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Refereed/Peer-reviewed COVID-19 infects via the respiratory system, but it can affect multiple systems and lead to multi system failure. There is growing evidence that smoking may be associated with higher rates of COVID-19 infections and worse outcomes due to increased levels of ACE2 in lung epithelial cells, but it is unknown whether E-cigarette use may lead to increased risk of COVID-19 infection from the SARS-CoV-2 virus. In this study, healthy donor bronchial epithelial cells (NHBE) and monocyte-derived macrophages (MDM) were exposed to cigarette smoke extract (CSE) or nicotine or flavoured E-cigarette vapour extract (EVE) before the assessment of SARS-CoV-2 recognition receptors ACE2 and TMPRSS2 genes. MDMs exposed to CSE and Tobacco EVE showed increased ACE2 expression; however, no treatment altered the TMPRSS2 expression. ACE2 was found to be upregulated by >2-fold in NHBE cells exposed to CSE, as well as nicotine, banana, or chocolate EVE, while TMPRSS2 was only upregulated by CSE or nicotine EVE exposure. These findings suggesting that flavourings can increase ACE2 expression in multiple cell types, while TMPRSS2 expression increases are limited to the epithelial cells in airways and may be limited to nicotine and/or cigarette smoke exposure. Therefore, increased risk of COVID-19 infection cannot be ruled out for vapers.

The growing interest in regulating flavored E-liquids must incorporate understanding of the “flavoring profile” of each E-liquid—which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1–64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1β secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.

Smoking continues to be a burden to economies and health-care systems across the world. One proposed solution to the problem has been e-cigarettes; however, because they are a relatively new product in the market, little is known about their potential health impacts. Furthermore, e-cigarettes continue to evolve at a rapid rate, making it necessary to regularly review and summarize available studies. Although e-cigarettes are marketed as a smoking cessation tool by some manufacturers, the reality is that many nonsmokers, including youth, are using them. This review focuses on two major demographic groups (smokers and nonsmokers) and evaluates the most recent data (early 2017 to mid 2019) regarding the potential health effects of e-cigarettes. We assessed peer-reviewed studies on the health impacts of e-cigarettes, with a particular focus on common questions asked by policy makers, clinicians, and scientists: (1) What are the effects of e-cigarettes compared with air/not smoking?; (2) Is there any direct evidence of harm or benefit to humans?; (3) Is there a risk from secondhand exposure?; (4) What are the risks and/or benefits of e-cigarettes compared with tobacco cigarette use?; (5) Are there risks or benefits to specific populations (eg, people with COPD or asthma, pregnant women [and their offspring])?; (6) What are the effects of flavoring chemicals?; (7) What are the effects of including nicotine in e-liquids?; (8) How often is nicotine concentration labeling incorrect?; and (9) What are the risks when e-cigarettes explode?

Background and objective E-cigarettes are often marketed and thought of as emitting harmless vapour; however, verification of their safety for non-smokers is scarce. We have previously shown that E-cigarettes cause decreased phagocytosis of bacteria by macrophages via reductions in surface bacterial recognition receptors. This study assessed the effect of E-cigarette constituents, 3 E-liquid apple flavours, nicotine, vegetable glycerine and propylene glycol, on bronchial epithelial cell viability, apoptosis and cytokine secretion and macrophage phagocytosis of apoptotic airway cells and phagocytic recognition molecules. Methods Cell necrosis and apoptosis were measured by Sytox Green stain and Annexin V. Efferocytosis was measured by internalization of pHrodo Green labelled apoptotic airway cells by macrophages. Expression of macrophage cell surface apoptotic cell receptors was measured by flow cytometry. Cytokine release by E-cigarette-exposed airway cells was measured by cytokine bead array. Results E-cigarette vapour increased primary bronchial epithelial necrosis and apoptosis. E-cigarette vapour reduced efferocytosis (lowest flavour 12.1%) versus control (20.2%, P = 0.032). The efferocytosis receptor CD44 was reduced by one flavour (MFI 1863 vs 2332 control, P = 0.016) and all components reduced expression of CD36, including the glycol bases (MFI 1067-12 274 vs 1415 control). Reduced secretion of TNF-α, IL-6, IP-10, MIP-1α and MIP-1β was observed for all flavour variants. Conclusion E-cigarettes can cause bronchial epithelial apoptosis and macrophage efferocytosis dysfunction via reduced expression of apoptotic cell recognition receptors. These data further show that E-cigarettes should not be considered harmless to non-smokers and their effects may go far beyond cytotoxicity to cells.

Introduction The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. Methods Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1β. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. Results Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1β, were demonstrated in COPD lungs (n = 9) vs. control (n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an > 8-fold increase (p 6-fold increase (p Conclusion The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.

In chronic obstructive pulmonary disease (COPD) apoptotic bronchial epithelial cells are increased, and their phagocytosis by alveolar macrophages (AM) is decreased alongside bacterial phagocytosis. Epithelial cellular lipids, including those exposed on uncleared apoptotic bodies, can become oxidized, and may be recognized and presented as non-self by antigen presenting cells. CD1b is a lipid-presenting protein, previously only described in dendritic cells. We investigated whether CD1b is upregulated in COPD AM, and whether lipid oxidation products are found in the airways of cigarette smoke (CS) exposed mice. We also characterise CD1b for the first time in a range of macrophages and assess CD1b expression and phagocytic function in response to oxidised lipid. Bronchoalveolar lavage and exhaled breath condensate were collected from never-smoker, current-smoker, and COPD patients and AM CD1b expression and airway 8-isoprostane levels assessed. Malondialdehyde was measured in CS-exposed mouse airways by confocal/immunofluorescence. Oxidation of lipids produced from CS-exposed 16HBE14o- (HBE) bronchial epithelial cells was assessed by spectrophotometry and changes in lipid classes assessed by mass spectrometry. 16HBE cell toxicity was measured by flow cytometry as was phagocytosis, CD1b expression, HLA class I/II, and mannose receptor (MR) in monocyte derived macrophages (MDM). AM CD1b was significantly increased in COPD smokers (4.5 fold), COPD ex-smokers (4.3 fold), and smokers (3.9 fold), and AM CD1b significantly correlated with disease severity (FEV1) and smoking pack years. Airway 8-isoprostane also increased in smokers and COPD smokers and ex-smokers. Malondialdehyde was significantly increased in the bronchial epithelium of CS-exposed mice (MFI of 18.18 vs 23.50 for control). Oxidised lipid was produced from CS-exposed bronchial epithelial cells (9.8-fold of control) and showed a different overall lipid makeup to that of control total cellular lipid. This oxidised epithelial lipid significantly upregulated MDM CD1b, caused bronchial epithelial cell toxicity, and reduced MDM phagocytic capacity and MR in a dose dependent manner. Increased levels of oxidised lipids in the airways of COPD patients may be responsible for reduced phagocytosis and may become a self-antigen to be presented by CD1b on macrophages to perpetuate disease progression despite smoking cessation.

IntroductionThe role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke.Methods Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1β. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells.Results Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1β, were demonstrated in COPD lungs (n=9) vs. control (n=5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 hrs stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an >8-fold increase (p6-fold increase (pConclusion The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.

The TSANZ develops position statements where insufficient data exist to write formal clinical guidelines. In 2018, the TSANZ addressed the question of potential benefits and health impacts of electronic cigarettes (EC). The working party included groups focused on health impacts, smoking cessation, youth issues and priority populations. The 2018 report on the Public Health Consequences of E-Cigarettes from the United States NASEM was accepted as reflective of evidence to mid-2017. A search for papers subsequently published in peer-reviewed journals was conducted in August 2018. A small number of robust and important papers published until March 2019 were also identified and included. Groups identified studies that extended, modified or contradicted the NASEM report. A total of 3793 papers were identified and reviewed, with summaries and draft position statements developed and presented to TSANZ membership in April 2019. After feedback from members and external reviewers, a collection of position statements was finalized in December 2019. EC have adverse lung effects and harmful effects of long-term use are unknown. EC are unsuitable consumer products for recreational use, part-substitution for smoking or long-term exclusive use by former smokers. Smokers who require support to quit smoking should be directed towards approved medication in conjunction with behavioural support as having the strongest evidence for efficacy and safety. No specific EC product can be recommended as effective and safe for smoking cessation. Smoking cessation claims in relation to EC should be assessed by established regulators Refereed/Peer-reviewed

Cigarette smoke (CS)-induced emphysema is an important contributor to chronic obstructive pulmonary disease (COPD). We have shown the efficacy of azithromycin in reducing airway inflammation in COPD and in reducing exacerbations in severe asthma; however, the effects of long-term azithromycin on emphysema development have not been shown. We employed live animal imaging to monitor emphysema-like development and the effects of interventional azithromycin treatment in CS-exposed mice. BALB/c mice (female, 10 weeks; n = 10) were exposed to CS for 1 hr twice daily, 5 days/week, and for 12 weeks (CS). Half were cotreated with low-dose azithromycin during weeks 7–12 (CS + Azi; 0.2 mg kg−1 day−1). Microcomputed tomography (CT) and magnetic resonance imaging (MRI) scans were acquired longitudinally. Histological examinations were performed post mortem (mean linear intercept (Lm) and leukocyte infiltration). CS increased median Lm (CS: 42.45 µm versus control: 34.7 µm; p =.0317), this was recovered in CS + Azi mice (33.03 µm). Average CT values were reduced in CS mice (CS: −399.5 Hounsfield units (HU) versus control: −384.9 HU; p =.0286) but not in CS + Azi mice (−377.3 HU). CT values negatively correlated with Lm (r = −.7972; p =.0029) and T2-weighted MRI (r = −.6434; p =.0278). MRI also showed significant CS-induced inflammatory changes that were attenuated by azithromycin in the lungs, and positively correlated with Lm (r =.7622; p =.0055) and inflammatory foci counts (r =.6503; p =.0257). Monitoring of emphysema development is possible via micro-CT and MRI. Interventional azithromycin treatment in CS-exposed mice attenuated the development of pulmonary emphysema-like changes Refereed/Peer-reviewed

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides—2′-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2′-desoxy molecule (GS-560660)—with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae. We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1β (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5–1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1β was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-A resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCE Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-Å resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis. Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells. IMPORTANCE Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

Ovarian cancer has a distinct tendency for metastasising via shedding of cancerous cells into the peritoneal cavity and implanting onto the peritoneum that lines the pelvic organs. Once ovarian cancer cells adhere to the peritoneal cells, they migrate through the peritoneal layer and invade the local organs. Alterations in the extracellular environment are critical for tumour initiation, progression and intra-peritoneal dissemination. To increase our understanding of the molecular mechanisms involved in ovarian cancer metastasis and to identify novel therapeutic targets, we recently studied the interaction of ovarian cancer and peritoneal cells using a proteomic approach. We identified several extracellular matrix (ECM) proteins including, fibronectin, TGFBI, periostin, annexin A2 and PAI-1 that were processed as a result of the ovarian cancer–peritoneal cell interaction. This review focuses on the functional role of these proteins in ovarian cancer metastasis. Our findings together with published literature support the notion that ECM processing via the plasminogen–plasmin pathway promotes the colonisation and attachment of ovarian cancer cells to the peritoneum and actively contributes to the early steps of ovarian cancer metastasis.

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [32P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10–100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease.

Bushfires are increasing in frequency and severity worldwide. Bushfire smoke contains organic/inorganic compounds including aldehydes and acrolein. We described suppressive effects of tobacco smoke on the phagocytic capacity of airway macrophages, linked to secondary necrosis of uncleared apoptotic epithelial cells, persistence of non-typeable H. influenzae (NTHi), and inflammation. We hypothesised that bushfire smoke extract (BFSE) would similarly impair macrophage function. THP-1 or monocyte-derived macrophages (MDM) were exposed to 1–10% BFSE prepared from foliage of 5 common Australian native plants (genus Acacia or Eucalyptus), or 10% cigarette smoke extract (CSE). Phagocytic recognition receptors were measured by flow cytometry; pro-inflammatory cytokines and caspase 1 by immunofluorescence or cytometric bead array; viability by LDH assay; and capsase-3/PARP by western blot. BFSE significantly decreased phagocytosis of apoptotic cells or NTHi by both THP-1 macrophages and MDM vs air control, consistent with the effects of CSE. BFSE significantly decreased MDM expression of CD36, CD44, SR-A1, CD206 and TLR-2 and increased active IL-1β, caspase-1 and secreted IL-8. BFSE dose-dependently decreased THP-1 macrophage viability (5-fold increase in LDH at 10%) and significantly increased active caspase-3. BFSE impairs macrophage function to a similar extent as CSE, highlighting the need for further research, especially in patients with pre-existing lung disease.

Over 80% of ovarian cancer patients develop chemoresistance which results in a lethal course of the disease. A well-established cause of chemoresistance involves the family of ATP-binding cassette transporters, or ABC transporters that transport a wide range of substrates including metabolic products, nutrients, lipids, and drugs across extra- and intra-cellular membranes. Expressions of various ABC transporters, shown to reduce the intracellular accumulation of chemotherapy drugs, are increased following chemotherapy and impact on ovarian cancer survival. Although clinical trials to date using ABC transporter inhibitors have been disappointing, ABC transporter inhibition remains an attractive potential adjuvant to chemotherapy. A greater understanding of their physiological functions and role in ovarian cancer chemoresistance will be important for the development of more effective targeted therapies. This article will review the role of the ABC transporter family in ovarian cancer progression and chemoresistance as well as the clinical attempts used to date to reverse chemoresistance. Refereed/Peer-reviewed

Introduction We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling. Methods Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling. Results Spns2 expression was significantly increased (p < 0.05) in alveolar macrophages from current-smokers/COPD patients (n = 5) compared to healthy nonsmokers (n = 8) and nonsmoker lung transplant patients (n = 4). Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p < 0.001 in 3 experiments), primary nasal epithelial cells (p < 0.01 in 2 experiments), and in smoke-exposed mice (p < 0.001, n = 6 animals per group). Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells. Conclusion Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure.

// Noor A. Lokman 1 , Alison SF. Elder 1 , Miranda P. Ween 2 , Carmen E. Pyragius 1 , Peter Hoffmann 3 , Martin K. Oehler 1,4,* and Carmela Ricciardelli 1,* 1 Robinson Institute, Research Centre for Reproductive Health, School of Paedriatrics and Reproductive Health, University of Adelaide, Adelaide, SA 5005, Australia 2 Research Centre for Infectious Diseases, School of Molecular Biosciences, University of Adelaide, Adelaide, SA 5005, Australia 3 Adelaide Proteomics Centre, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005, Australia 4 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, SA 5005, Australia * The last two authors share senior authorship. Correspondence: Carmela Ricciardelli, email: // Keywords : annexin A2, ovarian cancer, invasion, chick chorioallantoic membrane assay, xenograft model Received : June 25, 2013 Accepted : July 12, 2013 Published : July 14, 2013 Abstract Our recent research identified the protein annexin A2 to be regulated by ovarian cancer-peritoneal cell interactions. This study investigated the role of annexin A2 in ovarian cancer metastasis and its potential utility as a novel therapeutic target, using in vitro and in vivo ovarian cancer models. Annexin A2 expression was examined by qRT-PCR and western blotting in ovarian cancer cell lines and immunohistochemistry in serous ovarian carcinoma tissues. Annexin A2 siRNAs were used to evaluate the effects of annexin A2 suppression on ovarian cancer cell adhesion, motility, and invasion. Furthermore, annexin A2 neutralizing antibodies were used to examine the role of annexin A2 in tumor invasion and metastasis in vivo using a chick chorioallantoic membrane assay and an intraperitoneal xenograft mouse model. Strong annexin A2 immunostaining was observed in 90% (38/42) of the serous ovarian cancer cells and was significantly increased in the cancer-associated stroma compared to non-malignant ovarian tissues. Annexin A2 siRNA significantly inhibited the motility and invasion of serous ovarian cancer cells and adhesion to the peritoneal cells. Annexin A2 neutralizing antibodies significantly inhibited OV-90 cell motility and invasion in vitro and in vivo using the chick chorioallantoic membrane assay. The growth of SKOV-3 cells and their peritoneal dissemination in nude mice was significantly inhibited by annexin A2 neutralizing antibodies. Annexin A2 plays a critical role in ovarian cancer metastasis and is therefore a potential novel therapeutic target against ovarian cancer.

Annexin A2 is a calcium-dependent, phospholipid-binding protein found on various cell types. It is up-regulated in various tumor types and plays multiple roles in regulating cellular functions, including angiogenesis, proliferation, apoptosis, cell migration, invasion and adhesion. Annexin A2 binds with plasminogen and tissue plasminogen activator on the cell surface, which leads to the conversion of plasminogen to plasmin. Plasmin is a serine protease which plays a key role in the activation of metalloproteinases and degradation of extracellular matrix components essential for metastatic progression. We have recently found that both annexin A2 and plasmin are increased in conditioned media of co cultured ovarian cancer and peritoneal cells. Our studies suggest that annexin A2 is part of a tumor-host signal pathway between ovarian cancer and peritoneal cells which promotes ovarian cancer metastasis. Accumulating evidence suggest that interactions between annexin A2 and its binding proteins play an important role in the tumor microenvironment and act together to enhance cancer metastasis. This article reviews the current knowledge on the biological role of annexin A2 and its binding proteins in solid malignancies including ovarian cancer. Refereed/Peer-reviewed

There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer.

Ovarian cancer metastasis is characterized by the shedding of malignant cells from the surface of the ovary and their implantation onto the peritoneal surface, which lines the abdominal cavity. As the factors promoting this process are poorly understood, we investigated the ovarian cancer-peritoneal interaction by means of in vitro coculture experiments with ovarian cancer (OVCAR-5 and SKOV-3) and peritoneal (LP-9) cells. One of the proteins differentially expressed in the coculture secretome was identified by MALDI-TOF/TOF mass spectrometry as the extracellular matrix protein transforming growth factor-beta-induced protein (TGFBIp, also known as beta ig-H3). Immunohistochemistry showed high TGFBIp levels in normal surface ovarian epithelial and peritoneal cells, whereas TGFBIp levels in primary serous ovarian carcinomas and matching metastatic implants was very low. In functional in vitro experiments, treatment with recombinant TGFBIp significantly increased the motility and invasiveness of OVCAR-5 and SKOV-3 cells and significantly increased ovarian cancer cell (OVCAR-5, OVCAR-3 and SKOV-3) adhesion to LP-9 cells. TGFBIp was found to be processed at both the N-and C-terminus in the secretome of the ovarian cancer-peritoneal cell coculture. Plasmin inhibitors blocked TGFBIp processing and significantly reduced OVCAR-5 cell adhesion to peritoneal cells. We conclude that TGFBIp expressed by peritoneal cells increases the metastatic potential of ovarian cancer cells. TGFBIp is therefore a potential novel therapeutic target against ovarian cancer. Refereed/Peer-reviewed

// Carmela Ricciardelli 1 , Noor A. Lokman 1 , Carmen E. Pyragius 1 , Miranda P. Ween 2 , Anne M. Macpherson 1 , Andrew Ruszkiewicz 3 , Peter Hoffmann 4 , Martin K. Oehler 1, 5 1 Discipline of Obstetrics and Gynaecology, School of Medicine, Robinson Research Institute, University of Adelaide, Adelaide, 5000, South Australia, Australia 2 Lung Research Laboratory, Hanson Institute, Adelaide, South Australia, Australia, Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, 5000, South Australia, Australia 3 Centre of Cancer Biology, University of South Australia and Department of Anatomical Pathology, SA Pathology, Adelaide, 5000, South Australia, Australia 4 Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, 5005, South Australia, Australia 5 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, 5000, South Australia, Australia Correspondence to: Carmela Ricciardelli, email: carmela.ricciardelli@adelaide.edu.au Keywords: ovarian cancer, keratin 5, tumor progression, recurrence, chemoresistance Received: October 01, 2015 Accepted: January 16, 2017 Published: January 27, 2017 ABSTRACT This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 ( KRT6A , KRT6B & KRT6C ) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery ( n = 117) and after neoadjuvant chemotherapy ( n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03−2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12−3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Central to the ability of P. aeruginosa to colonise both environmental and host niches is the acquisition of zinc. Here we show that P. aeruginosa PAO1 acquires zinc via an ATP-binding cassette (ABC) permease in which ZnuA is the high affinity, zinc-specific binding protein. Zinc uptake in Gram-negative organisms predominantly occurs via an ABC permease and consistent with this expectation a P. aeruginosa ΔznuA mutant strain showed an ~60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. However, the effect of zinc limitation on the transcriptome of P. aeruginosa PAO1 revealed significant changes in gene expression that enable adaptation to low-zinc conditions. Genes significantly up-regulated included non-zinc-requiring paralogs of zinc-dependent proteins and a number of novel import pathways associated with zinc acquisition. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation.

E-cigarettes are perceived as harmless; however, evidence of their safety is lacking. New data suggests E-cigarettes discharge a range of compounds capable of physiological damage to users. We previously established that cigarette smoke caused defective alveolar macrophage phagocytosis. The present study compared the effect E-cigarette of components; E-liquid flavors, nicotine, vegetable glycerine, and propylene glycol on phagocytosis, proinflammatory cytokine secretion, and phagocytic recognition molecule expression using differentiated THP-1 macrophages. Similar to CSE, phagocytosis of NTHi bacteria was significantly decreased by E-liquid flavoring (11.65–15.75%) versus control (27.01%). Nicotine also decreased phagocytosis (15.26%). E-liquid, nicotine, and E-liquid+ nicotine reduced phagocytic recognition molecules; SR-A1 and TLR-2. IL-8 secretion increased with flavor and nicotine, while TNFα, IL-1β, IL-6, MIP-1α, MIP-1β, and MCP-1 decreased after exposure to most flavors and nicotine. PG, VG, or PG:VG mix also induced a decrease in MIP-1α and MIP-1β. We conclude that E-cigarettes can cause macrophage phagocytic dysfunction, expression of phagocytic recognition receptors and cytokine secretion pathways. As such, E-cigarettes should be treated with caution by users, especially those who are nonsmokers. Refereed/Peer-reviewed

In microaerophilic or anaerobic environments, Pseudomonas aeruginosa utilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO 4 2− ). Here, we show that molybdate acquisition in P. aeruginosa occurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of the modA gene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition of P. aeruginosa . Collectively, these data highlight the importance of molybdate for anaerobic growth of P. aeruginosa and reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.

Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a ‘toxic’ effect on bacterial pathogens, such as S. pneumoniae.

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer. Refereed/Peer-reviewed

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.