but neutrophils predominated. Characteristic Th2-responsive genes (Il-13, Eotaxin, Muc5ac, Periostin and SerpinB2 ) as well as genes more characteristic of Th17 responses (Cxcl1/Kc, Cxcl2 and Csf3) were significantly elevated. Treatment with Dex did not abrogate AHR, even though eosinophilia and the ‘Th2’ gene signature were significantly reduced. However, neutrophils and the ‘Th17’ signature remained elevated. Conclusion Although IL-13 promotes eosinophilic airways disease, it can also drive corticosteroid refractory inflammation characterised by persistent neutrophilia, Th17 cytokines and maintenance of AHR. These findings may help explain the beneficial effect of dupilumab in uncontrolled asthma. The Ccsp/Il-13 mouse may be a useful model for dissecting the molecular pathways and mechanisms associated with predominant neutrophilic, corticosteroid refractory disease.

Rational A disintegrin and metalloproteinase 33 (ADAM33) was discovered in 2002 as an asthma susceptibility gene. Genetic associations have been made between ADAM33 polymorphisms and asthma disease severity, bronchial hyperresponsivenss (BHR) and rate of lung function decline in both adults and children. A soluble, catalytically active form of the protein (sADAM33) has been identified in the bronchoalveolar lavage fluid of patients, levels of which correlate with disease severity (Lee JY et al, AJRCCM 2006). To study the role sADAM33, a lung specific, doxycycline (Dox) inducible, transgenic mouse, expressing the human pro and metalloproteinase domains of the full-length protein was generated (Ccsp-rtTA/Otet-ADAM33-Pro-MP). Induction of sADAM33, followed by house dust mite (HDM) sensitisation and challenge, resulted in increased BHR and airway inflammation (Davies ER et al, JCI-Insight 2016). The mechanisms by which ADAM33 promotes this susceptibility are unclear. The aim of this work is to identify pathways that are augmented by the induction of sADAM33, which promote increased sensitivity to allergen. Methods RNA samples from whole lung of adult mice, where sADAM33 had been induced for 4 or 8 weeks, were analysed by next generation RNA sequencing. Identified genes were confirmed across experimental time points (72 hour, 7 day, 4 and 8 weeks on Dox) in wider sample cohorts of Ccsp-rtTA/Otet-ADAM33-Pro-MP and control mice through RTqPCR. Results The predominant signal from the RNAseq output was for modulation of immune response genes at 4 weeks of sADAM33 expression (GO:0006955 Immune response: 31 genes, 58.49% coverage, FDR p value=7.09 E-22). Genes associated with an immune activation signature (Ccl5, Irgm1, Gm12250, Gzmb, Ncr1) were validated at least one time point. By 8 weeks of sADAM33 expression genes associated with negative regulation of the response were also validated (Ido1, Cd274). Conclusion Induction of sADAM33 in murine lungs, without allergic sensitisation, augmented underlying immune processes in this transgenic mouse model, which may contribute to increased susceptibility to allergic airway inflammation and BHR when challenged with allergen. Further work is required to delineate how sADAM33 affects immune cell populations and their behaviour in the lung.

Background: Increased tissue stiffness is a consequence and a driver of fibrosis. Tissue stiffness is modulated by the lysyl oxidase (LOX) family of enzymes, which cross-link collagen and elastin fibres. Increased expression of LOX-like-2 (LOXL2) is associated with the development of fibrosis and therefore a therapeutic target. We have profiled novel orally bioavailable LOXL2-selective small molecule inhibitors in in vitro and in vivo models of lung fibrosis. Methods: In the in vitro model, lung fibroblasts from IPF patients were cultured under optimised conditions for mature collagen matrix deposition. Following TGF-β1 treatment, multicellular foci formed which were histochemically similar in organization to fibroblastic foci in vivo (Jones et al., AJRCCM 191;2015:A4912). Transient overexpression of active TGF-β1 by adenovector gene transfer in rat lungs results in severe progressive fibrosis (Sime et al JCI 1997;100:768–776). The effects of treatment with a LOXL2-selective inhibitor (days 2-28) on histology and lung function were assessed at day 28. Results: Treatment with LOXL2-selective inhibitors dose-dependently reduced cross-link formation, altered the organisation of collagen matrix and reduced matrix stiffness (assessed by parallel plate compression) in the in-vitro model. In vivo, treatment at 15 or 30 mg/kg significantly lowered lung elastance compared to vehicle control from 2.2 (0.39) to 1.7 (0.31) or 1.6 (0.31) respectively (mean (SD) cmH2O/ml; p Conclusions The results suggest that inhibition of LOXL2 using these novel inhibitors has the potential to improve lung function in patients with lung fibrosis by reducing tissue stiffness.

Asthma is a common chronic disease characterized by variable respiratory distress with underlying airway inflammation and airflow obstruction. The incidence of asthma has risen inexorably over the past 50 years, suggesting that environmental factors are important in its etiology. All inhaled environmental stimuli interact with the lung at the respiratory epithelium, and it is a testament to the effectiveness of the airway innate defenses that the majority of inhaled substances are cleared without the need to elicit an inflammatory response. However, once this barrier is breached, effective communication with immune and inflammatory cells is required to protect the internal milieu of the lung. In asthma, the respiratory epithelium is known to be structurally and functionally abnormal. Structurally, the epithelium shows evidence of damage and has more mucus-producing cells than normal airways. Functionally, the airway epithelial barrier can be more permeable and more sensitive to oxidants and show a deficient innate immune response to respiratory virus infection compared with that in normal individuals. The potential of a susceptible epithelium and the underlying mesenchyme to create a microenvironment that enables deviation of immune and inflammatory responses to external stimuli may be crucial in the development and progression of asthma. In this review, we consider three important groups of environmental stimuli on the epithelium in asthma: oxidants, such as environmental pollution and acetaminophen; viruses, including rhinovirus; and agents that cause barrier disruption, such as house dust mite allergens. The pathology associated with each stimulus is considered, and potential future treatments arising from research on their effects are presented.

Aims: To (i) investigate the effects of Romidepsin in vitro using fibroblasts and alveolar cells (ATII); (ii) in vivo in Bleomycin treated mice; and (iii) to identify biomarkers in order to monitor response to therapy in a future clinical trial. Background: Idiopathic pulmonary fibrosis (IPF) is a complex chronic fibroproliferative disease of unknown aetiology and with limited therapeutic options.Numerous 9single pathway9 agents have failed to show efficacy in IPF clinical trial. By targeting multiple genes and pathways, HDAC inhibitors offer novel therapeutic strategies that could be used alone or in combination with existing agents (Pirfenidone and Nintedanib). Methods: Fibroblast and ATII cell proliferation were determined by cell counting and MTS assay. Myofibroblast differentiation was analysed by western blotting (WB) for α -SMA and Lysyl Oxidase (LOX). The antifibrotic effects of Romidepsin on bleomycin treated mice were assessed by RTqPCR, histology and WB. Broncho-alveolar-lavage (BAL) fluid from IPF patients was analysed by WB. Results: Romidepsin caused a strong inhibition of IPF fibroblast proliferation, myofibroblast differentiation and secretion of LOX. Comparison of the effect of Romidepsin on ATII cells and IPF fibroblasts showed that the ATII cells were significantly less sensitive. Romidepsin reduced bleomycin-induced lung fibrosis in mice and suppressed the expression of LOX. We detected elevated levels of LOX in the BALF of IPF patients. Conclusions: Romidepsin shows strong anti-fibrotic effects without harmful consequences on ATII cells. It is therefore a potential novel anti-fibrotic therapy and LOX may be a potential biomarker of response.

Aims: Investigation of epithelial barrier formation using PLLA membranes for application in bioengineering. Background: The development of functional and biocompatible substitutes for damaged tissue or organs is a major challenge in biomedical engineering. The epithelial barrier plays a central role in tissue homeostasis and immunity preventing damage and contamination of the interstitial tissues. Different in vitro models of the lung and intestinal epithelial barriers have been well characterized, however these tend to use non-biodegradable and/or poorly biocompatible scaffolds. Therefore, there is a need for better supports for epithelial cells for future applications in tissue engineering. Methods: Biodegradable PLLA membranes of differing morphologies were used as scaffolds to create an epithelial barrier model. Bronchiolar epithelial cells (H441) were cultured on PLLA membranes in transwell supports in order to assess barrier formation as measured by trans-epithelial electric resistance (TEER). Results: H441 cells readily attached to PLLA membranes and formed a confluent cell layer within two days. This was accompanied by a significant increase in TEER and correlated with the formation of intercellular tight junctions. Conclusions: A functional epithelial barrier can be established on biodegradable PLLA membranes that are not only biocompatible but be produced with different morphologies and mechanical properties. Therefore, PLLA membranes have potential utility in tissue engineering applications requiring bio-absorbable membranes in lung tissue or organ regeneration.

AimsTo quantify the extent of squamous metaplasia in bronchial biopsies and relate it to the presence of chronic obstructive pulmonary disease (COPD), a smoking-related pathology.MethodsBronchial biopsies (n = 15 in each group) from smokers with COPD GOLD stage1 and GOLD stage2, smokers without COPD and healthy non-smokers were stained immunohistochemically with a panel of antibodies that facilitated the identification of pseudostratified epithelium and distinction of squamous metaplasia and squamous epithelium from tangentially cut epithelium. The percentage length of each of these epithelial phenotypes was measured as a percent of total epithelial length using computerised image analysis. Sections were also stained for carcinoembryonic antigen and p53, early markers of carcinogenesis, and Ki67, and the percentage epithelial expression measured.ResultsThe extent of squamous metaplasia was significantly increased in both COPD1 and COPD2 compared to healthy smokers and healthy non-smokers. The amount of fully differentiated squamous epithelium was also increased in COPD1 and COPD2 compared to healthy non-smokers, as was the expression of carcinoembryonic antigen. These features correlated with one other.ConclusionIn subjects with COPD there is a loss of pseudostratified epithelium accompanied by an increase in squamous metaplasia with transition into a fully squamous epithelium and expression of early markers of carcinogenesis.

Since the activity of several conventional anticancer drugs is restricted by resistance mechanisms and dose-limiting side-effects, the design of formulations for local application on malignant lesions seems to be an efficient and promising drug delivery approach. In this study, the effect of locally applied 5-FU on cell death was evaluated both in a SCC4/HEK001 model and in a newly proposed 3D outgrowth model of oral squamous cell carcinoma (OSCC). Initially, the optimal drug dose was established by delivery of solutions containing different amounts of 5-FU. The solution containing 1% (w/v) of 5-FU resulted effective in inducing cell death with complete eradication of cell colonies. Buccal tablets were designed to deliver 5-FU locoregionally to the cancer lesions of the oral cavity. Tablets were prepared using a drug loaded matrix of acrylic/methacrylic acid copolymer containing 1% (w/w) of 5-FU and applied on 3D outgrowths. The drug release from tablets appeared to be sufficient to induce cell death as confirmed by transmission electron microscopy and enzymatic assay (TUNEL). After 120 h of treatment, when about 90% of the drug had been discharged from the tablets into the culture environment, 5-FU caused loss of cell-cell communications and apoptotic cell death. After 192 h, a complete disaggregation of the 3D oral outgrowths and the death of all the cells was observed. Buccal matrix tablets could be considered a promising new approach to the locoregional treatment of OSCC. Risks of systemic toxicity are avoided since very low drug doses are delivered.

Background and objective: Resistin-like molecule-b (RELM-b) is a necessary and sufficient stimulus for airway remodelling in animal models of asthma, but until recently, its role in human disease had not been investigated. The hypothesis that RELM-b expression would increase with increasing asthma severity and further increase following acute bronchoconstrictor challenges has been examined. Methods: Bronchial biopsies from healthy subjects and patients with mild and severe asthma were immunostained for RELM-b, as were airway biopsies obtained in mild asthmatics before and 4 days after repeated inhalation challenges with either allergen, methacholine or methacholine preceded by salbutamol as a control. Bronchial brushings were also evaluated for RELM-b mRNA. Results: RELM-b immunoreactivity, which colocalized to airway epithelial cells, increased with disease severity; healthy volunteers, median per cent epithelial area 1.98%, mild asthma 3.49% and severe asthma 5.89% (P < 0.001 between groups). RELM-b immunoreactivity significantly and inversely correlated in asthma with forced expiratory volume in 1 s % predicted (P = 0.005). Acute changes in immunoexpression were evident after repeated inhalation challenge with allergen (2.15 % to 4.35 % (P = 0.01)) and methacholine (4.21 % to 6.16 % (P = 0.01)) but did not change in the salbutamol/methacholine challenge group. These changes correlated with change in basement membrane thickness (r = 0.38, P = 0.02). Epithelial RELM-bgene expression was not altered in asthma.

Background and objective: a disintegrin and metalloproteinase (ADAM) 33 is a susceptibility gene associated with inflammatory lung and skin diseases. It is selectively expressed in mesenchymal cells, and its metalloprotease activity has been linked to angiogenesis and tissue remodelling. A soluble form of ADAM33 (sADAM33) has been identified in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, and its levels inversely correlate with lung function. Because of its association with inflammatory lung diseases, it was hypothesized that sADAM33 is elevated in BALF of patients with pulmonary sarcoidosis. Methods: after removal of Ig using Protein A/G and enrichment using Concanavalin A beads, sADAM33 was identified in BALF by Western blotting. A fluorescence resonance energy transfer peptide cleavage assay was used to assess ADAM33-like activity in BALF. Results: sADAM33 protein in BALF was detected as a 25 kDa fragment, and levels were significantly increased in samples from sarcoid patients when compared to healthy controls (P Conclusions: release of sADAM33 is increased in sarcoid and may be associated with abnormal lung function. sADAM33 may be a biomarker of lung tissue inflammation and remodelling in sarcoid

Background: Rhinovirus (RV)-induced chronic obstructive pulmonary disease (COPD) exacerbations exhibit TH2-like inflammation. We hypothesized that RV-infected bronchial epithelial cells (BEC) overproduce TH2-switching hub cytokine, thymic stromal lymphopoietin (TSLP) in COPD. Methods: Primary BEC from healthy (HBEC) and from COPD donors (COPD-BEC) were grown in 12-well plates, infected with RV16 (0.5–5 MOI) or stimulated with agonists for either toll-like receptor (TLR) 3 (dsRNA, 0.1–10 µg/ml) or RIG-I-like helicases (dsRNA-LyoVec, 0.1–10 µg/ml). Cytokine mRNA and protein were determined (RTqPCR; ELISA). Results: dsRNA dose-dependently evoked cytokine gene overproduction of TSLP, CXCL8 and TNF-α in COPD-BEC compared to HBEC. This was confirmed using RV16 infection. IFN-β induction did not differ between COPD-BEC and HBEC. Endosomal TLR3 inhibition by chloroquine dose-dependently inhibited dsRNA-induced TSLP generation and reduced generation of CXCL8, TNF-α, and IFN-β. Stimulation of cytosolic viral sensors (RIG-I-like helicases) with dsRNA-LyoVec increased production of CXCL8, TNF-α, and IFN-β, but not TSLP. Conclusions: Endosomal TLR3-stimulation, by dsRNA or RV16, induces overproduction of TSLP in COPD-BEC. dsRNA- and RV-induced overproduction of TNF-α and CXCL8 involves endosomal TLR3 and cytosolic RIG-I-like helicases and so does the generation of IFN-β in COPD-BEC. RV16 and dsRNA-induced epithelial TSLP may contribute to pathogenic effects at exacerbations and development of COPD.

Asthma is characterized pathologically by structural changes in the airway, termed airway remodeling. These changes are associated with worse long-term clinical outcomes and have been attributed to eosinophilic inflammation. In vitro studies indicate, however, that the compressive mechanical forces that arise during bronchoconstriction may induce remodeling independently of inflammation. We evaluated the influence of repeated experimentally induced bronchoconstriction on airway structural changes in patients with asthma.We randomly assigned 48 subjects with asthma to one of four inhalation challenge protocols involving a series of three challenges with one type of inhaled agent presented at 48-hour intervals. The two active challenges were with either a dust-mite allergen (which causes bronchoconstriction and eosinophilic inflammation) or methacholine (which causes bronchoconstriction without eosinophilic inflammation); the two control challenges (neither of which causes bronchoconstriction) were either saline alone or albuterol followed by methacholine (to control for nonbronchoconstrictor effects of methacholine). Bronchial-biopsy specimens were obtained before and 4 days after completion of the challenges.Allergen and methacholine immediately induced similar levels of bronchoconstriction. Eosinophilic inflammation of the airways increased only in the allergen group, whereas both the allergen and the methacholine groups had significant airway remodeling not seen in the two control groups. Subepithelial collagen-band thickness increased by a median of 2.17 μm in the allergen group (interquartile range [IQR], 0.70 to 3.67) and 1.94 μm in the methacholine group (IQR, 0.37 to 3.24) (P0.001 for the comparison of the two challenge groups with the two control groups); periodic acid-Schiff staining of epithelium (mucus glands) also increased, by a median of 2.17 percentage points in the allergen group (IQR, 1.03 to 4.77) and 2.13 percentage points in the methacholine group (IQR, 1.14 to 7.96) (P=0.003 for the comparison with controls). There were no significant differences between the allergen and methacholine groups.Bronchoconstriction without additional inflammation induces airway remodeling in patients with asthma. These findings have potential implications for management.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine that strongly activates dendritic cells and can initiate allergic inflammation. Since exposure to rhinovirus or double-stranded (ds) RNA (a surrogate of viral infection) induces TSLP expression in bronchial epithelial cells (BECs), this cytokine may link innate antiviral responses and the type 2 adaptive immune response.As BECs from donors with asthma have a deficient interferon (IFN) response to rhinovirus infection, a study was undertaken to test the hypothesis that their antiviral response shows a bias towards TSLP production.Primary BECs were grown from subjects with asthma and healthy volunteers. After exposure to dsRNA, interleukin (IL)-8, IFNbeta and TSLP mRNA and protein expression were measured by RT-qPCR and ELISA, respectively.dsRNA dose-dependently increased IL-8 expression in BECs with no significant difference between the groups. However, BECs from subjects with asthma expressed less IFNbeta and more TSLP mRNA and protein in response to dsRNA than BECs from those without asthma (median (IQR) 57 (38-82) pg/ml vs 106 (57-214) pg/ml for IFNbeta (p0.05) and 114 (86-143) pg/ml vs 65 (32-119) pg/ml for TSLP (p0.05) in response to 10 microg/ml dsRNA for 24 h). Induction of TSLP mRNA by dsRNA was blocked by Toll-like receptor 3 or protein kinase inhibitors or by preventing de novo protein synthesis, but not by neutralisation of type I IFN receptors.BECs from subjects with asthma are biased towards higher TSLP and lower IFNbeta production in response to dsRNA, suggesting that viral infection in asthma may lead to an altered mediator profile that biases towards a Th2 immune response.

Asthma is an inflammatory disorder of the airways dominated by a Th2-type pattern. Because of this, most research has focused on investigating the role of allergic pathways with the hope of discovering novel therapeutic targets. Unfortunately, this strategy (which has been extended to animal models) has failed to identify any therapeutic modalities other than anti-IgE and leukotriene modifiers directed to targets known about for many years. It seems that the problem lies in placing allergy at the center of disease pathogenesis, when in practice other environmental factors may be equally if not more important in the induction and then progression of asthma. An alternative view is that asthma is primarily a defect of epithelial barrier function that, as in atopic dermatitis, allows greater access of environmental allergens, microorganisms, and toxicants to the airway tissue. Evidence is provided to show that both the physical and functional barrier of the airway epithelium is defective in asthma with disrupted tight junctions, reduced antioxidant activity, and impaired innate immunity. This explains the remarkable susceptibility of asthmatic airways to respiratory viruses and the impact of air pollutants on asthma exacerbations. It also provides a mechanism for programming of dendritic cells to drive a Th2 response in the origins of asthma. Viewing asthma primarily as an epithelial disease with adoption of a chronic wound scenario also provides a route to airway wall remodeling and the varying asthma phenotypes over the life course.

Recent advances in the field of innate immunity have driven an important reappraisal of the role of these processes in airway disease. Various strands of evidence indicate that resident cells, such as macrophages and epithelial cells, have central importance in the initiation of inflammation. Macrophage activation has the potential to regulate not just typical aspects of innate immunity but also, via a variety of intricate cell-cell networks, adaptive responses and responses characterized by Th2-type cytokine production. In turn, such adaptive immune processes modify the phenotype and function of the innate immune system. Cooperative responses between monocytic cells and tissue cells are likely to be crucial to the generation of effective inflammatory responses, and a realization of the importance of these networks is providing a new way of identifying antiinflammatory therapies. Importantly, the repeated cycles of allergic and nonallergic inflammation that comprise chronic human airway disease are not necessarily well described by current terminology, and we propose and describe a concept of contiguous immunity, in which continual bidirectional cross-talk between innate and adaptive immunity describes disease processes more accurately.

Aims: Investigate the effect of the HADCI, FK228, on IPF fibroblasts differentiation and migration. Background: IPF is the most severe form of interstitial pneumonias with an unknown aetiology and without a successful therapy. Recent studies have demonstrated that HDAC inhibitors are good candidate for the treatment of fibrosis. These enzymes are specifically druggable, providing important therapeutic targets for IPF. Methods: The expression of FAK in IPF fibroblasts during FK228 treatment was assessed by western blot and real time PCR. The migration ability of IPF fibroblasts was assessed by scratch wound assay and transwell migration assay. Results: During FK228 treatment, FAK protein levels are down regulated in IPF fibroblasts resulting in the reduction of cell motility. Conclusions: FK228 is a good candidate for the treatment of IPF exhibiting a potent inhibition of fibroblast migration. By target FAK expression, FK228 could reduce fibroblasts dissemination resulting in a slower progression of lung fibrosis.