Early increase in blood supply (EIBS) is associated with tumor risk in the Azoxymethane model of colon cancer.

BACKGROUND: The present study aimed to investigate the role of blood supply in early tumorigenesis in colorectal cancer. We leveraged the renin angiotensin system (RAS) to alter colonic blood supply and determine the effect on tumor initiation and progression. METHODS: To test the effect of blood supply on tumorigenesis, 53 male A/J mice were randomly assigned to one of three RAS modulation groups and one of two AOM treatments. The RAS modulation groups were I) water (RAS-unmodulated) as a control group, II) angiotensin-II and III) the angiotensin receptor blocker, Losartan. The mice in each group were then randomly split into either the saline control condition or the AOM-treated condition in which tumors were induced with a standard protocol of serial azoxymethane (AOM) injections. To monitor microvascular changes in the rectal mucosa during the study, we used confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal hemoglobin concentration ([Hb]) and blood vessel radius (BVR). RESULTS: At 12 weeks post-AOM injections and before tumor formation, CLE images revealed many traditional hallmarks of angiogenesis including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting in the pericryptal capillaries of the rectal mucosa in AOM-Water tumor bearing mice. PGS measurements at the same time-point showed increased rectal [Hb] and decreased BVR. At later time points, CLE images showed pronounced angiogenic features including irregular networks throughout the colon. Notably, the AOM-Losartan mice had significantly lower tumor multiplicity and did not exhibit the same angiogenic features observed with CLE, or the increase in [Hb] or decrease in BVR measured with PGS. The AOM-AngII mice did not have any significant trends. CONCLUSION: In-vivo PGS measurements of rectal colonic blood supply as well as CLE imaging revealed angiogenic disruptions to the capillary network prior to tumor formation. Losartan demonstrated an effective way to mitigate the changes to blood supply during tumorigenesis and reduce tumor multiplicity. These effects can be used in future studies to understand the early vessel changes observed.

Fully automated fiber-based optical spectroscopy system for use in a clinical setting.

While there are a plethora of in vivo fiber-optic spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials with highly trained personnel familiar with the operation of experimental optical technologies, very few techniques show the same level of success in large multicenter trials. To meet the stringent requirements for a viable optical spectroscopy system to be used in a clinical setting, we developed components including an automated calibration tool, optical contact sensor for signal acquisition, and a methodology for real-time in vivo probe calibration correction. The end result is a state-of-the-art medical device that can be realistically used by a physician with spectroscopic fiber-optic probes. We show how the features of this system allow it to have excellent stability measuring two scattering phantoms in a clinical setting by clinical staff with ∼0.5 % standard deviation over 25 unique measurements on different days. In addition, we show the systems' ability to overcome many technical obstacles that spectroscopy applications often face such as speckle noise and user variability. While this system has been designed and optimized for our specific application, the system and design concepts are applicable to most in vivo fiber-optic-based spectroscopic techniques.

Use of volumetric laser endomicroscopy for dysplasia detection at the gastroesophageal junction and gastric cardia.

AIM: To determine specific volumetric laser endomicroscopy (VLE) imaging features associated with neoplasia at the gastroesophageal junction (GEJ) and gastric cardia. METHODS: During esophagogastroduodenoscopy for patients with known or suspected Barrett's esophagus, VLE was performed before biopsies were taken at endoscopists' discretion. The gastric cardia was examined on VLE scan from the GEJ (marked by top of gastric folds) to 1 cm distal from the GEJ. The NinePoints VLE console was used to analyze scan segments for characteristics previously found to correlate with normal or abnormal mucosa. Glands were counted individually. Imaging features identified on VLE scan were correlated with biopsy results from the GEJ and cardia region. RESULTS: This study included 34 cases. Features characteristic of the gastric cardia (gastric rugae, gastric pit architecture, poor penetration) were observed in all (100%) scans. Loss of classic gastric pit architecture was common and there was no difference between those with neoplasia and without (100% vs 74%, P = NS). The abnormal VLE feature of irregular surface was more often seen in patients with neoplasia than those without (100% vs 18%, P < 0.0001), as was heterogeneous scattering (86% vs 41%, P < 0.005) and presence of anomalous glands (100% vs 59%, P < 0.05). The number of anomalous glands did not differ between individual histologic subgroups (ANOVA, P = NS). CONCLUSION: The transition from esophagus to gastric cardia is reliably identified on VLE. Histologically abnormal cardia mucosa produces abnormal VLE features. Optical coherence tomography algorithms can be expanded for use at the GEJ/cardia.

High molecular weight polyethylene glycol (PEG 15-20) maintains mucosal microbial barrier function during intestinal graft preservation.

BACKGROUND: During organ transplantation, it is inevitable that tissues undergo cold ischemia during harvest and transport before implantation. Polyethylene-based polymers have been proposed and tested as preservation agents, with promising results. We have previously reported that a high molecular weight polyethylene glycol (PEG) (15-20,000 MW; PEG 15-20) protects the intestinal epithelium against a variety of cellular stresses, including radiation injury and microbial invasion, by mechanisms that appear to involve lipid rafts. The aim of this study was to determine the preservation effect of PEG 15-20 on the integrity of intestine grafts harvested for subsequent transplantation. MATERIALS AND METHODS: We harvested intestinal grafts from mice using a complete surgical technique for intestinal transplantation and assessed them for the effect of PEG on graft tissue integrity. We preserved half of the grafts in histidine-tryptophan-ketoglutarate solution (HTK) alone and half in HTK-PEG 15-20 solution at 4°C for 24 h. We examined gross morphology, wet to dry ratios, histology, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick end labeling assay for apoptosis, goblet cell numbers, and bacterial localization studies to evaluate the effect of PEG on tissue integrity. RESULTS: Results demonstrated that PEG 15-20 had a superior preservation effect over HTK alone in all parameters tested. The effect of PEG was notable on attenuation of epithelial apoptosis, preservation of mucus-producing cells, and bacterial adherence to the epithelium. CONCLUSIONS: Taken together, these studies suggest that use of PEG 15-20 as a potential adjuvant during intestinal transplant may offer significant promise to prolong graft survival during organ harvest.

Agent-based model of epithelial host-pathogen interactions in anastomotic leak.

BACKGROUND: There is a growing recognition of the significance of host-pathogen interactions (HPIs) in gut biology leading to a reassessment of the role of bacteria in intestinal anastomotic leak. Understanding the complexities of the early postsurgical gut HPI requires integrating knowledge of both epithelial and bacterial behaviors to generate hypotheses of potential mechanisms of interaction. Agent-based modeling is a computational method well suited to achieve this goal, and we use an agent-based model (ABM) to examine alterations in the HPI affecting reestablishment of the epithelial barrier that may subsequently lead to anastomotic leak. METHODS: Computational agents representing Pseudomonas aeruginosa were added to a previously validated ABM of epithelial restitution. Simulated experiments were performed examining the effect of radiation on bacterial binding to epithelial cells, plausibility of putative binding targets, and potential mechanisms of epithelial cell killing by virulent bacteria. RESULTS: Simulation experiments incorporating radiation effects on epithelial monolayers produced binding patterns akin to those seen in vitro and suggested that promotility integrin-laminin associations represent potential sites for bacterial binding and disruption of restitution. Simulations of potential mechanisms of epithelial cell killing suggested that an injected cytotoxin was the means by which virulent bacteria produced the tissue destruction needed to generate an anastomotic leak, a mechanism subsequently confirmed with genotyping of the virulent P aeruginosa strain. CONCLUSIONS: This study emphasizes the utility of ABM as an adjunct to traditional research methods and provides insights into the potentially critical role of HPI in the pathogenesis of anastomotic leak.

Intestinal tissues induce an SNP mutation in Pseudomonas aeruginosa that enhances its virulence: possible role in anastomotic leak.

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (≈ 10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.

Candida albicans isolates from the gut of critically ill patients respond to phosphate limitation by expressing filaments and a lethal phenotype.

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient.

Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration.

OBJECTIVE: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients. BACKGROUND DATA: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown. METHODS: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a µ opioid receptor antagonist. RESULTS: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone. CONCLUSIONS: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Pseudomonas aeruginosa potentiates the lethal effect of intestinal ischemia-reperfusion injury: the role of in vivo virulence activation.

BACKGROUND: Experimental models of intestinal ischemia-reperfusion (IIR) injury are invariably performed in mice harboring their normal commensal flora, even though multiple IIR events occur in humans during prolonged intensive care confinement when they are colonized by a highly pathogenic hospital flora. The aims of this study were to determine whether the presence of the human pathogen Pseudomonas aeruginosa in the distal intestine potentiates the lethality of mice exposed to IIR and to determine what role any in vivo virulence activation plays in the observed mortality. METHODS: Seven- to 9-week-old C57/BL6 mice were exposed to 15 minutes of superior mesenteric artery occlusion (SMAO) followed by direct intestinal inoculation of 1.0 × 10(6) colony-forming unit of P. aeruginosa PAO1 into the ileum and observed for mortality. Reiterative studies were performed in separate groups of mice to evaluate both the migration/dissemination pattern and in vivo virulence activation of intestinally inoculated strains using live photon camera imaging of both a constitutive bioluminescent P. aeruginosa PAO1 derivative XEN41 and an inducible reporter derivative of PAO1, the PAO1/lecA:luxCDABE that conditionally expresses the quorum sensing-dependent epithelial disrupting virulence protein PA 1 Lectin (PA-IL). RESULTS: Mice exposed to 15 minutes of SMAO and reperfusion with intestinal inoculation of P. aeruginosa had a significantly increased mortality rate (p < 0.001) of 100% compared with <10% for sham-operated mice intestinally inoculated with P. aeruginosa without SMAO and IIR alone (<50%). Migration/dissemination patterns of P. aeruginosa in mice subjected to IIR demonstrated proximal migration of distally injected strains and translocation to mesenteric lymph nodes, liver, spleen, lung, and kidney. A key role for in vivo virulence expression of the barrier disrupting adhesin PA-IL during IIR was established since its expression was enhanced during IR and mutant strains lacking PA-IL displayed attenuated mortality. CONCLUSIONS: The presence of intestinal P. aeruginosa potentiates the lethal effect of IIR in mice in part due to in vivo virulence activation of its epithelial barrier disrupting protein PA-IL.

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH.

BACKGROUND: During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation. RESULTS: In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with P. aeruginosa had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in C. elegans. Transcriptional analysis of P. aeruginosa demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses. CONCLUSION: Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by P. aeruginosa can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients.

Protective effects of nonionic triblock copolymers on bile acid-mediated epithelial barrier disruption.

Translocation of bacteria and other luminal factors from the intestine following surgical injury can be a major driver of critical illness. Bile acids have been shown to play a key role in the loss of intestinal epithelial barrier function during states of host stress. Experiments to study the ability of nonionic block copolymers to abrogate barrier failure in response to bile acid exposure are described. In vitro experiments were performed with the bile salt sodium deoxycholate on Caco-2 enterocyte monolayers using transepithelial electrical resistance to assay barrier function. A bisphenol A coupled triblock polyethylene glycol (PEG), PEG 15-20, was shown to prevent sodium deoxycholate-induced barrier failure. Enzyme-linked immunosorbent assay, lactate dehydrogenase, and caspase 3-based cell death detection assays demonstrated that bile acid-induced apoptosis and necrosis were prevented with PEG 15-20. Immunofluorescence microscopic visualization of the tight junctional protein zonula occludens 1 (ZO-1) demonstrated that PEG 15-20 prevented significant changes in tight junction organization induced by bile acid exposure. Preliminary transepithelial electrical resistance-based studies examining structure-function correlates of polymer protection against bile acid damage were performed with a small library of PEG-based copolymers. Polymer properties associated with optimal protection against bile acid-induced barrier disruption were PEG-based compounds with a molecular weight greater than 10 kd and amphiphilicity. The data demonstrate that PEG-based copolymer architecture is an important determinant that confers protection against bile acid injury of intestinal epithelia.

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function.

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3ß, and ERK1/2. There was also maintenance of cardiac myocyte ß-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.

Oral PEG 15-20 protects the intestine against radiation: role of lipid rafts.

Intestinal injury following abdominal radiation therapy or accidental exposure remains a significant clinical problem that can result in varying degrees of mucosal destruction such as ulceration, vascular sclerosis, intestinal wall fibrosis, loss of barrier function, and even lethal gut-derived sepsis. We determined the ability of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect the intestine against the early and late effects of radiation in mice and rats and to determine its mechanism of action by examining cultured rat intestinal epithelia. Rats were exposed to fractionated radiation in an established model of intestinal injury, whereby an intestinal segment is surgically placed into the scrotum and radiated daily. Radiation injury score was decreased in a dose-dependent manner in rats gavaged with 0.5 or 2.0 g/kg per day of PEG 15-20 (n = 9-13/group, P < 0.005). Complementary studies were performed in a novel mouse model of abdominal radiation followed by intestinal inoculation with Pseudomonas aeruginosa (P. aeruginosa), a common pathogen that causes lethal gut-derived sepsis following radiation. Mice mortality was decreased by 40% in mice drinking 1% PEG 15-20 (n = 10/group, P < 0.001). Parallel studies were performed in cultured rat intestinal epithelial cells treated with PEG 15-20 before radiation. Results demonstrated that PEG 15-20 prevented radiation-induced intestinal injury in rats, prevented apoptosis and lethal sepsis attributable to P. aeruginosa in mice, and protected cultured intestinal epithelial cells from apoptosis and microbial adherence and possible invasion. PEG 15-20 appeared to exert its protective effect via its binding to lipid rafts by preventing their coalescence, a hallmark feature in intestinal epithelial cells exposed to radiation.

Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa.

There is now substantial evidence that compounds released during host stress directly activate the virulence of certain opportunistic pathogens. Here, we considered that endogenous opioids might function as such compounds, given that they are among the first signals to be released at multiple tissue sites during host stress. We tested the ability of various opioid compounds to enhance the virulence of Pseudomonas aeruginosa using pyocyanin production as a biological readout, and demonstrated enhanced virulence when P. aeruginosa was exposed to synthetic (U-50,488) and endogenous (dynorphin) kappa-agonists. Using various mutants and reporter strains of P. aeruginosa, we identified involvement of key elements of the quorum sensing circuitry such as the global transcriptional regulator MvfR and the quorum sensing-related quinolone signaling molecules PQS, HHQ, and HQNO that respond to kappa-opioids. The in vivo significance of kappa-opioid signaling of P. aeruginosa was demonstrated in mice by showing that dynorphin is released from the intestinal mucosa following ischemia/reperfusion injury, activates quinolone signaling in P. aeruginosa, and enhances the virulence of P. aeruginosa against Lactobacillus spp. and Caenorhabditis elegans. Taken together, these data demonstrate that P. aeruginosa can intercept opioid compounds released during host stress and integrate them into core elements of quorum sensing circuitry leading to enhanced virulence.

Recognition of intestinal epithelial HIF-1alpha activation by Pseudomonas aeruginosa.

Human intestinal epithelial cell monolayers (Caco-2) subjected to hypoxia and reoxygenation release soluble factors into the apical medium that activate the virulence of the opportunistic pathogen Pseudomonas aeruginosa to express the potent barrier-dysregulating protein PA-I lectin/adhesin. In this study, we defined the role of hypoxia-inducible factor (HIF)-1alpha in this response. We tested the ability of medium from Caco-2 cells with forced expression of HIF-1alpha to increase PA-I expression in P. aeruginosa and found that medium from Caco-2 cells overexpressing HIF-1alpha increased PA-I expression compared with medium from control cells (P < 0.001, ANOVA). To identify the components responsible for this response, medium was fractionated by molecular weight and subjected to mass spectroscopy, which identified adenosine as the possible mediator. Both adenosine and its immediate downstream metabolite inosine induced PA-I expression in P. aeruginosa in a dose-dependent fashion. Because inosine was not detectable in the medium of Caco-2 cells exposed to hypoxia or overexpressing HIF-1alpha, we hypothesized that P. aeruginosa itself might metabolize adenosine to inosine. Using mutant and parental strains of P. aeruginosa, we demonstrated that P. aeruginosa metabolized adenosine to inosine via adenosine deaminase and that the conditioned medium enhanced the extracellular accumulation of inosine. Together, these results provide evidence that P. aeruginosa can recognize and respond to extracellular end products of intestinal hypoxia that are released after activation of HIF-1alpha. The ability of P. aeruginosa to metabolize adenosine to inosine may represent a subversive microbial virulence strategy that deprives the epithelium of the cytoprotective actions of adenosine.