Regulation of murine intestinal inflammation by reactive metabolites of oxygen and nitrogen: divergent roles of superoxide and nitric oxide.

Several reports have implicated reactive oxygen and nitrogen metabolites (RONS) in the initiation and/or progression of inflammatory bowel diseases (IBDs). We have investigated the role of three key RONS-metabolizing enzymes (inducible nitric oxide synthase [iNOS], superoxide dismutase [SOD], nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in a murine model of IBD. Mice genetically deficient ((-/-)) in either iNOS or the p47phox subunit of NADPH oxidase, transgenic (Tg) mice that overexpress SOD, and their respective wild-type (WT) littermates were fed dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. In addition, the specific iNOS inhibitor 1400W was used in DSS-treated WT and p47phox(-/-) mice. WT mice responded to DSS feeding with progressive weight loss, bloody stools, elevated serum NO(X) and colonic mucosal injury with neutrophil infiltration. Both the onset and severity of colitis were significantly attenuated in iNOS(-/-) and 1400W-treated WT mice. While the responses to DSS did not differ between WT and p47phox(-/-) mice, enhanced protection was noted in 1400W-treated p47phox(-/-) mice. Interestingly, SOD(Tg) mice exhibited more severe colitis than their WT littermates. These findings reveal divergent roles for superoxide and iNOS-derived NO in intestinal inflammation.

Influence of hypercholesterolemia and hypertension on ischemia-reperfusion induced P-selectin expression.

Risk factors for cardiovascular disease have been shown to exacerbate the inflammatory response and microvascular dysfunction that is normally associated with ischemia-reperfusion. The objective of this study was to determine whether hypercholesterolemia and/or hypertension alter I/R-induced expression of P-selectin in the intestinal vasculature. Male control and hypertensive (HTN) rats were placed on either a normal diet or high cholesterol diet (HCD) for at least 3 weeks resulting in hypercholesterolemia (HC). Ischemia was induced by occlusion of the superior mesenteric artery for 15 min, followed by either 30 min or 4 h of reperfusion. The dual radiolabeled antibody technique was used to quantify the rapid (30 min) and transcription-dependent (4 h) expression of P-selectin. Tissue myeloperoxidase (MPO) was used to quantify neutrophil infiltration. The constitutive (basal) expression of P-selectin did not differ among the experimental groups. Although I/R significantly increased P-selectin expression in control, HC, and HTN+HC, P-selectin expression did not increase in HTN. The HC group exhibited the largest increments in P-selectin expression and tissue MPO after I/R. The increment in P-selectin expression was not significantly attenuated in HC rats that were rendered thrombocytopenic with anti-platelet serum. Treatment with an anti-P-selectin antibody largely prevented the exaggerated MPO increase noted in HC. These findings indicate that hypercholesterolemia in contrast to hypertension enhances the expression of P-selectin in the postischemic intestinal vasculature.

Hypercholesterolemia alters endotoxin-induced endothelial cell adhesion molecule expression.

While there is a growing body of evidence suggesting that hypercholesterolemia prior to the onset of atherosclerosis renders tissues more susceptible to inflammation, the mechanisms that underlie this exaggerated inflammatory response remain poorly defined. The overall objective of this study was to assess the influence of hypercholesterolemia on endotoxin-induced endothelial cell adhesion molecule (CAM) expression in different vascular beds. Another objective was to determine whether the altered endothelial CAM expression in hypercholesterolemic animals is associated with a corresponding change in plasma cytokine levels. Male Sprague/Dawley rats (SD) were placed either on a normal (control) or high cholesterol (HC) diet for 3 weeks. The dual radiolabeled monoclonal antibody (mAb) technique was used to measure the expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 in different vascular beds after intraperitoneal injection of endotoxin (LPS) derived from Salmonella abortus equi. LPS induced a significant increase in the expression of all endothelial CAMs in both normocholesterolemic and hypercholesterolemic groups. However, hypercholesterolemia enhanced LPS-induced expression of P-selectin, E-selectin, and ICAM-1 in several vascular beds, while VCAM-1 expression was unaffected. Thrombocytopenia, induced with anti-platelet serum, did not alter LPS-induced P-selectin expression in either group, suggesting that platelets do not contribute to this response. Hypercholesterolemia was associated with an exaggerated increase in plasma TNF-alpha, but not IL-1beta, after LPS treatment. These results indicate that hypercholesterolemia in rats may render tissues more vulnerable to the inflammatory effects of LPS by enhancing the expression of certain endothelial CAMs.

Comparison of magnetic resonance urography to dimercaptosuccinic acid scan for the identification of renal parenchyma defects in children with vesicoureteral reflux.

OBJECTIVE: The objective of this study was to compare the accuracy of dimercaptosuccinic acid (DMSA) renal scan to magnetic resonance urography (MRU) in the identification of renal parenchyma defects (RPD). MATERIALS AND METHODS: Twenty-five children with history of acute pyelonephritis and vesicoureteral reflux underwent DMSA scan and MRU to determine the presence of RPD. DMSA scans and MRUs were each evaluated by two radiologists and agreement achieved by consensus. Discordant DMSA-MRU findings were re-evaluated in a side-by-side comparison and an ultimate consensus reached. RESULTS: The ultimate consensus diagnosis was 18 kidneys with RPDs in 15 patients, of which five were classified as mild RPDs, six as moderate RPDs, and seven as severe RPDs. Although DMSA scan and MRU were similar in their ability to diagnose RPDs, MRU was considered to represent the true diagnosis in 11 of the 12 discordant cases in consensus review by four pediatric radiologists. MRU showed a much higher inter-observer agreement with a weighted kappa of 0.96 for both kidneys compared to 0.71 for the right kidney and 0.86 for the left kidney by DMSA scan. CONCLUSIONS: Our results suggest that MRU is superior to DMSA scan in the identification of renal parenchyma defects.

Splanchnic ischaemia-reperfusion injury: mechanistic insights provided by mutant mice.

Reperfusion of ischaemic tissues often leads to microvascular dysfunction that is manifested as impaired endothelium-dependent dilation of arterioles, enhanced fluid filtration and leucocyte plugging in capillaries, and the trafficking of leucocytes and plasma protein extravasation in postcapillary venules. Efforts to define the mechanisms that underlie these microvascular responses to ischaemia and reperfusion have largely relied on pharmacological agents and monoclonal antibodies. Gene-targeting technology has been applied to the production of transgenic and knockout mice that are rapidly gaining acceptance as tools for mechanistic studies of ischaemia-reperfusion (I/R) injury that obviate some of the concerns (e.g. specificity) raised about previously employed experimental strategies. This review summarizes some of our efforts to apply gene-targeted mice to the study of I/R injury in the splanchnic vascular bed. A role for endothelial cell adhesion molecules (CAMs) and reactive oxygen metabolites is supported by results from mutant mice. Low density lipoprotein receptor mice also reveal that the microvascular and inflammatory responses to I/R are greatly exaggerated during chronic hypercholesterolaemia. The wide variety of mutant mice that have been produced for inflammation-related research makes this experimental strategy particularly promising for mechanistic investigations of the tissue responses to I/R.

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis.

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.

Role of appendix and spleen in experimental colitis.

There is growing clinical evidence suggesting that certain secondary lymphoid tissues (e.g., appendix and spleen) contribute to the initiation and/or perpetuation of ulcerative colitis. In this study, the importance of secondary lymphoid tissues in inducing colitis was assessed experimentally by removing the spleen and/or appendix (or sham operation) prior to inducing colitis in mice. Feeding 2.5% dextran sulphate sodium (DSS) in drinking water over 7 days induced colitis. Clinical disease activity was assessed based on weight loss, stool consistency, and presence of blood in stools. Additional measurements included white blood cell count and hematocrit, and myeloperoxidase activity (MPO) in colon samples. Colonic injury was assessed by histology and computerized image analysis. DSS treatment in sham-operated mice produced colitis associated with weight loss, bloody diarrhea, and mucosal ulceration. Clinical assessment of DSS-treated mice subjected to appendectomy or combined appendectomy/splenectomy exhibited a delayed onset and course of disease activity. Histomorphologic examination revealed significantly lower damage scores and a reduction in ulcerated mucosal surface area. Colonic MPO activity, which correlated with tissue injury and disease activity, was lowest in appendectomized mice. No beneficial effects of splenectomy were observed after 7 days of colitis. These findings support the hypothesis that appendicular lymphoid tissue, but not the spleen, contributes to the development of colitis.