Higher Prevalence of Bacteroides fragilis in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance.

Bacteroides fragilis has previously been linked to Crohn's disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of B. fragilis and its virulence factors bft (enterotoxin) and ubiquitin among 181 CD patients and the impact on the intestinal epithelial barrier in vitro. The prevalence of B. fragilis was significantly higher in active (n = 69/88, 78.4%) as compared to remissive (n = 58/93, 62.4%, p = 0.018) CD patients. Moreover, B. fragilis was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, increased when exposed to secretomes of bft-positive (bft-1 and bft-2 isotype; increased TEER ∼160%, p < 0.001) but not when exposed to bft-negative strains. Whole metagenome sequencing and metabolomics, respectively, identified nine coding sequences and two metabolites that discriminated TEER-increasing from non-TEER-increasing strains. This study revealed a higher B. fragilis prevalence during exacerbation. Surprisingly, bft-positive secretomes increased epithelial resistance, but we excluded Bft as the likely causative factor.

Characterization of Feces-Derived Bacterial Membrane Vesicles and the Impact of Their Origin on the Inflammatory Response.

The human gastrointestinal tract harbors a diverse and complex microbiome, which interacts in a variety of ways with the host. There is compelling evidence that gut microbial dysbiosis, defined as an alteration of diversity and abundance in intestinal microbes, is an etiological factor in inflammatory bowel disease (IBD). Membrane vesicles (MVs), which are nano-sized particles released by bacteria, have been found to interact with the host and modulate the development and function of the immune system. As a result MVs have been suggested to play a critical role in both health and disease. In this study we developed a method to isolate, characterize and assess the immunoreactivity of heterogeneous populations of MVs from fecal samples (fMVs) of healthy volunteers. We successfully isolated 2*109-2*1010 particles/ml from 0.5 gram of feces by using a combination of ultrafiltration and size exclusion chromatography (SEC) from 10 fecal samples. Bead-based flowcytometry in combination with tunable resistive pulse sensing (TRPS) provided a reliable method for (semi-)quantitative determination of fMVs originating from both Gram-positive and Gram-negative bacteria, while transmission electron microscopy confirmed the presence of fMVs. Real time 16s PCR on bacterial cell fractions or isolated fMVs DNA of the most common phyla (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria) revealed differences in the relative abundance between bacteria and the fMVs. Moreover, fMVs evoke the release of TNF- by THP-1 cells in a dose-dependent matter. Also, a significant positive correlation was found between Actinobacteria/-Proteobacteria derived vesicles and the release of TNF-. It has become increasingly clear that fMVs could provide an additional layer to the definition of homeostasis or dysbiosis of the microbiota. The current study supports their potential involvement in the intestinal homeostasis or inflammatory disorders and provides putative interesting incentives for future research.

How to Count Our Microbes? The Effect of Different Quantitative Microbiome Profiling Approaches.

Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g., flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extent different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: (1) microbial cell counting using flow cytometry (QMP), (2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and (3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.

Non-steroidal anti-inflammatory drugs increase urinary neutrophil gelatinase-associated lipocalin in recreational runners.

OBJECTIVES: To study the effects of running with/without the use of pain killers on urinary neutrophil gelatinase-associated lipocalin (uNGAL) and other parameters of kidney function in recreational runners. METHODS: Participants of the 10- and 21.1-km Weir Venloop race were enrolled and their urine samples collected before and after the run. Urine dipstick and other conventional tests used to assess kidney function were performed. The presence of ibuprofen, diclofenac, naproxen, and/or paracetamol was assessed by LC-MS/MS. uNGAL was measured with a two-step chemiluminescent immunoassay. RESULTS: NSAIDs/analgesics were detected in urine of 5 (14.4%) 10-km runners and 13 (28.9%) 21.1-km runners. Only half-marathon participants showed significant increases in uNGAL (pre: 11.7 [7.1-34.3] ng/mL; post: 33.4 [17.4-50.4] ng/mL; P = .0038). There was a significant effect of NSAID/analgesic use on uNGAL increase (F2, 76  = 4.210, P = .004). Post hoc tests revealed that uNGAL increased significantly in runners who tested positive for ibuprofen/naproxen compared to runners who did not use any medications (P = .045) or those who tested positive for paracetamol (P = .033). Running distance had a significant influence on the increase in uNGAL (F1, 53  = 4.741, P < .05), specific gravity (F1, 60  = 9.231, P < .01), urinary creatinine (F1, 61  = 10.574, P < .01), albumin (F1, 59  = 4.888, P < .05), and development of hematuria (χ2 (4) = 18.44, P = .001). CONCLUSIONS: Running distance and use of ibuprofen/naproxen were identified as risk factors for uNGAL increase in recreational runners.

Host-microbe cross-talk in the lung microenvironment: implications for understanding and treating chronic lung disease.

Chronic respiratory diseases are highly prevalent worldwide and will continue to rise in the foreseeable future. Despite intensive efforts over recent decades, the development of novel and effective therapeutic approaches has been slow. However, there is new and increasing evidence that communities of micro-organisms in our body, the human microbiome, are crucially involved in the development and progression of chronic respiratory diseases. Understanding the detailed mechanisms underlying this cross-talk between host and microbiota is critical for development of microbiome- or host-targeted therapeutics and prevention strategies. Here we review and discuss the most recent knowledge on the continuous reciprocal interaction between the host and microbes in health and respiratory disease. Furthermore, we highlight promising developments in microbiome-based therapies and discuss the need to employ more holistic approaches of restoring both the pulmonary niche and the microbial community.

Cell Type- and Exposure-Specific Modulation of CD63/CD81-Positive and Tissue Factor-Positive Extracellular Vesicle Release in response to Respiratory Toxicants.

Chronic exposure to respiratory stressors increases the risk for pulmonary and cardiovascular diseases. Previously, we have shown that cigarette smoke extract (CSE) triggers the release of CD63+CD81+ and tissue factor (TF)+ procoagulant extracellular vesicles (EVs) by bronchial epithelial cells via depletion of cell surface thiols. Here, we hypothesized that this represents a universal response for different pulmonary cell types and respiratory exposures. Using bead-based flow cytometry, we found that bronchial epithelial cells and pulmonary fibroblasts, but not pulmonary microvascular endothelial cells or macrophages, release CD63+CD81+ and TF+ EVs in response to CSE. Cell surface thiols decreased in all cell types upon CSE exposure, whereas depletion of cell surface thiols using bacitracin only triggered EV release by epithelial cells and fibroblasts. The thiol-antioxidant NAC prevented the EV induction by CSE in epithelial cells and fibroblasts. Exposure of epithelial cells to occupational silica nanoparticles and particulate matter (PM) from outdoor air pollution also enhanced EV release. Cell surface thiols were mildly decreased and NAC partly prevented the EV induction for PM10, but not for silica and PM2.5. Taken together, induction of procoagulant EVs is a cell type-specific response to CSE. Moreover, induction of CD63+CD81+ and TF+ EVs in bronchial epithelial cells appears to be a universal response to various respiratory stressors. TF+ EVs may serve as biomarkers of exposure and/or risk in response to respiratory exposures and may help to guide preventive treatment decisions.

Proteomic analysis reveals procoagulant properties of cigarette smoke-induced extracellular vesicles.

Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers.

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease.

Extracellular vesicles (EV) are secreted signaling entities that enhance various pathological processes when released in response to cellular stresses. Respiratory exposures such as cigarette smoke and air pollution exert cellular stresses and are associated with an increased risk of several chronic diseases. The aim of this review was to examine the evidence that modifications in EV contribute to respiratory exposure-associated diseases. Publications were searched using PubMed and Google Scholar with the search terms (cigarette smoke OR tobacco smoke OR air pollution OR particulate matter) AND (extracellular vesicles OR exosomes OR microvesicles OR microparticles OR ectosomes). All original research articles were included and reviewed. Fifty articles were identified, most of which investigated the effect of respiratory exposures on EV release in vitro (25) and/or on circulating EV in human plasma (24). The majority of studies based their main observations on the relatively insensitive scatter-based flow cytometry of EV (29). EV induced by respiratory exposures were found to modulate inflammation (19), thrombosis (13), endothelial dysfunction (11), tissue remodeling (6), and angiogenesis (3). By influencing these processes, EV may play a key role in the development of cardiovascular diseases and chronic obstructive pulmonary disease and possibly lung cancer and allergic asthma. The current findings warrant additional research with improved methodologies to evaluate the contribution of respiratory exposure-induced EV to disease etiology, as well as their potential as biomarkers of exposure or risk and as novel targets for preventive or therapeutic strategies.

Redox-dependent thiol modifications: implications for the release of extracellular vesicles.

Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation. As the redox state of protein thiols greatly impacts three-dimensional protein structure and, consequently, function, redox modifications of protein thiols may directly modulate EV release in response to changes in the cell's redox environment. In this review article, we discuss targets of redox-dependent thiol modifications that are known or expected to be involved in the regulation of EV release, namely redox-sensitive calcium channels, N-ethylmaleimide sensitive factor, protein disulfide isomerase, phospholipid flippases, actin filaments, calpains and cell surface-exposed thiols. Thiol protection is proposed as a strategy for preventing detrimental changes in EV signaling in response to inflammation and oxidative stress. Identification of the thiol-containing proteins that modulate EV release in pro-oxidant environments could provide a rationale for broad application of thiol group-containing antioxidants in chronic inflammatory diseases.

Arthroscopic soft tissue reconstruction in anterior shoulder instability.

Anterior glenohumeral instability remains a difficult problem in the athletic and working patient populations. Treatment strategies are variable and range from nonoperative approaches incorporating immobilization and rehabilitation to surgical management. Surgical decision-making can be challenging, especially with a high-level patient who wishes to return to high-demand activities. Operative options range from open soft tissue stabilization to arthroscopic soft tissue stabilization, with both open and arthroscopic options for bony reconstruction in the setting of clinically significant anterior glenoid bone loss. In all cases, understanding appropriate indications and utilizing sound surgical techniques are critical for achieving a successful result. For the majority of patients with anterior shoulder instability, arthroscopic soft tissue reconstruction with suture anchors is a successful, minimally invasive approach that results in a stable shoulder with excellent outcomes and low complication rates.

Gram-negative bacterial membrane vesicle release in response to the host-environment: different threats, same trick?

Bacteria are confronted with a multitude of stressors when occupying niches within the host. These stressors originate from host defense mechanisms, other bacteria during niche competition or result from physiological challenges such as nutrient limitation. To counteract these stressors, bacteria have developed a stress-induced network to mount the adaptations required for survival. These stress-induced adaptations include the release of membrane vesicles from the bacterial envelope. Membrane vesicles can provide bacteria with a plethora of immediate and ultimate benefits for coping with environmental stressors. This review addresses how membrane vesicles aid Gram-negative bacteria to cope with host-associated stress factors, focusing on vesicle biogenesis and the physiological functions. As many of the pathways, that drive vesicle biogenesis, confer we propose that shedding of membrane vesicles by Gram-negative bacteria entails an integrated part of general stress responses.

Evaluation of fever in the immediate post-operative period following shoulder arthroplasty.

AIMS: To determine the incidence and timing of post-operative fevers following shoulder arthroplasty and the resulting investigations performed. PATIENTS AND METHODS: A retrospective review was conducted of all patients undergoing shoulder arthroplasty over a nine-year period. The charts of all patients with a post-operative fever (≥ 38.6°C) were reviewed and the results of all investigations were analysed. RESULTS: A total of 2167 cases (in 1911 patients) were included of whom 92 (4.2%) had a documented fever. Obese cases had a significantly greater risk for fever (relative risk 1.53; 95% confidence interval 1.02 to 2.32; p = 0.041). Investigations were performed in 43/92 cases (46.7%), with a diagnosis being made in six cases (6.6% of the total, two of whom had their diagnosis made post-discharge). CONCLUSION: Around one in 25 cases develop a fever following shoulder arthroplasty; most have no infective aetiology. These patients may be being over-investigated; investigations should be performed in patients with persistent fever or on those with an identifiable source of infection on clinical examination. Cite this article: Bone Joint J 2017;99-B:1515-19.

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

BACKGROUND: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. RESULTS: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. CONCLUSIONS: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

Comparative outcomes of outpatient and inpatient total shoulder arthroplasty: an analysis of the Medicare dataset.

AIMS: The aim of the present study was to compare the 30- and 90-day re-admission rates and complication rates of outpatient and inpatient total shoulder arthroplasty (TSA). PATIENTS AND METHODS: The United States Medicare Standard Analytical Files database was questioned to identify patients who had undergone outpatient or inpatient TSA between 2005 and 2012. Patient characteristics were compared between the two groups using chi-squared analysis. Multivariate logistic regression analysis was used to control for differences in baseline patient characteristics and to compare the two groups in terms of post-operative complications within 90 days and re-admission within 30 days and 90 days. RESULTS: A total of 123 347 Medicare subscribers underwent TSA between 2005 and 2012; 3493 (2.8%) had the procedure performed as an outpatient. A significantly greater proportion of patients who underwent TSA as inpatients were women, had a history of smoking, and had a greater incidence of medical comorbidity including diabetes, coronary artery disease, congestive heart failure, and chronic kidney disease (p < 0.05 for all). Re-admission rates were significantly higher for inpatients at both 30 days (0.83% versus 0.60%, p = 0.016, odds ratio 1.8) and 90 days (2.87% versus 2.04%, p < 0.001, odds ratio 1.8). Complications, including thromboembolic events (p < 0.001) and surgical site infection (p = 0.002), were significantly higher in inpatients. CONCLUSION: Patients who underwent TSA on an outpatient basis were overall younger and healthier than those who had inpatient surgery, which suggests that patient selection was taking place. After controlling for age, gender, and medical conditions, patients who underwent TSA as outpatients had lower rates of 30- and 90-day re-admission and a lower rate of complications than inpatients. Cite this article: Bone Joint J 2017;99-B:934-8.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.

Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection.

Patients with more severe chronic obstructive pulmonary disease frequently experience exacerbations and it is estimated that up to 50% of these exacerbations are associated with bacterial infections. The mainstay treatment for these infection-related exacerbations constitutes the administration of glucocorticoids, alone or in combination with antibiotics. A recent line of evidence demonstrates that many hormones including the steroid beclomethasone can also directly affect bacterial growth, virulence, and antibiotic resistance. The effect of these regimens on the release of potentially virulent and toxic membrane vesicles (MVs) is at present unclear. In this study, we determined the effect of several pharmacological agents on MVs release by and bacterial growth of common respiratory pathogens. We found that neither the release of MVs nor the bacterial growth was affected by the glucocorticoids budesonide and fluticasone. The macrolide antibiotic azithromycin only inhibited the growth of Moraxella catarrhalis but no effects were observed on bacterial MV release at a concentration that is achieved locally in the epithelial lining on administration. The macrophage pro-inflammatory response to MVs was significantly reduced after treatment with budesonide and fluticasone but not by azithromycin treatment. Our findings suggest that these glucocorticoids may have a positive effect on infection-related inflammation although the bacterial growth and MV release remained unaffected.

Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections.

Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants.

INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment.

Viral-bacterial interactions in the respiratory tract.

In the respiratory tract, viruses and bacteria can interact on multiple levels. It is well known that respiratory viruses, particularly influenza viruses, increase the susceptibility to secondary bacterial infections. Numerous mechanisms, including compromised physical and immunological barriers, and changes in the microenvironment have hereby been shown to contribute to the development of secondary bacterial infections. In contrast, our understanding of how bacteria shape a response to subsequent viral infection is still limited. There is emerging evidence that persistent infection (or colonization) of the lower respiratory tract (LRT) with potential pathogenic bacteria, as observed in diseases like chronic obstructive pulmonary disease or cystic fibrosis, modulates subsequent viral infections by increasing viral entry receptors and modulating the inflammatory response. Moreover, recent studies suggest that even healthy lungs are not, as had long been assumed, sterile. The composition of the lung microbiome may thus modulate responses to viral infections. Here we summarize the current knowledge on the co-pathogenesis between viruses and bacteria in LRT infections.