Differential neutrophil activation in viral infections: Enhanced TLR-7/8-mediated CXCL8 release in asthma

© 2015 The Authors. Respirology published by Wiley Publishing Asia Pty Ltd on behalf of Asian Pacific Society of Respirology. Background and objective Respiratory viral infections are a major cause of asthma exacerbations. Neutrophils accumulate in the airways and the mechanisms that link neutrophilic inflammation, viral infections and exacerbations are unclear. This study aims to investigate anti-viral responses in neutrophils from patients with and without asthma and to investigate if neutrophils can be directly activated by respiratory viruses. Methods Neutrophils from peripheral blood from asthmatic and non-asthmatic individuals were isolated and stimulated with lipopolysaccharide (LPS) (1 μg/mL), f-met-leu-phe (fMLP) (100 nM), imiquimod (3 μg/mL), R848 (1.5 μg/mL), poly I:C (10 μg/mL), RV16 (multiplicity of infection (MOI)1), respiratory syncytial virus (RSV) (MOI1) or influenza virus (MOI1). Cell-free supernatants were collected after 1 h of neutrophil elastase (NE) and matrix metalloproteinase (MMP)-9 release, or after 24 h for CXCL8 release. Results LPS, fMLP, imiquimod and R848 stimulated the release of CXCL8, NE and MMP-9 whereas poly I:C selectively induced CXCL8 release only. R848-induced CXCL8 release was enhanced in neutrophils from asthmatics compared with non-asthmatic cells (P < 0.01). RSV triggered the release of CXCL8 and NE from neutrophils, whereas RV16 or influenza had no effect. Conclusion Neutrophils release CXCL8, NE and MMP-9 in response to viral surrogates with R848-induced CXCL8 release being specifically enhanced in asthmatic neutrophils. Toll-like receptor (TLR7/8) dysregulation may play a role in neutrophilic inflammation in viral-induced exacerbations. We aimed to investigate and compare neutrophil responses to bacterial compounds and viral mimetics as well as compare responses between people with and without asthma. We also investigated neutrophil responses to live respiratory viruses. Here we provide a novel comprehensive comparison showing differential and specific activation in innate immune cells. See Editorial, page 1

Activity and Process Stability of Purified Green Pepper (Capsicum annuum) Pectin Methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 °C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 °C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55?57 °C) and a biphasic model for higher temperatures (58?70 °C). The enzyme showed a stable behavior toward high-pressure/temperature treatments. Keywords: Capsicum annuum; pepper; pectin methylesterase; purification; characterization; thermal and high-pressure stabilit

Effects of β2 Agonists, Corticosteroids, and Novel Therapies on Rhinovirus-Induced Cytokine Release and Rhinovirus Replication in Primary Airway Fibroblasts

Rhinovirus-(RV-) induced asthma exacerbations account for high asthma-related health costs and morbidity in Australia. The cellular mechanism underlying this pathology is likely the result of RV-induced nuclear-factor-kappa-B-(NF-κB-) dependent inflammation. NF-κB may also be important in RV replication as inhibition of NF-κB inhibits replication of other viruses such as human immunodeficiency virus and cytomegalovirus. To establish the role of NF-κB inhibitors in RV-induced IL- 6 and IL-8 and RV replication, we used pharmacological inhibitors of NF-κB, and steroids and/or β2 agonists were used for comparison. Primary human lung fibroblasts were infected with RV-16 in the presence of NF-κB inhibitors: BAY-117085 and dimethyl fumarate; β2 agonist: salmeterol; and/or corticosteroids: dexamethasone; fluticasone. RV-induced IL-6 and IL-8 and RV replication were assessed using ELISAs and virus titration assays. RV replicated and increased IL-6 and IL-8 release. Salmeterol increased, while dexamethasone and fluticasone decreased RV-induced IL-6 and IL-8 (P<0.05). The NF-κB inhibitor BAY-117085 inhibited only RV-induced IL-6 (P<0.05) and dimethyl fumarate did not alter RV-induced IL-6 and IL-8. Dimethylfumarate increased RV replication whilst other drugs did not alter RV replication. These data suggest that inhibition of NF-κB alone is unlikely to be an effective treatment compared to current asthma therapeutics

Tissue is the issue-sarcoidosis following ABVD chemotherapy for Hodgkin's lymphoma: a case report

Thirty two year old Caucasian female presented 2 months post partum with fevers, cough and shortness of breath. CT scan of the chest to rule out pulmonary embolism revealed mediastinal lymphadenopathy. Biopsy of the nodes revealed classic Hodgkin's lymphoma and she received ABVD chemotherapy. She was in remission as confirmed by a PET/CT scan. Five months later she had another PET/CT scan which showed areas of hypermetabolism indicating a possible relapse. Biopsy revealed sarcoidosis. She received steroids and 18 months later remained in clinical remission. This rare case of sarcoid following classic Hodgkin's lymphoma illustrates that clinical presentation, physical exam, lab investigations and even PET/CT scans may not be able to discriminate between Hodgkin's lymphoma and sarcoidosis. Tissue biopsy and pathological diagnosis remain the gold standard

BET proteins are associated with the induction of small airway fibrosis in COPD.

RationaleIn COPD, small airway fibrosis occurs due to increased extracellular matrix (ECM) deposition in and around the airway smooth muscle (ASM) layer. Studies of immune cells and peripheral lung tissue have shown that epigenetic changes occur in COPD but it is unknown whether airway mesenchymal cells are reprogrammed.ObjectivesDetermine if COPD ASM cells have a unique epigenetic response to profibrotic cytokine transforming growth factor β1 (TGF-β1).MethodsPrimary human ASM cells from COPD and non-COPD smoking patients were stimulated with TGF-β1. Gene array analysis performed to identify differences in ECM expression. Airway accumulation of collagen 15α1 and tenascin-C proteins was assessed. Aforementioned ASM cells were stimulated with TGF-β1 ± epigenetic inhibitors with qPCR quantification of COL15A1 and TNC. Global histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity were assessed. chromatin immunoprecipitation (ChIP)-qPCR for histone H3 and H4 acetylation at COL15A1 and TNC promoters was carried out. Effects of bromoterminal and extraterminal domain (BET) inhibitor JQ1(+) on expression and acetylation of ECM target genes were assessed.Measurements and main resultsCOPD ASM show significantly higher COL15A1 and TNC expression in vitro and the same trend for higher levels of collagen 15α1 and tenascin-c deposited in COPD airways in vivo. Epigenetic screening indicated differential response to HDAC inhibition. ChIP-qPCR revealed histone H4 acetylation at COL15A1 and TNC promoters in COPD ASM only. ChIP-qPCR found JQ1(+) pretreatment significantly abrogated TGF-β1 induced histone H4 acetylation at COL15A1 and TNC.ConclusionsBET protein binding to acetylated histones is important in TGF-β1 induced expression of COL15A1 and TNC and maintenance of TGF-β1 induced histone H4 acetylation in cell progeny

A novel immunomodulatory function of neutrophils on rhinovirus-Activated monocytes in vitro

© 2016 Published by the BMJ Publishing Group Limited. Background Rhinovirus (RV) infections are the major precipitant of asthma exacerbations. While neutrophilic lung inflammation occurs during such infections, its role remains unclear. Neutrophilic inflammation is associated with increased asthma severity and steroid refractory disease. Neutrophils are vital for controlling infections but also have immunomodulatory functions. Previously, we found that neutrophils respond to viral mimetics but not replication competent RV. We aimed to investigate if neutrophils are activated and/or modulate immune responses of monocytes during RV16 infection. Methods Primary human monocytes and autologous neutrophils were cocultured with or without RV16, in direct contact or separated by transwells. RV16-stimulated monocytes were also exposed to lysed neutrophils, neutrophil membrane components or soluble neutrophil intracellular components. Interleukin 6 (IL-6) and C-X-C motif (CXC)L8 mRNA and proteins were measured by quantitative PCR and ELISA at 24â €..hours. Results RV16 induced IL-6 and CXCL8 in monocytes, but not neutrophils. RV16-induced IL-6 and CXCL8 from monocytes was reduced in the presence of live neutrophils. Transwell separation abolished the inhibitory effects. Lysed neutrophils inhibited RV16-induced IL-6 and CXCL8 from monocytes. Neutrophil intracellular components alone effectively inhibited RV16-induced monocyte-derived IL-6 and CXCL8. Neutrophil intracellular components reduced RV16-induced IL-6 and CXCL8 mRNA in monocytes. Conclusions Cell contact between monocytes and neutrophils is required, and preformed neutrophil mediator(s) are likely to be involved in the suppression of cytokine mRNA and protein production. This study demonstrates a novel regulatory function of neutrophils on RV-Activated monocytes in vitro, challenging the paradigm that neutrophils are predominantly proinflammatory

Mathematical modelling of polyamine metabolism in bloodstream-form trypanosoma brucei: An application to drug target identification

© 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis article has been made available through the Brunel Open Access Publishing Fund.We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.The work was carried out under a PhD programme partly funded by Prof. Ray Welland, School of Computing Science, University of Glasgo

Cancer cells exploit an orphan RNA to drive metastatic progression.

Here we performed a systematic search to identify breast-cancer-specific small noncoding RNAs, which we have collectively termed orphan noncoding RNAs (oncRNAs). We subsequently discovered that one of these oncRNAs, which originates from the 3' end of TERC, acts as a regulator of gene expression and is a robust promoter of breast cancer metastasis. This oncRNA, which we have named T3p, exerts its prometastatic effects by acting as an inhibitor of RISC complex activity and increasing the expression of the prometastatic genes NUPR1 and PANX2. Furthermore, we have shown that oncRNAs are present in cancer-cell-derived extracellular vesicles, raising the possibility that these circulating oncRNAs may also have a role in non-cell autonomous disease pathogenesis. Additionally, these circulating oncRNAs present a novel avenue for cancer fingerprinting using liquid biopsies

Towards Compliance of Cross-Organizational Processes and their Changes

Businesses require the ability to rapidly implement new processes and to quickly adapt existing ones to environmental changes including the optimization of their interactions with partners and customers. However, changes of either intra- or cross-organizational processes must not be done in an uncontrolled manner. In particular, processes are increasingly subject to compliance rules that usually stem from security constraints, corporate guidelines, standards, and laws. These compliance rules have to be considered when modeling business processes and changing existing ones. While change and compliance have been extensively discussed for intra-organizational business processes, albeit only in an isolated manner, their combination in the context of cross-organizational processes remains an open issue. In this paper, we discuss requirements and challenges to be tackled in order to ensure that changes of cross-organizational business processes preserve compliance with imposed regulations, standards and laws