allopurinol and Escherichia-coli-Infections

In previous work, we identified xanthine oxidase (XO) as an important enzyme in the interaction between the host and enteropathogenic Escherichia coli(EPEC) and Shiga-toxigenic E. coli(STEC). Many of the biological effects of XO were due to the hydrogen peroxide produced by the enzyme. We wondered, however, if uric acid generated by XO also had biological effects in the gastrointestinal tract. Uric acid triggered inflammatory responses in the gut, including increased submucosal edema and release of extracellular DNA from host cells. While uric acid alone was unable to trigger a chloride secretory response in intestinal monolayers, it did potentiate the secretory response to cyclic AMP agonists. Uric acid crystals were formed in vivo in the lumen of the gut in response to EPEC and STEC infections. While trying to visualize uric acid crystals formed during EPEC and STEC infections, we noticed that uric acid crystals became enmeshed in the neutrophilic extracellular traps (NETs) produced from host cells in response to bacteria in cultured cell systems and in the intestine in vivo Uric acid levels in the gut lumen increased in response to exogenous DNA, and these increases were enhanced by the actions of DNase I. Interestingly, addition of DNase I reduced the numbers of EPEC bacteria recovered after a 20-h infection and protected against EPEC-induced histologic damage.

Topics: Animals; Cell Line; Colforsin; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Gastrointestinal Hormones; Humans; Intestines; Natriuretic Peptides; Rabbits; Shiga-Toxigenic Escherichia coli; Uric Acid; Xanthine Oxidase

The aim of this study was to evaluate if xanthine oxidase and myeloperoxidase levels quantitation method may alternate routine culture method, which takes more time in the diagnosis of urinary tract infections.. Five hundred and forty-nine outpatients who had admitted to Clinic Microbiology Laboratory were included in the study. The microorganisms were identified by using VITEK System. The urine specimens that were negative from the quantitative urine culture were used as controls. The activities of MPO and XO in spot urine were measured by spectrophotometric method.. Through the urine cultures, 167 bacteria were isolated from 163 urine specimens; 386 cultures yielded no bacterial growth. E. coli was the most frequent pathogen. In infection with E. coli both XO and MPO levels were increased the most. The sensitivity, specificity, positive predictive value, and negative predictive value for XO were 100%, 100%, 100%, and 100%, respectively. These values for MPO were 87%, 100%, 100%, and 94%, respectively.. These data obtained suggest that urine XO and MPO levels may be new markers in the early detection of UTI.

Topics: Adolescent; Adult; Aged; Biomarkers; Child; Child, Preschool; Early Diagnosis; Escherichia coli Infections; Female; Humans; Infant; Male; Middle Aged; Peroxidase; Proteinuria; Sensitivity and Specificity; Urinary Tract Infections; Xanthine Oxidase; Young Adult

Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

Topics: Animals; Bodily Secretions; Cell Line; Disease Models, Animal; Enteropathogenic Escherichia coli; Escherichia coli Infections; Host-Pathogen Interactions; Humans; Hydrogen Peroxide; Intestines; Rabbits; Shiga-Toxigenic Escherichia coli; Uric Acid; Virulence; Xanthine Oxidase

Transactivation of the DNA-binding proteins nuclear factor-kappa B (NF-kappa B) and activator protein (AP)-1 by de novo oxyradical generation is a stereotypic redox-sensitive process during hypoxic stress of the liver. Systemic trauma is associated with splanchnic hypoxia-reoxygenation (H/R) followed by intraportal gram-negative bacteremia, which collectively have been implicated in posttraumatic liver dysfunction and multiple organ damage. We hypothesized that hypoxic stress of the liver before stimulation by Escherichia coli serotype O55:B5 (EC) amplifies oxyradical-mediated transactivation of NF-kappa B and AP-1 as well as cytokine production compared with noninfectious H/R or gram-negative sepsis without prior hypoxia. Livers from Sprague-Dawley rats underwent perfusion for 180 min with or without 0.5 h of hypoxia (perfusate PO(2), 40 +/- 5 mmHg) followed by reoxygenation and infection with 10(9) EC or 0.9% NaCl infusion. In H/R + EC livers, nuclear translocation of NF-kappa B and AP-1 was unexpectedly reduced in gel shift assays vs. normoxic EC controls, as were perfusate TNF-alpha and IL-1 beta levels. Preceding hypoxic stress paradoxically increased postbacteremic reduced-to-oxidized glutathione ratios plus nuclear localization of I kappa B alpha and phospho-I kappa B alpha, but not JunB/FosB profiles. Notably, xanthine oxidase inhibition increased transactivation as well as cytokine production in H/R + EC livers. Thus brief hypoxic stress of the liver before intraportal gram-negative bacteremia potently suppresses activation of canonical redox-sensitive transcription factors and production of inflammatory cytokines by mechanisms including xanthine oxidase-induced oxyradicals functioning in an anti-inflammatory signaling role. These results suggest a novel multifunctionality of oxyradicals in decoupling hepatic transcriptional activity and cytokine biosynthesis early in the posttraumatic milieu.

Topics: Animals; Cell Nucleus; Cytokines; Cytoplasm; Escherichia coli Infections; Glutathione; Glutathione Disulfide; Hypoxia; I-kappa B Proteins; Liver; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Xanthine Oxidase

Reductions in hepatic O(2) delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-kappaB (NF-kappaB) via generation of reactive O(2) species (ROS), the combination of these stimuli downregulates hepatic TNF-alpha gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-alpha gene expression is transcriptionally mediated by reduced activation of NF-kappaB. Buffer-perfused rat livers (n = 52) were studied over 180 min after intraportal infection at t = 0 with 10(9) live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (PO(2) approximately 41 +/- 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia (t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-kappaB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using (32)P-labeled oligonucleotides specific for NF-kappaB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-kappaB nuclear translocation and TNF-alpha bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O(2) consumption. XO inhibition reversed the hypoxic suppression of NF-kappaB nuclear translocation and ameliorated decreases in cell-associated TNF-alpha. Thus decreases in hepatic O(2) delivery reduce postbacteremic nuclear translocation of NF-kappaB and hepatic TNF-alpha biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

Topics: Allopurinol; Animals; Bacteremia; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Escherichia coli Infections; Gene Expression; Hypoxia; In Vitro Techniques; Liver; Liver Function Tests; Male; NF-kappa B; Perfusion; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stress, Physiological; Tumor Necrosis Factor-alpha; Xanthine Oxidase

Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O2- is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O2-, H2O2, and ONOO- was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O2- (from 35+/-2 cpm/ring at baseline to 166+/-21 cpm/ring at 12 hours and 225+/-16 cpm/ring at 30 hours) and H2O2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO- formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O2- with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O2- and ONOO-. Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely. Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved.

Topics: Analysis of Variance; Animals; Aorta; Blotting, Northern; Down-Regulation; Endothelium, Vascular; Endotoxemia; Enzyme Inhibitors; Escherichia coli; Escherichia coli Infections; Immunoblotting; In Vitro Techniques; Lipopolysaccharides; Luminescent Measurements; Muscle Relaxation; Muscle, Smooth, Vascular; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Onium Compounds; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxide Dismutase; Superoxides; Xanthine Oxidase

This study focuses upon two discrete components of posttransplant hepatic reticuloendothelial system (RES) function-phagocytosis and killing of bacteria-under various conditions of ischemic preservation. We had previously reported that, following intravenous injection of rats with 51Cr and 125I double-labeled Escherichia coli, hepatic 51Cr levels can be used to reliably quantify hepatic phagocytic clearance of the bacteria from the blood (HPC), while the subsequent release of 125I from the liver accurately parallels hepatic bacterial killing. Here, Wistar rats were transplanted with syngeneic livers perfused with either normal saline (NS) or University of Wisconsin solution (UW) and stored at 4 degrees C for 1, 2, or 3 hr prior to implantation. Control rats underwent laparotomy and hepatic artery ligation. Using the double-labeled E coli assay, HPC was decreased in all transplanted animals when compared with controls, reaching a nadir on the third postoperative day (P < 0.05). In rats transplanted with livers preserved in NS, the fraction of phagocytosed organisms that were subsequently killed (hepatic killing efficiency=HKE) was increased to 142%, 129%, or 112% of normal following 1, 2, or 3 hr of cold ischemia, respectively; P < 0.05). Conversely, preservation of donor allografts in UW was associated with marked depression of HKE. Moreover, rats receiving NS- or UW-preserved livers tolerated an intravenous challenge with Streptococcus pneumoniae poorly (50% mortality) compared with hepatic artery ligated controls (12% mortality) at 7 days. Ischemic preservation of rat livers in NS resulted in a dose (of ischemia)-dependent reduction of hepatic phagocytosis coupled with a potentiation of HKE. Preservation in UW, however, produced a striking suppression of both components of hepatic RES function. Following a septic challenge survival was reduced in both groups of transplanted rats.

Topics: Adenosine; Allopurinol; Animals; Escherichia coli; Escherichia coli Infections; Glutathione; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Preservation

We tested the hypothesis that reducing the hepatic O2 supply by 30 min of constant-flow hypoxia (PO2, approximately 45 Torr) following gram-negative bacteremia downregulates tumor necrosis factor-alpha (TNF-alpha) in buffer-perfused rat lives (total n = 44). Eight groups were studied after intraportal 10(9) viable E. coli serotype 055:B5 (EC) or 0.9% NaCl (NS) at t = 0:1) normoxic EC; 2) normoxic NS controls; 3) EC+hypoxia (H)-reoxygenation (R) in which H began 30 min after EC followed by 120 min of R; and 4) NS+H/R. To assess the role of cyclooxygenase vs. xanthine oxidase activation, the effects of 10(-5) M indomethacin (Indo) in 5) Indo+EC+H/R and 6) Indo+NS+H/R were compared with allopurinol (Allo) in 7) Allo+EC+H/R and 8) Allo+NS+H/R groups. Bacterial clearance, bioactive and antigenic TNF-alpha, and hepatic O2 uptake and performance were serially assessed, as was prostaglandin (PG) E2 at baseline and peak hypoxia in EC-challenged groups. Intrahepatic bacterial killing and TNF-alpha mRNA were determined at t = 180 min. Bioactive venous TNF-alpha did not increase in normoxic NS controls (6 +/- 3 U/ml at t = 180 min; mean +/- SE), whereas levels rose in NS4H/R by 180 min (111 +/- 34 U/ml; P < 0.01) without increases in TNF-alpha mRNA. In contrast, EC-induced increases in TNF-alpha transcripts during normoxia were attenuated in EC+H/R, as were protein levels (57 +/- 20 U/ml; P < 0.05), despite similar bacterial clearance. Neither Indo-mediated reductions in PGE2 nor allopurinol increased TNF-alpha after EC+H/R.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bacteremia; Cyclooxygenase Inhibitors; Cytokines; Down-Regulation; Escherichia coli Infections; Hypoxia; Liver; Male; Oxygen; Perfusion; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Xanthine Oxidase

Previous studies show that chronic pyelonephritis and end stage renal disease may follow acute pyelonephritis in children and adolescents when improperly or inadequately treated. Our study shows that there is a significant decrease in renal function following untreated acute bacterial pyelonephritis due to nephron loss. The acute inflammatory response is responsible for much of the renal damage, although damage from renal ischemia is an additional significant factor. The present study used a combination of an antibiotic and a xanthine oxidase inhibitor (allopurinol) as compared to antibiotic therapy alone begun 72 hours after infection. Both were successful in eradicating the infection rapidly, but did not entirely prevent renal damage. Treatment prior to 72 hours thus is important. It appears that the combined treatment, designed to eradicate the bacteria as well as reduce the post-ischemic reperfusion damage and the phagocytic burst of phagocytosis is ideal, as this combined treatment was effective in preventing almost all renal damage and loss of renal function.

Topics: Allopurinol; Animals; Antibodies, Bacterial; Cefonicid; Drug Therapy, Combination; Escherichia coli; Escherichia coli Infections; Female; Kidney; Macaca mulatta; Organ Size; Pyelonephritis

Topics: Animals; Dogs; Dopamine; Escherichia coli Infections; Fluid Therapy; Free Radicals; Hemodynamics; Ibuprofen; Lipid Peroxidation; Nitriles; Oxamic Acid; Sepsis; Shock, Septic; Superoxides; Tromethamine; Xanthine Oxidase

Reactive oxygen species have been found to be responsible for the tissue injury caused in experimental pyelonephritis in mice. The extent of lipid peroxidation (as assayed by malondialdehyde formation) was found to be increased significantly (p less than .001) in the infected group as compared to the normal mice. Superoxide dismutase and catalase (oxygen free radical scavengers) showed a significant decrease (p less than .001) in the extent of lipid peroxidation even in the presence of infection. Dimethyl sulfoxide, a hydroxyl ion scavenger, was however found to be effective only at 4 and 7 days postinfection (p less than .001). Allopurinol, an inhibitor of xanthine oxidase, did not significantly (p greater than .05) inhibit the formation of lipid peroxides, even upto 7 days postinfection. There was a significant decrease (p less than .05) in the activities of renal brush border membrane enzymes used as markers of renal tissue damage (i.e. alkaline phosphatase, leucine amino-peptidase and gamma-glutamyl transpeptidase) in the infected group as compared to the normal group. In the presence of superoxide dismutase, dimethylsulfoxide and catalase except allopurinol, the activities of all the enzymes but maltase were found to be increased significantly (p less than .05) as compared to the infected group. There was a significant increase (p less than .01) in the bacterial count in the presence of superoxide dismutase and DMSO in infected mice as compared to the infected control mice. However, no significant difference was observed in the catalase and allopurinol treated groups.

Topics: Allopurinol; Animals; Catalase; Dimethyl Sulfoxide; Escherichia coli Infections; Free Radicals; Kidney; Lipid Peroxides; Mice; Mice, Inbred BALB C; Microvilli; Pyelonephritis; Superoxide Dismutase; Xanthine Oxidase

The inflammatory response and the respiratory burst of bacterial phagocytosis have been shown to be at least partially responsible for the renal damage from infection. In addition, we have shown that renal blood flow decreases following infection. Hypoxanthine is produced in ischemic tissue during the anaerobic metabolism of adenosine monophosphate (AMP). During reperfusion hypoxanthine is metabolized to uric acid and superoxide in the presence of xanthine oxidase. The toxicity of this oxygen radical was prevented by preventing its formation with pretreatment with allopurinol, an xanthine oxidase inhibitor. The data suggest that xanthine oxidase may be the enzyme responsible for the respiratory burst of phagocytosis, as well as preventing reperfusion damage which occurs after ischemia.

Topics: Adenosine Monophosphate; Allopurinol; Animals; Escherichia coli Infections; Female; Hypoxanthine; Hypoxanthines; Kidney; Macaca fascicularis; Phagocytosis; Pyelonephritis; Xanthine Oxidase