Necrotising enterocolitis (NEC) has a complex pathophysiology but the common end-point is ischaemia reperfusion injury (IRI) and intestinal necrosis. We have previously reported that RIC significantly reduces the intestinal injury in a rat model of NEC. Here we describe the changes in intestinal mRNA occurring in the intestine of animals exposed to IRI, both with and without RIC. Related rat-pups were randomly assigned to four groups: SHAM, IRI only, RIC only and RIC + IRI. IRI animals, underwent 40 min of intestinal ischaemia, and 90 min of reperfusion. Animals that underwent RIC had three cycles of 5 min of alternating ischaemia/reperfusion by means of a ligature applied to the hind limb. Samples from the terminal ileum were immediately stored in RNA-preserving media for later next generation sequencing and transciptome analysis using R v 3.6.1. Differential expression testing showed that 868 genes differentially expressed in animals exposed to RIC alone compared to SHAM and 135 in the IRI and RIC group compared to IRI alone. Comparison between these two sets showed that 25 genes were differentially expressed in both groups. Pro-inflammatory molecules: NF-ĸβ2, Cxcl1, SOD2 and Map3k8 all show reduced expression in response to RIC. Targeted gene analysis revealed increased expression in PI3K which is part of the so-called RISK-pathway which is a key part of the protective mechanisms of RIC in the heart. Overall, this transcriptomic analysis shows that RIC provides a protective effect to the intestine via anti-inflammatory pathways. This could be particularly relevant to treating and preventing NEC.

Objective: Remote ischaemic conditioning (RIC) has been shown to reduce ischaemia-reperfusion injury(IRI) in multiple organ systems. IRI is seen in multiple bowel pathologies in the newborn, including NEC. We investigated the potential of RIC as a novel therapy for various intestinal pathologies in the newborn. Methods: We used an established intestinal IRI model in rat pups which results in similar intestinal injury to necrotising enterocolitis (NEC). Animals were randomly allocated to IRI only(n = 14), IRI + RIC(n = 13) or sham laparotomy(n = 10). The macroscopic extent of intestinal injury is reported as a percentage of total small bowel. Injury severity was measured using Chiu-Park scoring. Neutrophil infiltration/activation was assayed by myeloperoxidase activity. Immunohistochemistry was used to assess the expression of hypoxia-inducible factor alpha (HIF-1a). Data are median (interquartile range). Results: Animals that underwent RIC showed a decreased extent of macroscopic injury from 100%(85 -100%) in the IRI only group to 58%(15-84%, p = 0.003) in the IRI + RIC group. Microscopic injury score was significantly lower in animals that underwent RIC compared to IRI alone (3.5[1.25-5] vs 5.5[4-6], p = 0.014). Intestinal myeloperoxidase activity in animals exposed to IRI was 3.4 mU/mg of tissue (2.5 -3.7) and 2.1 mU/mg(1.5-2.8) in the IRI + RIC group(p = 0.047). HIF-1a expression showed a nonsignificant trend towards reduced expression in the IRI + RIC group. Conclusions: RIC reduces the extent and severity of bowel injury in this animal model, supporting the hypothesis that RIC has therapeutic potential for intestinal diseases in the newborn. & COPY; 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Large structures are introduced into deep-water marine environments by several industrial activities, including hydrocarbon exploitation. Anthropogenic structures can alter ecosystem structure and functioning in many marine ecosystems but the responses on continental margins are poorly known. Here, we investigate the short-term response of benthic megafauna to the installation of a 56 km-long 30 cm diameter pipeline on the Angolan Margin (Block 31) from 700 to 1800 m water depth using remotely operated vehicle imagery. Clear depth-related patterns exist in the density, diversity and community structure of megafauna observed in 2013 prior to pipeline installation. These patterns are altered in a subsequent survey in 2014, three-months after pipeline installation. Significant increases in density, particularly in mid-slope regions are observed. Diversity is generally, but not consistently, enhanced, particularly in the shallower areas in 2014. Clear changes are noted in community structure between years. These changes are primarily caused by increases in the abundance of echinoderms, particularly the echinoid Phormosoma sp. indet. There was no evidence of colonisation of the pipeline in three months by visible fauna. The few large anemones observed attached to the pipe may be able to move as adults. The pipeline appeared to trap organic material and anthropogenic litter, and may enhance available food resources locally as well as providing hard substratum. These results indicate complex and ecosystem-dependent responses to structure installation and caution against simplistic approaches to environmental management.

Abstract Background Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. Methods We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. Results Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). Conclusions Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program—as proposed by some reports—may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

BACKGROUND AND AIMS Mucosal healing is important in Crohn's disease therapies. Epithelial homeostasis becomes dysregulated in Crohn's, with increased permeability, inflammation, and diarrhoea. MicroRNAs are small non-coding RNAs that regulate gene expression and show changes in inflammatory bowel disease. Tumour necrosis factor alpha [TNFα] inhibitor protein 3 is raised in Crohn's and regulates TNFα-mediated activation of NFκB. We investigated TNFα regulation by microRNA in Crohn's disease [CD], and studied effects on epithelial permeability and inflammation. METHODS Colonic epithelium from CD and healthy donor biopsies was isolated using laser capture microdissection, and microRNA was quantified. Tumour necrosis factor alpha inhibitor protein 3 was characterised immunohistochemically on serial sections. Expression effect of microRNA was confirmed with luciferase reporter assays. Functional barrier permeability studies and innate cytokine release were investigated with cell and explant culture studies. RESULTS MicroRNA23a levels significantly increased in colonic Crohn's epithelium compared with healthy epithelium. Luciferase reporter assays in transfected epithelial cells confirmed that microRNA23a repressed expression via the 3' untranslated region of tumour necrosis factor alpha inhibitor protein 3 mRNA, coinciding with increased NFκB-mediated transcription. Immunohistochemical staining of TNFAIP3 protein in colonic biopsies was reduced or absent in adjacent Crohn's sections, correlating inversely with microRNA23a levels and encompassing some intercohort variation. Overexpression of microRNA23a increased epithelial barrier permeability in a colonic epithelial model and increased inflammatory cytokine release in cultured explant biopsies, mimicking Crohn's disease characteristics. CONCLUSIONS MicroRNA23a overexpression in colonic Crohn's epithelium represses tumour necrosis factor alpha inhibitor protein 3, enhancing sensitivity to TNFα, with increased intestinal permeability and cytokine release.

Interleukin-13 (IL-13) is an important Type 2 T helper (Th2) cytokine, controlling biological functions in epithelium and has been linked to asthma, atopic dermatitis and ulcerative colitis (UC). Interleukin-13 signals through IL-13 receptor α-1 (IL13RA1 (gene) and IL13Rα1 (protein)), a receptor that can be regulated by microRNAs (miRs). MicroRNAs are small non-coding single-stranded RNAs with a role in several pathologies. However, their relevance in the pathophysiology of UC, a chronic inflammatory condition of the colonic mucosa, is poorly characterised. Here, we determined the expression of IL13Rα1 in UC, its potential regulation by miRs and the subsequent effect on IL-13 signalling. Inflamed mucosa of UC patients showed decreased mRNA and protein expression of IL13RA1 when compared to healthy controls. We show that miR-31 and miR-155 are upregulated in inflamed UC mucosa and that both directly target the 3′ untranslated region of IL13RA1 mRNA. Transfection of miR-31 and miR-155 mimics reduced the expression of IL13RA1 mRNA and protein, and blocked IL-13-dependent phosphorylation of signal transducer and activator of transcription 6 (STAT6) in HT-29 cells, a gut epithelium cell line. Interleukin-13 activation of suppressor of cytokine signaling 1 (SOCS1) and eotaxin-3 (CCL26) expression was also diminished. MicroRNA-31/microRNA-155 mimics also downregulated IL13RA1 in ex vivo human inflamed UC biopsies. We propose that miR-31 and miR-155 have an important role in limiting IL-13 signalling in UC disease.

The contribution of epithelial–mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR–RAS–ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial–mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

BACKGROUND: Cortisol is a prototypical human stress hormone essential for life, yet the precise role of cortisol in the human stress response to injury or infection is still uncertain. Glucocorticoids (GCs) such as cortisol are widely understood to suppress inflammation and immunity. However, recent research shows that GCs also induce delayed immune effects manifesting as immune stimulation. In this study, we show that cortisol enhances the immune-stimulating effects of a prototypical proinflammatory cytokine, interferon-ϒ (IFN-ϒ). We tested the hypothesis that cortisol enhances IFN-ϒ–mediated proinflammatory responses of human mononuclear phagocytes (monocyte/macrophages [MOs]) stimulated by bacterial endotoxin (lipopolysaccharide [LPS]). METHODS: Human MOs were cultured for 18 hours with or without IFN-ϒ and/or cortisol before LPS stimulation. MO differentiation factors granulocyte-macrophage colony stimulating factor (GM-CSF) or M-CSF were added to separate cultures. We also compared the inflammatory response with an acute, 4-hour MO incubation with IFN-ϒ plus cortisol and LPS to a delayed 18-hour incubation with cortisol before LPS exposure. MO activation was assessed by interleukin-6 (IL-6) release and by multiplex analysis of pro- and anti-inflammatory soluble mediators. RESULTS: After the 18-hour incubation, we observed that cortisol significantly increased LPS-stimulated IL-6 release from IFN-ϒ–treated undifferentiated MOs. In GM-CSF–pretreated MOs, cortisol increased IFN-ϒ–mediated IL-6 release by >4-fold and release of the immune stimulant IFN-α2 (IFN-α2) by >3-fold, while suppressing release of the anti-inflammatory mediator, IL-1 receptor antagonist to 15% of control. These results were reversed by either the GC receptor antagonist RU486 or by an IFN-ϒ receptor type 1 antibody antagonist. Cortisol alone increased expression of the IFN-ϒ receptor type 1 on undifferentiated and GM-CSF–treated MOs. In contrast, an acute 4-hour incubation of MOs with IFN-ϒ and cortisol showed classic suppression of the IL-6 response to LPS. CONCLUSIONS: These results reveal a surprisingly robust proinflammatory interaction between the human stress response hormone cortisol and the immune activating cytokine IFN-ϒ. The results support an emerging physiological model with an adaptive role for cortisol, wherein acute release of cortisol suppresses early proinflammatory responses but also primes immune cells for an augmented response to a subsequent immune challenge. These findings have broad clinical implications and provide an experimental framework to examine individual differences, mechanisms, and translational implications of cortisol-enhanced immune responses in humans.

The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross‐talk are not fully understood.

Background.Accumulation of unfolded or misfolded proteins in the ER lumen leads to activate Unfolded Protein Response (UPR) to maintain intestinal epithelium homeostasis.Recent studies have reported that deregulation of UPR or ER stress (ERS) is observed in the colonic mucosa of Ulcerative Colitis (UC) patients.Colon from UC patients is also characterized by an altered epithelium homeostasis linked to an excessive proteolytic activity.Aims.We aimed to investigate the impact of the ER stress on the cysteine and serine proteolytic (trypsin-like) activity in enterocytes and goblet cells, and its consequences on the intestinal barrier function (IBF).Methods.To study the impact of the ERS on proteolytic activity, human cells lines including Caco-2 (enterocyte) and HT29MTX (goblet cells) were stimulated with ERS inducers such as Thapsigargin (10µg/mL) and Tunicamycin (20µg/mL).At 2, 4, 6 and 24 hours after ERS induction.The activity and expression of Trypsin-Like activity and cysteine-Like Activity proteases in the supernatant of the cell culture and in cells were investigated by enzyme assay (Z-Gly-Pro-Arg trypsin-and Z-Phe-Arg cysteine-substrates) and by qPCR respectively.To analyze the impact on IBF, we monitored, using transwell systems, paracellular permeability, IL-8 secretion and the expression of the resistin like molecule b (RLMb) at 6 and 24 hours after ERS.Trypsin-like inhibitor (AESBF, 20uM) was used to check the Trypsin-like proteases involvement in IBF.Results.Activation of ERS by thapsigargin or tunicamycin enhanced the secretion of Trypsin-like proteases by Caco-2 cells and HT29MTX cells at 2, 4, and 6 hours, losing the effect at 24 hours, probably because of inducer depletion.Moreover, at 6 and 24 hours, after ERS induction, mRNA expression of the three Trypsin-like proteases (PRSS1, 2 and 3) were increased.By using transwell system, we observed that the increased Trypsin-like proteases triggered by ERS only affected the apical side of the enterocyte monolayers.Concerning the impact of ERS on IBF, we have evidenced that induction of ERS increased the paracellular permeability, IL-8 secretion in apical and basolateral compartments as well as RLM bexpression.Trypsinlike protease inhibitor (AESBF) treatment inhibited ERS-increased permeability and RLM b mRNA overexpression, while AESBF aggravated IL-8 secretion in both compartments.Conclusions.Our data demonstrate, that ERS increases the expression of Trypsin-like activity at the apical side of enterocytes and muco-secreting cells.This enhanced proteases activity played a key role in the destabilization of the epithelial homeostasis such as the permeability, RLMb expression and the production of inflammatory mediators.These results evidence, for the first time, a crosstalk between ERS and proteolytic activity, two cardinal features of the UC.

AbstractMillions of people throughout the tropics consume wild meat. Overhunting reduces food security for people and large predators, yet little is known of the impact of hunting in systems where people and predators target the same prey species. We collate published data on predator diet in Belize with interview data about the consumption of wild and domestic meat by Belizeans, to compare the wild-meat diets of humans, jaguarsPanthera oncaand pumasPuma concolorand assess the sustainability of the combined offtake by humans and jaguars. Six wild mammal species (nine-banded armadilloDasypus novemcinctus, pacaCuniculus paca, collared peccaryPecari tajacu, white-lipped peccaryTayassu pecari, red brocket deerMazama americanaand white-tailed deerOdocoileus virginianus) comprised 7% of the animal-protein meals eaten by Belizeans. Overall, 80% of these meals were eaten by 20% of interviewees, suggesting a necessary role of wild meat for the minority. The same species were found in 69 and 86% of jaguar and puma scats, respectively. We estimate a national annual harvest of c. 4,000 tonnes of these six wild mammals by humans and jaguars, of which 78% is hunted by people. Sustainability is difficult to evaluate because prey population data are lacking in Belize. However, simple models suggest that a sustainable harvest at this rate would require higher prey population densities than averages recorded in hunted Neotropical forests. We emphasize the need for robust regional estimates of game species densities, to improve assessments of sustainability and inform hunting regulations. We recommend that the requirements of predators as well as those of people be considered when assessing wild meat harvests.

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

Introduction: Respiratory viruses infect the airway epithelial barrier and cause inflammation. This process involves cellular crosstalk between different cell types. By using a dynamic 3D co-culture model of airway epithelial cells (AECs) and endothelial cells we aimed to analyse the cellular crosstalk regulating airway inflammation during viral infections. Methods: Human AECs were cultured on the apical side of permeable filter supports while endothelial cells were cultured on the basolateral side facing the microfluidic flow. The 3D model was challenged apically with double stranded RNA (Poly(I:C)), a mimic of viral infections. Basal supernatants were collected in 2h intervals and mediator secretion determined by ELISA. Results: In contrast to static cultures, the microfluidic systemallowed analysis of airway barrier responses time-dependently at shorter intervals and with greater sensitivity. Poly(I:C) induced release of TNF-α by epithelial cells with a peak 4h after stimulation; this was followed temporally by release of fractalkine (CX3 CL1), a chemokine regulating immune cell migration, from the endothelial cells. Release of fractalkine could be prevented by neutralisation of TNF-α. Conclusion: Using adynamic 3D model offers the potential to investigate mechanisms of airway inflammation and cellular crosstalk under conditions closer to the in vivo situation. During viral infections, endothelial-derived CX3 CL1 release is triggered by epithelial derived TNF-α. This cellular crosstalk might play an important role in the regulation of immune cell infiltration.

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.

Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner. Conclusion: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status. (Hepatology 2014;59:1492-1504)

BACKGROUND:Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. OBJECTIVES:We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. METHODS:Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. RESULTS:TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. CONCLUSIONS:Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.

Identification of pathogenic bacteria in ascites correlates with poor clinical outcomes. Ascites samples are commonly reported culture-negative, even where frank infection is indicated. Culture-independent methods have previously reported bacterial DNA in ascites, however, whether this represents viable bacterial populations has not been determined. We report the first application of 16S rRNA gene pyrosequencing and quantitative PCR in conjunction with propidium monoazide sample treatment to characterise the viable bacterial composition of ascites. Twenty five cirrhotic patients undergoing paracentesis provided ascites. Samples were treated with propidium monoazide to exclude non-viable bacterial DNA. Total bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance determined by 16S rRNA gene pyrosequencing. Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed. Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing. Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species. Substantial variation in the species detected was observed between patients. Statistically significant relationships were identified between the bacterial community similarity and clinical measures, including ascitic polymorphonuclear leukocyte count and Child-Pugh class. Viable bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection. Microbiota composition significantly correlates with clinical measures. Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising fundamental questions about the processes that underlie the development of spontaneous bacterial peritonitis.