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Background and Aim: Controversy exists as to whether rats after bile duct ligation (BDL) are more susceptible to gastric mucosal damage (GMD) induced by irritants. In the present study we characterize GMD after intragastric instillation of either ethanol or hydrochloric acid (HCL), 3 and 21 days after the surgical procedure. Methods: Bile duct ligation and sham operated (SO) rats were studied. Results: Three days after surgery, BDL rats exhibited a reduction in gastric mucosal nitric oxide synthase (NOS) activity but an increase in ethanol-induced GMD. Twenty-one days after surgery gastric mucosal prostaglandin (PG) E2 generation in BDL rats was increased while NOS activity in both groups was similar. Ethanol-induced GMD in SO rats was higher. Pretreatment with NG-nitro-L-arginine methyl ester, prior to ethanol administration was associated with an increase in gastric mucosal PGE2 generation: (147% in SO and 104% in BDL rats) and in GMD (176% in SO and 303% in BDL rats). HCL induced GMD was of similar magnitude in both groups in both time periods. Conclusions: The gastric resistance to damage by irritants in rats with BDL is not a static phenomenon. This may result from sequential changes that occur in the gastric mucosal defense mechanisms during the evolution of liver disease.

Rats with portal hypertension and experimental liver disease may exhibit increased susceptibility of the gastric mucosa to damage by noxious agents, and increased bacterial translocation through the bowel wall. The aim of this study was to determine mucosal gastric and colonic generation of vasoactive substances, because they may contribute to the altered mucosal function. Rats with partial vein ligation (n = 7), complete bile duct ligation (n = 6) and sham-operated rats (n = 10) were studied. Three weeks following surgery rats were anesthetized, splenic pulp pressure was measured, stomachs and colons were removed and mucosa was extracted for determination of prostaglandin E2, thromboxane B2, leukotriene B4, leukotriene C4 and endothelin-1 by radioimmunoassay (ng/g) and platelet activating factor activity (pg/10 mg) by platelet aggregation. Pulp pressure was > 13 mmHg in partial vein ligated rats and bile duct ligated rats and 6 mmHg in sham-operated rats. No macroscopic or microscopic lesions were seen any of the removed tissues. Gastric mucosal prostaglandin E2 and thromboxane B2 generation were decreased by 35% and 7%, respectively, in bile duct ligated rats (bile duct ligated versus sham-operated, p < 0.05 for prostaglandin E2 and thromboxane B2). Gastric leukotriene B4 and C4 generation, platelet activating factor activity and endothelin-1 content did not differ significantly among the three groups. A different pattern of changes was observed in the colon. Colonic leukotriene B4 generation and endothelin-1 content were increased in bile duct ligated rats by 105% and 210%, respectively (bile duct ligated versus sham-operated, p < 0.05 for leukotriene B4 and endothelin-1). The decreased gastric mucosal prostaglandin E2 generation of bile duct ligated rats may render the gut mucosa of these animals relatively ischemic and vulnerable to damage by noxious agents. The increased colonic leukotriene B4 generation and the increased endothelin-1 content of the colonic mucosa of bile duct ligated rats may promote inflammatory and ischemic changes in the colonic mucosa and may enable bacterial translocation.

SUMMARY We determined endogenous gastric prostaglandin synthesis and its correlation with the prevention of duodenal ulcer relapse by misoprostol and ranitidine. Sixty-one patients with recent endoscopically healed duodenal ulcer were randomly allocated in a double-blind fashion for one year of treatment with misoprostol 400 μmUg nocte, ranitidine 150 mg nocte or placebo. Patients were followed every two months. Endoscopy was repeated at six and 12 months or beforehand, if relapse was suspected. Antral and fundic biopsies, 3–4 from each region, were obtained at each endoscopy for determination of prostaglandin synthesis. During the one year of treatment, 11 out of the 12 placebo treated patients flared up, as opposed to 10 out of 25 and four out of 24 misoprostol and ranitidine treated patients, respectively. The difference between all treatment groups was significant (P < 0.0001). In all subjects who flared up, pretrial endogenous antral and fundic prostaglandin E2 synthesis were not different from their respective synthesis in those who did not relapse. These results indicate that one year of treatment with both ranitidine and misoprostol prevents ulcer relapse better than placebo. However, ranitidine is significantly better than misoprostol. Both at six and 12 months there was no significant difference between the two treatment groups in the trend towards enhanced synthesis of gastric prostanoids. These results indicate that one year of treatment with both ranitidine and misoprostol prevents ulcer relapse better than placebo. However, ranitidine is significantly better than misoprostol. Both at six and 12 months there was no significant difference between the two treatment groups in the trend towards enhanced synthesis of gastric prostanoids.

Platelet activating factor, a potent inflammatory mediator, has been reported to induce gastrointestinal damage, whereas its inhibition or antagonism is associated with mucosal protection. The aim of the present study was to elucidate the association between acute experimental gastric damage and mucosal platelet activating factor generation in the rat, and to evaluate the protective effect of sucralfate in relation to mucosal platelet activating factor formation. Gastric damage in the rat was induced by either subcutaneous injection of indomethacin 30 mg/kg or intragastric administration of aspirin 100 mg/kg, hydrochloric acid 0.6 N, taurocholate 30 mM, ethanol 96%, or sodium chloride 25%. All agents induced a significant increase in mucosal platelet activating factor levels concomittently with induction of mucosal damage. Pretreatment with sucralfate 150 mg/rat provided a significant macroscopic and microscopic mucosal protection in all experimental models. This protection was associated with a significant decrease in mucosal platelet activating factor level in the hydrochloric acid, taurocholate, ethanol and hyperosmolar sodium chloride treated rats, whereas it remained unchanged in the aspirin and indomethacin treated rats. The data imply that platelet activating factor may have a limited role in the pathogenesis of indomethacin or aspirin induced damage, where other mechanisms such as cyclooxygenase inhibition dominate. In the damage induced by topical strong irritants, platelet activating factor may have a major pathogenetic role.

SUMMARY The effect of 1 week of treatment with indomethacin 150 mg/day on human gastric prostanoid synthesis was correlated with its effect on gastric and duodenal mucosa. Before and following 1 week of treatment, endoscopic appearance of the mucosa was evaluated and scored. Following 1 week of treatment with indomethacin, antral PGE2 and 6-keto-PGF1α were significantly lower than in normal subjects, but similar in patients with significant or with no mucosal damage. Co-treatment with ranitidine 150 mg b.d. or with cimetidine 400 mg b.d. reduced the mean mucosal damage score but did not affect gastric prostanoid synthesis, which was similar irrespective of the presence or absence of mucosal damage. It is therefore suggested that there is no correlation between indomethacin-induced inhibition of gastric prostanoid synthesis and its induction of mucosal damage.

Patients with advanced cirrhosis and rats with short-term bile duct ligation (BDL) are prone to develop nephrotoxicity from aminoglycosides. In this study, we characterized the renal response to gentamicin in rats with chronic BDL. BDL and sham-operated (SO) rats were given gentamicin (20 and 40 mg/kg/d) for 7 consecutive days, starting on the 18th postoperative day. Administration of gentamicin to SO group caused a decrease in cortical and medullary prostaglandin E(2)(PGE(2)) generation. However, mild reduction in creatinine clearance and an increase in fractional excretion of sodium occurred only in the BDL rats given the high gentamicin dose. This was accompanied by a reduction in cortical and medullary PGE(2) generation and a reduction in plasma nitric oxide production. In conclusion, gentamicin administration to rats with chronic BDL causes impairment of renal function. This happens only after the occurrence of simultaneous multiple insults to the renal protective mechanisms.

Substantial evidence suggests a negative association between cigarette smoking and the incidence and severity of ulcerative colitis, a common human in£ammatory bowel disease. Nicotine has been implicated in this association.The detection of nicotinic acetylcholine receptors in colonic epithelium, the primary tissue affected in ulcerative colitis, suggests a role for these receptors in the bene¢cial eiect of nicotine on colonic in£ammation. Using an animal model, we demonstrate for the ¢rst time that a5 nicotinic acetylcholine receptor knockout mice have signi¢cantly more severe experimental colitis than wild-type controls and that nicotine signi¢cantly ameliorates its course when compared with wild-type controls. These ¢ndings suggest that a5-containing nicotinic acetylcholine receptors participate in the modulation of colitis in mice, but other nicotinic acetylcholine receptor subunits also mediate the antiin£ammatory eiects of nicotine. NeuroReport 16:1123^1127 � c 2005 Lippincott Williams & Wilkins.

The constitutive cyclooxygenase (COX)-1 enzyme has been considered the physiologically important isoform for prostaglandin synthesis in the normal kidney. It has, therefore, been suggested that selective inhibitors of the ‘inducible’ isoform (COX-2) may be free from renal adverse effects. We studied the renal effects of the predominantly COX-2 antagonist nabumetone in isolated perfused kidneys. As compared with controls, kidneys removed after in vivo administration of oral nabumetone (15 mg/kg) disclosed altered renal function with reduced glomerular filtration rate, filtration fraction, and urine volume and enhanced hypoxic outer medullary tubular damage. By contrast, renal function and morphology were not affected in vivo by nabumetone or its active metabolite 6-methoxy-2-naphthylacetic acid. The latter agent (10–20 mg/kg i.v.) did not significantly alter renal microcirculation, as opposed to a selective substantial reduction in medullary blood flow noted with the nonselective COX inhibitor indomethacin (5 mg/kg i.v.). In a rat model of acute renal failure, induced by concomitant administration of radiocontrast, nitric oxide synthase, and COX inhibitors, the decline in kidney function and the extent of hypoxic medullary damage with oral nabumetone (80 mg/kg) were comparable to a control group, and significantly less than those induced by indomethacin. In rats subjected to daily oral nabumetone for 3 consecutive weeks, renal function and morphology were preserved as well. Both nabumetone and 6-methoxy-2-naphthylacetic acid reduced renal parenchymal prostaglandin E2 to the same extent as indomethacin. It is concluded that while nabumetone adversely affects renal function and may intensify hypoxic medullary damage ex vivo, rat kidneys are not affected by this agent in vivo, both in acute and chronic studies. COX selectivity may not explain the renal safety of nabumetone.

Ozone is one of the most powerful oxidants available, with many applications in industry and medicine. Medically relevant features of ozone include bacterial and virucidal properties, disinfection, sterilization, circulatory stimulation, and disruption of malignant cells. Ozone therapy is administered in various ways, including intravenously, intramuscularly, and intrarectally. The latter modality is used for the treatment of colitis and hepatitis. Our aim was to examine the effect of ozone water enema on normal and inflamed rat colonic mucosa. Ozone water (20 μg/ml) was prepared via ozone generator and administered intrarectally (0.5 ml) daily. Rats were killed one, three, and seven days after rectal ozone water administration, and their colons resected, rinsed, and weighed (grams per 10 cm). Damage was assessed macro- and microscopically and tissue processed for myeloperoxidase and nitric oxide synthase activity. Rats receiving saline served as controls. In an additional experiment colitis was induced by intrarectal iodoacetamide. Ozone therapy caused no macroscopic damage. Ozone therapy induced microscopic colitis, which lasted for at least a week and was accompanied by increase in segmental weight, myeloperoxidase and nitric oxide activity, and prostaglandin E2 generation. Ozone therapy had no protective effect on inflamed mucosa. In conclusion, ozone water therapy had a deleterious effect on normal colonic mucosa, suggesting intrarectal administration be reevaluated. Ozone water enema may serve as a model of microscopic colitis.

Reduced bone mineral density (BMD) is common in patients with inflammatory bowel disease (IBD), but the factors associated with its longitudinal rate of change have not been established. We prospectively assessed the rate of change in BMD, and its association with biochemical markers of bone turnover.Twenty-two patients with Crohn's disease and 14 ulcerative colitis patients age 37.1 +/- 11.6 yr were followed for 2 yr. Lumbar spine (L2-L4) and femoral neck BMD were measured by dual x-ray absorptiometry at baseline and 24 months. Bone-specific alkaline phosphatase, osteocalcin, urinary N-telopeptide crosslinked type 1 collagen (NTx), parathyroid hormone, and 25-hydroxyvitamin-D were determined at baseline.At baseline, 59% of Crohn's patients and 43% of ulcerative colitis patients were osteoporotic, with spine or femoral neck BMD T-score-2.5. Spine BMD, and spine and femoral neck T-scores were lower and disease duration was longer in nine patients with ileal resection compared with nonoperated patients (0.84 +/- 0.15 g/cm2 vs 0.96 +/- 0.11 g/cm2, -3.0 +/- 1.5 vs -1.7 +/- 1.3, -3.2 +/- 1.5 vs -2.2 +/- 1.0, respectively; all p0.05). At 24 months, 13/36 (36%) and 14/36 (39%) patients experienced spinal and femoral neck bone loss, respectively, with mean annual percent BMD changes of -2.0% and -1.5%, respectively. NTx, a bone resorption marker, inversely correlated with spinal BMD rate of change (r = -0.4, p0.05). Using quartiles analysis, patients with the highest NTx (Q4) experienced the greatest decrease in spine BMD compared with patients with the lowest NTx (Q1).Spine and femoral neck bone loss continues over time in more than one-third of IBD patients. Increased NTx level predicts spinal bone loss in IBD patients.

Both in experimental colitis and in inflammatory bowel disease, colonic eicosanoid generation is enhanced and may contribute to the pathogenesis of the inflammatory response. To evaluate the effect of selective cyclo-oxygenase-2 (COX-2) inhibitors on the extent and severity of two models of experimental colitis. Colitis was induced by intra-caecal administration of 2 ml 5% acetic acid or intra-colonic administration of 0.1 ml 3% iodoacetamide. Rats were treated intra-gastrically with nimesulide 2 × 10 mg/kg/day, or once with SC-236 6 mg/kg, and killed 1 or 3 days after damage induction. The colon was isolated, weighed, macroscopic damage was measured, and mucosal samples were obtained for histology and for determination of myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities and eicosanoid generation. The serum levels of thromboxane B2 (TXB2), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were determined. Nimesulide significantly decreased the extent of colitis induced by acetic acid. Both nimesulide and SC-236 significantly decreased the extent of iodoacetamideinduced colonic damage. The decrease in the extent of colitis induced by nimesulide was accompanied by a significant decrease in mucosal MPO and NOS activities. Nimesulide and SC-236 decreased the enhanced colonic eicosanoid generation in acetic acid and iodoacetamideinduced colitis, and, in iodoacetamide-treated rats, nimesulide also decreased the elevated serum TNF-α and IL-1β levels. The effective nimesulide and SC-236-induced amelioration of the severity of the colitis in acetic acid and iodoacetamide-treated rats confirms the role of eicosanoids in their pathogenesis and suggests that COX-2 inhibitors may be of value in the treatment of inflammatory bowel disease.Eur J Gastroenterol Hepatol12:223-231 © 2000 Lippincott Williams & Wilkins European Journal of Gastroenterology & Hepatology 2000, 12:223-231

Iron is pivotal is producing tissue-damaging reactive oxygen metabolites. Our aim is to determine the antiinflammatory activity of deferiprone, an oral iron chelator, in experimental colitis and gastritis. Colitis was induced by intraceccal administration of 2 ml 5% acetic acid or by intracolonic administration of 0.1 ml 3% iodoacetamide, with or without cotreatment with deferiprone. Gastritis was induced by intragastric administration of ethanol or hydrochloric acid (HCl) and by subcutaneous injection of indomethacin, with and without deferiprone. Rats were killed 24 hours after acetic acid and iodoacetamide, 30 minutes after ethanol, one hour after HCl, and three hours after indomethacin administration. The colon or stomach was isolated, macroscopic damage was measured, and mucosal samples were obtained for determination of eicosanoid generation, myeloperoxidase (MPO), and nitric oxide synthase (NOS) activities. Deferiprone decreased iodoacetamide and acetic acid-induced macroscopic colonic damage by 67% and 69%, respectively, and macroscopic gastric damage by 91%, 68%, and 46% induced by ethanol, HCl, and indomethacin, respectively. The effect of deferiprone was accompanied by significant decrease in colonic and gastric, MPO and NOS activities, and colonic prostaglandin E2 (PGE2) generation, in acetic acid, ethanol, and indomethacin models, whereas in the iodoacetamide and HCl models attenuation of the decrease in PGE2 generation was seen. Deferiprone is protective in experimental colitis and gastritis, probably due to decreased production of iron-dependent oxygen-free radicals. Oral iron chelators may constitute a novel approach to ameliorate gastrointestinal inflammatory disorders.

A rat model of experimental colitis and jejunitis induced by iodoacetamide (IA), a sulphydryl blocker is accompanied by increased leukotriene, prostaglandin E2 (PGE2) generation, and nitric oxide synthase (NOS) activity. Rat small intestinal and colonic surfactant-like particles (SLP) that accumulate on the apical surface of mucosal cells have been identified and specific antibodies to them have been produced. The aim of this study was to evaluate a possible role of SLP in IA-induced colitis and jejunitis. Inflammation was induced in Sprague-Dawley rats either by intracolonic administration of 3% IA (0.1 ml) or by intrajejunal administration of 2% IA (0.1 ml). Antisera raised against either colonic SLP, pulmonary SP-A (a major protein associated with colonic SLP), or small intestinal SLP were injected into the tail vein of rats 48 h before, simultaneous with, or 24 h after IA administration. Rats were killed 2 or 10 days after IA was given, their colon or small intestine was isolated and rinsed, and a segment of colon (10 cm) or small bowel (30 cm) was weighed and processed for microscopy, NOS and myeloperoxidase (MPO) activities, and PGE2 generation. Intracolonic or jejunal IA resulted after 48 h in extensive macroscopic and microscopic damage, accompanied by increased segmental weight, MPO and NOS activity, and PGE2 generation. Colonic SLP antibody administration, either 48 h before or at the time of damage induction, significantly decreased macroscopic as well as microscopic damage, segmental weight, MPO activity, and PGE2 generation, but had no effect on NOS activity. Neither control sera nor antisera against SP-A had any protective effect, nor did injection of anti-colonic SLP antisera given 24 h after IA. Small bowel SLP antibody offered no protection against IA jejunitis. IA-induced colitis but not jejunitis is ameliorated by intravenous injection of SLP antibody by a mechanism yet to be determined. These data provide further evidence of a physiologic role for gastrointestinal SLP.

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration. Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(II) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamnine but did not provide protection. The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O2*- and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.

Background Smoking, probably due to nicotine, has a bivalent effect on inflammatory bowel disease, ameliorating disease activity in ulcerative colitis and with a deleterious effect on Crohn's disease. The effect of nicotine patches in ulcerative colitis is controversial. Aim To investigate the effect of chronic nicotine use in a rat model of colitis. Methods Colitis was induced in Sprague-Dawley rats by rectal administration of 30 mg trinitrobenzene sulphonic acid (TNBS) in 50% ethanol. Nicotine was dissolved in drinking water (2.5, 12.5, 25 and 250 μg/ml), with rats drinking ad libitum. Nicotine administration started 10 days prior to damage induction and had no effect on weight gain or daily food intake of rats. Rats were sacrificed 1 and 5 days after TNBS administration, their colons resected, rinsed, weighed, damage assessed macroscopically (mm 2 ) and microscopically and tissue processed for myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities, leukotriene B 4 (LTB 4 ), prostaglandin E 2 (PGE 2 ) generation and interleukin-1 (IL-1) serum levels. Results Nicotine, by itself, caused no damage to the colon. Nicotine had a dose-dependent bivalent effect on colitis, significantly reducing macroscopic damage from 983 ± 10 mm 2 on TNBS alone to 429 ± 118 mm 2 on TNBS plus 12.5 μg/ml of nicotine, and escalating to 1086 ± 262 mm 2 on 250 μg/ml of nicotine. Segmental weight declined significantly (from 2.4 ± 0.2 to 1.65 ± 0.20 g/10 cm), on 12.5 μg/ml nicotine, as did MPO activity (from 3.2 ± 0.4 to 0.7 ± 0.1 units/g). All these parameters returned to the levels of TNBS alone when the dose of nicotine was increased to 250 μg/ml. Nicotine had no effect on NOS activity, PGE 2 generation and serum IL-1 levels, but increased LTB 4 generation. Conclusions Nicotine has a dose-dependent bivalent effect on TNBS-induced colitis which is not due to reduction in IL-1 serum levels or PGE 2 generation, and is not NOS-mediated.

Background—Hyperbaric oxygen (HBO) has been suggested to be beneficial in inflammatory bowel disease but the mechanisms responsible for its therapeutic effects have not been elucidated.Aim—To assess the effect of HBO treatment on colonic damage in two models of experimental colitis, and to examine whether this effect is mediated by modulation of NO synthesis.Methods—Colitis was induced by either flushing the colon with 2 ml 5% acetic acid or intracolonic administration of 30 mg trinitrobenzenesulphonic acid (TNB) dissolved in 0.25 ml 50% ethanol. Rats were exposed to HBO (100% oxygen at 2.4 atmosphere absolute) for one hour twice on the day of colitis induction and once daily thereafter. Control rats were treated only with acetic acid or TNB. Rats were killed 24 hours after acetic acid administration or one and seven days after TNB treatment. The colon was isolated, washed, and weighed, the lesion area was measured, and mucosal scrapings were processed for determination of myeloperoxidase (MPO) and NO synthase (NOS) activities, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) generation.Results—In control rats exposed for seven days to HBO, colonic NOS activity was significantly decreased by 61%, compared with its activity in untreated rats (2.93 (0.17) nmol/g/min). HBO significantly reduced by 51 and 62% the extent of injury induced by acetic acid and TNB respectively. The protection provided by HBO was accompanied by a significant decrease in colonic weight, PGE2 generation, MPO, and NOS activities. In acetic acid colitis, LTB4 generation was also significantly decreased.Conclusions—(1) HBO effectively decreases colitis induced by acetic acid and TNB. (2) The decreased NOS activity induced by HBO suggests that reduction in NO generation may be among the mechanisms responsible for the anti-inflammatory effect of HBO. (3) HBO may be considered in the treatment of patients with refractory inflammatory bowel disease.

Objective: The mechanism of bone loss in patients with inflammatory bowel disease (IBD) is not completely understood. The aim of this study was to assess indices of bone turnover and bone mineral density (BMD) in the lumbar spine and femoral neck in IBD patients. Methods: Sixty-three patients with Crohn’s disease and 41 with ulcerative colitis were studied. Serum bone-specific alkaline phosphatase (B-ALP), osteocalcin, parathyroid hormone (PTH), 25 hydroxyvitamin D, interleukin-6 (IL-6), and urinary N-telopeptide cross linked type 1 collagen (NTX) were determined. BMD of the lumbar spine and femoral neck was determined by dual x-ray absorptiometry in 59 patients. Results: In the femoral neck 42% of the patients had osteopenia (−2.5 SD < BMD T score < −1 SD) and another 41% had osteoporosis (BMD T score < −2.5). In the spine 34% of the patients had osteopenia and additional 42% had osteoporosis. BMD T scores were lower in the femoral neck compared to the spine. Reduced BMD was unrelated to gender, disease type, lifetime corticosteroid dose, but inversely correlated with disease duration (r = −0.36, p < 0.05). Serum IL-6 was higher in IBD patients compared to controls. A reduced level of osteocalcin, a marker of bone formation, was present in 7% of patients and an increase in NTX, a marker of bone resorption, in 25% of them. Osteoporotic IBD patients (spine or hip BMD T score < −2.5) had increased serum IL-6, osteocalcin and PTH level compared to nonosteoporotic patients. Conclusions: There is a high prevalence of reduced BMD at the spine and femoral neck in IBD patients, which is more severe in the hip. Bone turnover in osteoporotic IBD patients is associated with an increase in osteocalcin, PTH and IL-6. IL-6 may play a role in the pathogenesis of bone loss in IBD.

Background—Sulphydryl compounds are essential for maintaining mucosal integrity in the gastrointestinal tract.Aim—To characterise a model of experimental inflammation in the small intestine induced by a sulphydryl blocker.Methods—Inflammation in the small intestine was induced in rats by intrajejunal administration of 0.1 ml 2% iodoacetamide. The possible amelioration of the damage induced was modulated by intragastric administration of TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; 50 mg/100 g body weight), ketotifen (200 μg/100 g body weight) or by addition ofl-NAME (NG-nitro-l-arginine methyl ester; 0.1 mg/ml) or apocynin (120 μg/ml) to the drinking water. Rats were sacrificed at various time intervals, the small intestine resected, weighed, macroscopic lesions were assessed, and mucosal generation of inflammatory mediators and nitric oxide synthase activity were determined.Results—Intrajejunal administration of iodoacetamide induced, after one week, multifocal mucosal erosions, ulcerations with granulomas and giant Langhans cells. At two weeks, the mucosa was almost macroscopically intact but histologically epithelial granuloma and giant cells were present. Myeloperoxidase activity was increased in the first 24 hours, one week later mucosal nitric oxide synthase activity and generation of leukotriene B4, leukotriene C4 and thromboxane B2 were increased, whereas prostaglandin E2 generation was decreased notably. Ketotifen and apocynin significantly decreased the extent of injury which was not affected by TEMPOL orl-NAME.Conclusions—Jejunal inflammation induced by the sulphydryl blocker, iodoacetamide, resembles the pathological changes in Crohn’s disease. The protective effect of ketotifen and apocynin indicates the contribution of O2− and pro-inflammatory mediators to the pathogenesis of the damage, and may be a novel approach to the treatment of inflammatory bowel disease.

Nitric oxide (NO) exerts profound effects on renal physiology [1—41. Renal NO generation occurs in endothelial cells by the constitutive isoform of nitric oxide synthase (cNOS) and in macrophages, smooth muscle and mesangial cells by the inducible isoform (iNOS). The former system plays an important role in basal renal vascular tone and hemodynamic regulation, in mesangial contraction, renin release and tubuloglomerular feedback. The inducible NOS, widely distributed in the rat kidney, predominantly in the outer medulla and in cortical glomeruli [5], counterbalances vasoconstrictive, thrombogenic and proliferative stimuli associated with cytokine stimulation and inflammatory responses [6]. Intact nitrovasodilation is particularly important at the outer medulla, where tissue oxygen tension as low as 30 mm Hg normally exists [7]. Inhibition of NOS results in profound renal hypoperfusion, both at the cortex and outer medulla [8, 9], associated with substantial aggravation of medullary hypoxia [10]. Blockade of NO synthesis potentiates medullary hypoxic injury from radiocontrast and non-steroidal anti-inflammatory agents [9—111 and augments medullary hypoperfusion and damage during acute ureteral obstruction [121. Recently developed Clark-type selective NO microelectrodes enable real-time monitoring of NO concentrations in vitro in cell lines and incubated tissues [13—17], in organs maintained ex vivo [18] or in the living organism [19, 20]. With concomitant determination of intrarenal blood flow it could be a potent experimental tool for the evaluation of the role of NO in renal hemodynamics. In preliminary studies with the NO electrode, a paradoxical increase in medullary NO reading was noted following NOS inhibition. This led us to evaluate potential artifacts of NO monitoring, such as fluctuations in tissue oxygenation (since oxygen and reactive oxygen species scavenges NO with the production of NO2/NO3 , not sensed by the electrode) and temperature (which markedly affect the electrode current) [21]. Methods

We studied the effect of ketotifen, a second generation H1-receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo. Ketotifen (100 μg ml−1) increased human colonic NOS activity from 3.7±0.6 to 14.5±1.3 nmol g−1 min−1 (P

Bisacodyl and colchicine affect smooth-muscle contractility, intestinal water, and electrolyte transport. Nitric oxide (NO) stimulates intestinal electrolyte secretion and has an important role as a mediator of intestinal motility. We therefore studied, in rats, the effects of these agents on nitric oxide synthase (NOS) activity and gastrointestinal transit.Rats were treated with bisacodyl (10 mg/kg intragastrically) or colchicine (5 mg/kg intraperitoneally) with or without pretreatment with ketotifen (1 mg/kg intragastrically). Rats were killed after 1, 2, and 4 h. The intestine was isolated and rinsed, the mucosa scraped, and NOS activity determined. In all rats small-intestinal transit was measured 15 min after intragastric administration of charcoal.Bisacodyl (10 mg/kg) and colchicine (5 mg/kg) induced a significant decrease in jejunal NOS activity. Pretreatment with the mast cell stabilizer ketotifen, which has been shown to attenuate the increased permeability induced by NO inhibition, prevented the decrease in colonic and jejunal NOS activity induced by bisacodyl and colchicine. Bisacodyl and colchicine significantly decreased intestinal transit time. Their effect on transit time was similar to that induced by intravenous administration of NG-nitro-L-arginine methyl ester (10 mg/kg).It is suggested that the effect of bisacodyl and colchicine on intestinal transport is, at least partly, mediated through NO inhibition.

Enhanced nitric oxide (NO) generation by stimulated NO synthase (NOS) activity may, through its oxidative metabolism contribute to tissue injury in experimental colitis. In this study the possible amelioration of experimental colitis by NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS activity, was evaluated. Colitis was induced in rats by intracolonic administration of 30 mg trinitrobenzene sulphonic acid (TNB) dissolved in 0.25 ml 50% ethanol or by flushing the colon of capsaicin pretreated rats with 2 ml of 5% acetic acid. In several experiments, L-NAME 0.1 mg/ml was added to the drinking water at the time of colitis induction with TNB or seven days before acetic acid treatment. Rats were killed at various time intervals after induction of colitis. A 10 cm distal colonic segment was isolated, weighed, lesion area measured, and explants organ cultured for 24 hours for determination of NO generation by the Greiss reaction. The rest of the mucosa was scraped for determination of myeloperoxidase and NOS activities and leukotriene generation. In TNB treated rats mean arterial pressure was also determined up to 72 hours after damage induction, with or without cotreatment with nitroprusside. L-NAME significantly decreased the extent of tissue injury in TNB treated rats. Seven days after TNB treatment lesion area was reduced by 55%, colonic weight by 37%, and myeloperoxidase and NOS activity by 59% and 42%, respectively. Acetic acid induced colitis in capsaicin pretreated rats was also significantly decreased by L-NAME. Twenty four hours after acetic acid treatment lesion area was reduced by 61%, colonic weight by 21% and NOS activity by 39%. Mean (SEM) arterial blood pressure in TNB+L-NAME treated rats was 37.6 (8.1) mm Hg higher than in TNB treated rats, an effect that was only partially abolished by nitroprusside. These results show that inhibition of NO synthesis by an L-arginine analogue significantly ameliorates the extent of tissue injury in two models of experimental colitis, an effect that is not due only to its vasoconstrictor properties. Modulation of NO generation may be a novel therapeutic approach in inflammatory bowel disease.

To assess the role of nerves that synthesize nitric oxide (NO) in Hirschsprung's Disease (HD), the authors studied the distribution of the enzyme NADPH diaphorase (NADPHd) in normal and diseased bowel segments. In the proximal (ganglionic) segment of the colon, NADPHd-positive neurons were present in both myenteric and submucosal plexuses. In the distal involved colonic segments from HD patients, the typical pattern of the neuronal network was completely missing in the regions of the two plexuses; instead, only disorganized NADPHd-positive nerve fibers were present and NADPHd-reactive neurons were absent. Mucosal NO synthase activity was 2.76 +/- 0.38 nmol/g/min in the proximal segment and only 0.83 +/- 0.49 nmol/g/min in the distal segment (P.05, N = 3).

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.

Systemic administration of capsaicin aggravates ethanol-induced injury of rat gastric mucosa. We evaluated the effect of subcutaneous administration of capsaicin on the gastric mucosa and on inflammatory mediators in saline- and ethanol-treated rats. Functional ablation of primary afferent C-fibers by capsaicin (total 100 mg/kg subcutaneous) tripled ethanol-induced damage. Pretreatment with ketotifen, a mast cell stabilizer (1 mg/kg) protected rat gastric mucosa from the amplified injury induced by capsaicin and ethanol. Tempol, a selective nontoxic cell-permeable nitroxide, completely prevented the amplified gastric ulceration induced by capsaicin and ethanol. This was accompanied by a significant decrease in leukotriene B4 and C4 generation. It is therefore suggested that mast cells and free radicals contribute to the amplified injury observed in rats pretreated with capsaicin and ethanol and that the pharmacological modulation of mast cell release and scavenging of free radicals may be of therapeutic efficacy in the prevention of gastric injury.

Capsaicin-sensitive afferent neurons are involved in maintaining the integrity of the gastrointestinal mucosa. These neurons are closely apposed to mast cells and could, therefore, lead to their activation. In the present study, the role of capsaicin-sensitive neurons in the pathogenesis of experimental colitis and the possible involvement of mast cells and nitric oxide were evaluated. Rats were treated with capsaicin subcutaneously, 20, 30, and 50 mg/kg, on three consecutive days, a regimen shown to ablate primary afferent neurons. Colitis was induced two weeks later by flushing 2 ml 5% acetic acid into the proximal colon. Control rats received saline, acetic acid, or capsaicin alone. Another group of rats received ketotifen (100 micrograms/100 g body wt x 2/day) intragastrically 48 hr prior to damage induction and thereafter. Rats were sacrificed 24 hr after damage induction, the colon isolated, damage assessed, explants were organ-cultured for 24 hr for determination of nitric oxide generation, and mucosa extracted for determination of leukotriene B4 generation and nitric oxide synthase activity. Significant increases in colonic weight, nitric oxide synthase activity, and nitric oxide and leukotriene B4 generation accompanied the near tripling of acetic acid-induced damage in capsaicin-pretreated rats. Ketotifen pretreatment significantly decreased the macroscopic damage and the increase in colonic weight. The protection provided by ketotifen was accompanied by a significant decrease in leukotriene B4 generation and nitric oxide synthase activity (80%).(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide, the product of nitric oxide synthase in inflammatory cells, may have a role in tissue injury through its oxidative metabolism. Nitric oxide may have a role in the pathogenesis of duodenal ulcer and may be one of the mechanisms responsible for the association between gastric infection with Helicobacter pylori and peptic disease. In this study, calcium independent nitric oxide synthase activity was detected in human gastric mucosa suggesting expression of the inducible isoform. In 17 duodenal ulcer patients gastric antral and fundic nitric oxide synthase activity was found to be two and 1.5-fold respectively higher than its activity in the antrum and fundus of 14 normal subjects (p < 0.05). H pylori was detected in the antrum of 15 of 17 duodenal ulcer patients and only in 7 of 14 of the control subjects. Antral nitric oxide synthase activity in H pylori positive duodenal ulcer patients was twofold higher than in H pylori positive normal subjects (p < 0.05). In duodenal ulcer patients antral and fundic nitric oxide synthase activity resumed normal values after induction of ulcer healing with ranitidine. Eradication of H pylori did not further affect gastric nitric oxide synthase activity. These findings suggest that in duodenal ulcer patients stimulated gastric mucosal nitric oxide synthase activity, though independent of the H pylori state, may contribute to the pathogenesis of the disease.

The pathogenesis of gastric mucosal injury is still poorly understood. Recent reports implicate redox active metals and reactive oxygen species as mediators of gastric damage induced by ethanol or non-steroidal anti-inflammatory drugs. Attempts were made therefore to prevent gastric injury using chelators and the antioxidant enzymes catalase and superoxide dismutase. These attempts, at best, would only detoxify extracellular reactive species, such as those produced by activated circulating granulocytes and macrophages. This study utilises another strategy by pre-emption of both intra and extracellular reactive species using radical-radical annihilation reactions and by detoxifying redox active transition metals. Nitroxide, stable free radicals were shown to enter mucous cells, protect against the ethanol induced damage, and prevent gastric lesions induced by aspirin, indomethacin, 25% NaCl, or 0.6 N HCl. These findings confirm that gastric mucosal damage from the above agents is mediated by free radicals and, moreover, introduce a prototypical agent within a potential new class of gastric ulcer preventing drugs.

Background:Clostridium difficile toxin A is the principal mediator of inflammatory enterocolitis in experimental animals. The purpose of this study was to explore the effect of ketotifen, an anti-inflammatory drug, on toxin A-induced enterotoxicity in rat ileum. Methods: The effects of intragastric administration of ketotifen on secretion, mannitol permeability, histological damage, and mucosal levels of leukotriene B4, leukotriene C4, and platelet activating factor in toxin A-exposed rat ileal loops were measured in vivo. The effects of ketotifen on toxin A-mediated release of rat mast cell protease II (rat mucosa mast cell product) release were also measured in rat ileal explants in vitro. The effect of ketotifen on neutrophil migration in vitro was also evaluated. Results: Ketotifen pretreatment inhibited toxin A-associated intestinal secretion by 42.5% and mannitol permeability by 56.3% and reduced epithelial cell inflammation and necrosis. These effects were associated with reduced levels of leukotriene B4 by 65.8%, leukotriene C4 by 88.8%, platelet activating factor by 77.8%, and inhibition of rat mast cell protease II by 58.4%. In addition, pretreatment of neutrophils with ketotifen inhibited neutrophil migration in vitro. Conclusions: The protective effect of ketotifen in this animal model was associated with significant inhibition of release of mast cells and neutrophil derived mediators, supporting their involvement in C. difficile enteritis.

To elucidate the possible role of vasoactive intestinal peptide (VIP) in the pathogenesis of acute gastric mucosal damage, rats were treated intragastrically with 1.0 ml 96% ethanol with or without intravenous or intraperitoneal coadministration of VIP (1 nmol/liter to 1 μmol/liter/100 g). VIP was found to double the mean lesion area when compared with that induced by ethanol alone (P

Controversy exists as to whether rats after bile duct ligation (BDL) are more susceptible to gastric mucosal damage (GMD) induced by irritants. In the present study we characterize GMD after intragastric instillation of either ethanol or hydrochloric acid (HCL), 3 and 21 days after the surgical procedure.Bile duct ligation and sham operated (SO) rats were studied.Three days after surgery, BDL rats exhibited a reduction in gastric mucosal nitric oxide synthase (NOS) activity but an increase in ethanol-induced GMD. Twenty-one days after surgery gastric mucosal prostaglandin (PG) E(2) generation in BDL rats was increased while NOS activity in both groups was similar. Ethanol-induced GMD in SO rats was higher. Pretreatment with NG-nitro-L-arginine methyl ester, prior to ethanol administration was associated with an increase in gastric mucosal PGE(2) generation: (147% in SO and 104% in BDL rats) and in GMD (176% in SO and 303% in BDL rats). HCL induced GMD was of similar magnitude in both groups in both time periods.The gastric resistance to damage by irritants in rats with BDL is not a static phenomenon. This may result from sequential changes that occur in the gastric mucosal defense mechanisms during the evolution of liver disease.

The effects of ketotifen, a 'mast cell stabiliser,' on two models of experimental colitis were examined. The inflammatory response elicited by either trinitrobenzene sulphonic acid or acetic acid resulted in increased colonic synthesis of platelet activating factor, prostaglandin E2, thromboxane B2, leukotrienes B4 and C4, and myeloperoxidase activity. Intragastric administration of ketotifen 100 micrograms/100 grams twice daily significantly decreased mucosal damage when given prophylactically 48 hours before the induction of colitis and then throughout the experiment. This effect was consistent in both models and was accompanied by a significant reduction in mucosal generation of platelet activating factor, prostaglandin E2, thromboxane B2, and leukotrienes C4 and B4. Myeloperoxidase activity was reduced as well, reaching significance only in the acetic acid model. This study shows that both trinitrobenzene sulphonic acid and acetic acid colitis can be pharmacologically manipulated by ketotifen. The mechanism of action of ketotifen has not yet been determined. Ketotifen's potential in the treatment of active inflammatory bowel disease or in the prevention of exacertations, or both, remains to be elucidated.

The effect of immunosuppressive drugs, 4-aminosalicylic acid (4-ASA), acetyl 5-aminosalicylic acid (5-ASA), and ketotifen on human colonic eicosanoid accumulation was evaluated in view of enhanced accumulation in patients with active ulcerative colitis. Azathioprine (100 micrograms/ml), cyclosporin (100 micrograms/ml), and methotrexate (100 micrograms/ml) significantly inhibited, by 25-35%, prostaglandin E2 (PGE2) accumulation by organ-cultured colonic mucosa of ulcerative colitis patients. Methotrexate was the only immunosuppressive drug that inhibited leukotriene B4 (LTB4) accumulation (50%), whereas azathioprine inhibited the accumulation of leukotriene C4 (LTC4) (25%). 5-ASA and its metabolite, acetyl 5-ASA, inhibited by 20-70% PGE2, LTB4, and LTC4 accumulation in the culture, supporting the contention that acetyl 5-ASA is as active as 5-ASA in these respects. 4-ASA had no effect on any of the eicosanoids. Ketotifen, a mast cell stabilizer, significantly inhibited the accumulation of PGE2, LTB4, and LTC4 by 33-60%. These results suggest a potential, new, unrecognized mode by which the immunomodulators induce part of their therapeutic effects in inflammatory bowel disease and support the contention that acetyl 5-ASA is as active as 5-ASA. The results obtained also indicate that ketotifen, used effectively in the prevention of bronchial asthma, inhibits the accumulation of colonic eicosanoids and, thus, may be of value in the treatment of inflammatory bowel disease.

Nausea and vomiting are symptoms sometimes associated with motor dysfunction. We compared a group of young patients suffering from chronic nausea and/or vomiting and normal upper gastrointestinal x-ray series with a control group. The members of both groups underwent isotopic examinations of their stomachs. The aim of the study was to find a simple method of checking the stomach and proving a motor dysfunction in a group of patients with chronic, inexplicable nausea and vomiting. Patients and controls fasted for at least 6 hr were given 0.5 mCI of [99mTc] diethylene triaminopentaacetic acid orally in 150 cc milk with 50 g cornflakes. A time-activity curve was obtained and radioactivity over the stomach was recorded exponentially. The parameter of the T1/2 emptying time was used. In normal controls T1/2 emptying time ranged from 18 to 26 min. Twenty-five symptomatic patients were examined, three of whom achieved normal values, but 22 patients showed pathologic results ranging from 36 to 184 min. In patients with chronic nausea and/or vomiting an isotopic examination of the stomach may provide a simple and rapid diagnostic method of evaluation.

To elucidate the possible role of substance P in the pathogenesis of acute gastric mucosal damage, rats were treated intragastrically with 1.0 mL 96% ethanol, 0.6N HCl, or 25% NaCl, with or without IP coadministration of substance P, senktide, or septide (1 mumol/L per 100 g). All three peptides were found to double the mean lesion area when compared with that induced by ethanol, whereas substance P antagonist (1 mumol/L per 100 g) prevented the expansion of damage extent. The increased damage was associated with increased gastric mucosal levels of platelet activating factor, leukotriene B4, and leukotriene C4. Substance P antagonists also reduced by half the extent of the gastric damage induced by ethanol when administered by itself. WEB 2086 (platelet-activating factor antagonist; Boehringer Ingelheim KG, Germany), hydroxyzine (H1 blocker), and cimetidine (H2 blocker) reduced lesion area by 50%, but only in rats treated with both substance P and ethanol. Ketotifen (mast-cell stabilizer) (100 micrograms/100 g), administered orally 30 minutes before damage induction, totally abolished the extent of the damage induced by either ethanol or the coadministration of ethanol and peptides in the surface epithelium of the entire mucosa. The protective effect of ketotifen was accompanied by significant reduction in mucosal generation of platelet-activating factor, leukotriene C4, and leukotriene B4. Similar mucosal protection was afforded by ketotifen against damage induced by 0.6N HCl, 25% NaCl, or indomethacin. Therefore, it is suggested that substance P is involved in the pathogenesis of acute ethanol-induced gastric mucosal damage. The effective mucosal protection provided by ketotifen indicates the important role of mast cells and their mediators in the pathogenesis of acute gastric mucosal damage and may have therapeutic implications.

Introduction Osteopenia is a common complication of human inflammatory bowel disease (IBD). We evaluated the contribution of colonic inflammation to osteopenia and its mechanism in a murine colitis model. Methods Colitis was induced by adding dextran sodium sulfate (DSS) to the drinking water for 2 weeks to nine-week-old Balb/C male mice. 5% DSS was added on the first week and was reduced to 2.5% on the second week. Age- and sex-matched Balb/C mice served as the control group. Indices of femoral bone mass and architecture were determined by micro computed tomography (μCT). Bone formation parameters and osteoclast number were determined by dynamic histomorphometry. The degree of colonic inflammation was assessed by a clinical disease activity index, and colonic mucosal myeloperoxidase activity. Results DSS-treated mice exhibited a significantly lower bone mass compared to controls as indicated by decreased trabecular bone volume (BV/TV) of 32%. This reduction was accompanied by decreased trabecular number (23%) and connectivity density (37%) compared to the controls. No changes were observed in cortical bone indices. Osteopenia resulted from suppressed bone formation, as indicated by decreased trabecular double-labeled surface (dL%) of 90%, mineralizing surface (MS) of 62%, and bone formation rate (BFR) of 67%, and increased bone resorption as indicated by a 34% increase in osteoclast number in DSS-treated mice compared to the controls. Myeloperoxidase activity inversely correlated with trabecular BV/TV ( r = − 0.67, p = 0.02), trabecular number ( r = − 0.86, p = 0.0008) and connectivity density ( r = − 0.63, p = 0.03). Myeloperoxidase activity inversely correlated with the bone formation indices: dL%, MS, and BFR ( r = − 0.79, p = 0.007, r = − 0.84, p = 0.002, r = − 0.83, p = 0.003, respectively). Conclusions DSS-induced colitis is associated with reduced femoral bone mass and altered micro architecture, which results from suppressed bone formation and increased bone resorption. The decrease in indices of bone mass, structure and formation are directly linked to the degree of colonic mucosal inflammation. DSS-induced colitis can be used to study pharmacological interventions for bone loss in colitis.

Oral administration of nonsteroidal antiinflammatory drugs induces gastroduodenal mucosal damage in experimental animals as well as in humans. The aim of the present study was to evaluate the effect of rectally administered nonsteroidal antiinflammatory drugs on gastric mucosal damage, as well as on mucosal prostaglandin E2 synthesis. Fasting male rats were treated intrarectally with 0.5 ml 1% NaHCO3 solution containing several concentrations of either indomethacin, aspirin, ibuprofen, diclofenac, ketoprofen, or sulindac and concomitantly received 1 ml of 150 mM HCL intragastrically. Control rats received intrarectally the vehicle only. After 4 h, lesions in the secretory part of the stomach were scored and mucosal prostaglandin E2 synthesis was determined by the ex vivo prostaglandin generation technique. Dose-dependent mucosal damage was observed in indomethacin- and diclofenac-treated rats. Ketoprofen damage did not show dose dependency. In sulindac- and aspirin-treated rats, as well as in controls, no damage was detected. All drugs induced a significant and comparable degree of inhibition of prostaglandin E2 synthesis. There was no correlation between the severity of the mucosal damage and the inhibition of prostaglandin E2 synthesis. The ulcerogenicity of rectally administered nonsteroidal antiinflammatory drugs is therefore not directly related to the degree of inhibition of prostaglandin E2 synthesis and is probably related to the specific chemical and pharmacokinetic properties of each individual drug.

Background: We previously showed that Toll-like receptor-9 (TLR-9) ligands ameliorate experimental colitis. In this study, we evaluated the effect of TLR-9 ligands on the generation of proinfiammatory cytokines by human colonic mucosa. Materials and Methods: Colonoscopic biopsies were obtained from patients with active ulcerative colitis (UC) and from normal subjects. The tissue was organ cultured for 24 hours in the presence or absence of different types of immunostimulatory (ISS) (CpO)­ oligonucleotides (ODNs). Tumor necrosis factor-a (TNF-a) and interleukin-l ~ (IL-l~) levels in the medium were determined by enzyme-linked immunosorbent assay. Results: In active UC, hTNF-a and hIL-I~ generation by inflamed colonic mucosa is 7- and 3-fold higher, respectively, than their generation by normal mucosa. Class B CpO ODNs inhibited colonic TNF-a and IL-I~ generation by 50%, whereas class A or C ODNs had a partial or no effect, respectively. A novel class ofODNs that is based on multiple TCG repeats was as effective as class B ODNs. This inhibition resulted from the transcriptional suppression of IL­ l ~ that occurred within the first 2 hours after ISS-ODN incubation. The addition of chloroquine abolished the inhibitory effects of ISS­ ODNs on colonic TNF-a and IL-IJ3 generation. Conclusions: Only certain classes of ISS-ODNs inhibit the enhanced TNF-a and IL-I~ generated ex vivo by inflamed colonic mucosa of patients with UC. The effect ofISS-ODNs is mediated by triggering of TLR-9. These results suggest a potential therapeutic value for ISS-ODNs in uc.

Background & Aims: Bone loss is a common complication of human inflammatory bowel disease (IBD), but its mechanisms are not understood completely. We investigated bone metabolism in interleukin-10-deficient ( IL-10−/− ) mice, an animal model with IBD features. Methods: IL-10−/− male mice (8- and 12-weeks-old) and their age-matched wild-type counterparts (C57BL/6J) were studied. Bone mass of the femur was determined by ashing. Tibial cancellous and cortical bone mass and formation was measured by static and dynamic histomorphometry. Biomechanical strength of the femur was tested. Primary bone marrow stromal cell cultures were used to assess osteoblast generation. Serum levels of 25-OH vitamin D 3 , insulin-like growth factor-1 (IGF-1), parathyroid hormone, osteocalcin, and deoxy-pyridinoline cross-links were measured. The presence of colitis was determined histologically, and by IL-12 and interferon-γ (IFN-γ) secretion from cultured colonic explants. Results: Eight- and 12-week-old IL-10−/− mice developed osteopenia of both cancellous and cortical bone, evidenced by lower femoral ash weight, cancellous bone area and surface, trabecular number, and decreased cortical bone area and width. Osteopenia was associated with mechanical fragility, manifested by decreased stiffness and mechanical load at fracture, and was caused by suppressed bone formation, indicated by decreased cancellous double-labeled surface, mineralizing surface, serum osteocalcin level, and mineralized nodule number in bone marrow stromal cell cultures. IL-10−/− mice with colitis had significantly less bone mass compared with IL-10−/− mice without colitis. Conclusions: IL-10−/− mice develop the hallmarks of osteoporosis, that is, reduced bone mass, increased mechanical fragility, and suppressed bone formation. The presence of colitis is an important contributor to osteoporosis in IL-10−/− mice.

Background & Aims: We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. Methods: Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated probiotics and Escherichia coli (DH5) genomic DNA were administered intragastricly (i.g.) or subcutaneously (sc) to mice prior to the induction of colitis. Viable or -irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. Results: Intragastric and sc administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable -irradiated or viable probiotics. Mice deficient in MyD88 did not respond to -irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of -irradiated probiotics, whereas, in TLR9-deficient mice, -irradiated probiotics had no effect. Conclusions: The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.

Chronic nicotine administration has a dual effect on inflammatory bowel disease: augmentation of jejunitis and amelioration of colitis. We previously showed that chronic nicotine administration has divergent regional effects on small bowel and colonic mucosal mediators and blood flow.To examine the effects of nicotine administration on cytokine levels in normal rat small bowel mucosa, colonic mucosa, and blood.Male Sprague-Dawley rats weighing 200-250 g were given nicotine (12.5 micrograms/ml) that was dissolved in tap water. Rats were sacrificed on days 1, 2, 7 and 14 after nicotine initiation; blood was withdrawn, and small bowel and colon were resected, washed and weighed. Mucosal scrapings were extracted in 2 ml Krebs-Hemselest buffer for determination of interleukins-2, 6 and 10 using the Biosource International Immunoassay Kit.Nicotine decreased IL-10 and increased IL-6 levels in small bowel mucosa (from 3.5 +/- 0.5 to 0.4 +/- 0.1 pg/ml and from 1.9 +/- 0.4 to 13.6 +/- 0.4 pg/ml respectively; P0.05). Nicotine decreased IL-2 levels in the colon (from 15.8 +/- 3.0 to 7.9 +/- 1.0 pg/ml; P0.05), having no effect on IL-10 or IL-6 levels. Rats treated with nicotine had lower IL-6 and IL-2 blood levels compared to control rats.Nicotine has different regional effects on small bowel and colonic cytokine mucosal levels, which might explain some of its opposite effects on small bowel and colonic inflammation.

Within the colonic mucosa of rats with portal hypertension and liver cirrhosis, there is an increased generation of inflammatory mediators, such as leukotriene B4 and endothelin-1, and increased generation of nitric oxide. Nitric oxide overproduction may induce tissue injury. This study was undertaken to assess whether the colonic mucosa of rats with portal hypertension and liver disease have increased susceptibility to damage by noxious agents. In this study, acetic acid colitis was induced in rats with portal vein ligation and in control groups, and iodoacetamide colitis was induced in rats with partial portal vein ligation and cirrhosis due to bile duct ligation and in control groups. Rats with acetic acid colitis and those with iodoacetamide-induced colitis were studied 24 and 72 hours, respectively, after induction of colitis. Portal hypertension alone and portal hypertension with cirrhosis were present in the portal vein ligation and bile duct ligation models, respectively. In the rats with acetic acid, colitis lesion area, colonic mucosal myeloperoxidase activity, and prostaglandin E2 generation were not different between the portal vein ligation groups with and without colitis. Nitric oxide activity was higher only in the groups with colitis, irrespective of the presence of portal hypertension. In the group of rats with iodoacetamide colitis, colonic lesion area and colonic mucosal myeloperoxidase activity were similar in all groups with colitis. Colonic mucosal prostaglandin E2 generation was lower in the portal vein ligation and bile duct ligation rats with colitis compared with a control group. We concluded that rats with experimental portal hypertension do not have increased damage when induced by either acetic acid or iodoacetamide.

N-acetylcysteine ameliorates renal microcirculation: Studies in rats.BackgroundN-acetylcysteine (NAC) administration has been shown to ameliorate experimental acute renal failure induced by ischemia-reflow, and was found to prevent radiocontrast nephropathy in high-risk patients. While the protective effect of NAC has been primarily attributed to scavenging oxygen free radicals, improving renal microcirculation also may play a role in the prevention of acute renal failure.MethodsThis study was designed to explore the effect of NAC on renal microcirculation. Blood pressure, total renal blood flow and selective regional cortical and outer medullary blood flow were continuously monitored in anesthetized Sprague Dawley rats with ultrasonic and laser-Doppler probes during the infusion of NAC (60 mg/kg).ResultsIn control intact rats blood pressure and renal microcirculation were unaffected by NAC. By contrast, following renal vasoconstriction induced by the radiocontrast agent iothalamate meglumine, NAC decreased total, cortical and medullary vascular resistance by 7 to 10% (P < 0.05). NAC also reduced renal vascular resistance by 16% when given during angiotensin II infusion (P < 0.05). Altered renal microcirculation, induced by the cyclooxygenase inhibitor indomethacin, by the nitric oxide synthase-inhibitor, Nωnitro-l-arginine (L-NAME), or with their combination was partially restored by NAC. Nevertheless, NAC administration failed to attenuate renal function and morphology in a rat model of acute renal failure with selective outer medullary hypoxic injury, induced by indomethacin, L-NAME and iothalamate.ConclusionsNAC ameliorates renal vasoconstriction, an effect that seems to be mediated by mechanisms other than prostaglandins and nitric oxide. The potential renoprotective outcome of NAC and the role of its vasodilating effect on the pre-constricted renal vasculature should be evaluated further.

Background and Aim Clinical and experimental data suggest an important role for intestinal microflora in the pathogenesis of inflammatory bowel disease, and probiotics have been shown to ameliorate pouchitis. We evaluated the effect of different preparations of probiotic bacteria on experimental colitis in rats. Methods Rats were treated daily intragastrically with two probiotic preparations, VSL#3 or Lactobacillus strain GG (LGG), 7 days before induction of colitis and for another week thereafter. Colitis was induced by intracolonic administration of either dinitrobenzene sulfonic acid (DNBS) or iodoacetamide. Rats were killed 7 days after induction of colitis, the colon isolated, washed, weighed, lesion area measured, and mucosa processed for determination of myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities and prostaglandin E2 (PGE2) generation. Results In rats cotreated with VSL#3 or LGG and iodoacetamide, there was a significant decrease in the lesion area, 98 ± 37 mm2 and 142 ± 43 mm2, respectively, as compared with 342 ± 66 mm2 in the control group. Colonic wet weight significantly decreased to 1.3 ± 0.1 g/10 cm and 1.4 ± 0.1 g/10 cm, respectively, as compared with 1.7 ± 0.1 g/10 cm. There was also a significant decrease in PGE2 generation, MPO, and NOS activities in the VSL#3 and LGG treatment groups. Presence of VSL#3 bacteria in the rat's colon was confirmed by culture and polymerase chain reaction (PCR) amplification. Neither probiotic preparation had an effect on the extent of colonic damage in DNBS-induced colitis. Conclusion Both VSL#3 and LGG significantly ameliorated colitis induced by the sulfhydryl-blocker iodoacetamide, but had no effect on the immune-mediated DNBS-induced colitis. The results suggest a possible role for sulfhydryl compounds in the protective effect of probiotic bacteria, and support their use in patients with inflammatory bowel disease.

It has been suggested that the arteriolar vasodilatation and hyperdynamic circulation observed in rats with partial portal vein ligation (PPVL) is caused by increased splanchnic and systemic delivery of vasodilator substances. The aims of our study were to determine organ-specific generation of prostaglandin E 2 (PGE 2 ) in rats with PPVL during the evolution of portal hypertension. Rats with PPVL and sham-operated (S) rats were studied in the first, third, fourth and 14th postoperative days. They were anesthetized and splenic pulp pressure and blood pressure were measured. Spleen, colon and lungs were removed and the splenic, pulmonary and mucosal colonic PGE 2 were determined. All PPVL rats developed sequential hemodynamic changes compatible with evolving portal hypertension. Splenic pulp pressure was higher in PPVL rats compared with S rats during all days of the study. Within the group of PPVL the splenic pulp pressure was higher in the first postoperative day and decreased in the ensuing days. No changes in splenic and colonic PGE 2 generation were noted during the study period. Pulmonary PGE 2 generation increased significantly in the first postoperative day in PPVL rats compared with S rats. However, similar increase was observed on the third postoperative day in S rats. PGE 2 probably has no role in splanchnic hemodynamic changes during evolution of portal hypertension. Pulmonary PGE 2 generation may increase as a response to increased portal pressure, or to abdominal surgery.

Background & Aims: Impaired mucosal barrier, cytokine imbalance, and dysregulated CD4 + T cells play important roles in the pathogenesis of experimental colitis and human inflammatory bowel disease. Immunostimulatory DNA sequences (ISS-DNA) and their synthetic oligonucleotide analogs (ISS-ODNs) are derived from bacterial DNA, are potent activators of innate immunity at systemic and mucosal sites, and can rescue cells from death inflicted by different agents. We hypothesized that these combined effects of ISS-DNA could inhibit the damage to the colonic mucosa in chemically induced colitis and thereby limit subsequent intestinal inflammation. Methods: The protective and the anti-inflammatory effect of ISS-ODN administration were assessed in dextran sodium sulfate–induced colitis and in 2 models of hapten-induced colitis in Balb/c mice. Similarly, these effects of ISS-ODN were assessed in spontaneous colitis occurring in IL-10 knockout mice. Results: In all models of experimental and spontaneous colitis examined, ISS-ODN administration ameliorated clinical, biochemical, and histologic scores of colonic inflammation. ISS-ODN administration inhibited the induction of colonic proinflammatory cytokines and chemokines and suppressed the induction of colonic matrix metalloproteinases in both dextran sodium sulfate– and hapten–induced colitis. Conclusions: As the colon is continuously exposed to bacterial DNA, these findings suggest a physiologic, anti-inflammatory role for immunostimulatory DNA in the GI tract. Immunostimulatory DNA deserves further evaluation for the treatment of human inflammatory bowel disease. GASTROENTEROLOGY 2002;122:1428-1441

Recent studies have shown that maternal hyperinsulinaemia is a risk factor for the development of hypertension in pregnancy. Experimentally, pregnant rats with chronic exogenously induced hyperinsulinaemia (P-INS rats) have increased blood pressure at the end of gestation. This is associated with a blunted elevation of the excretion of the urinary metabolites of nitrate (UNOx). In the present study, we aimed to evaluate the mechanism(s) of the increase in blood pressure in this model. Four groups were studied: normal pregnant rats (P rats), P-INS rats, P-INS rats treated with l-arginine (2 g/l in the drinking water) (l-ARG rats) and hyperinsulinaemic virgin rats (V-INS rats). Systolic blood pressure (SBP), UNOx excretion (on ingestion of a controlled low-nitrate diet), urine noradrenaline (norepinephrine) and plasma endothelin levels were evaluated. Rats were killed on day 22 of pregnancy. Five P-INS rats were not killed at this time, in order to measure SBP 30 and 60 days after delivery. Fetal number and fetal body weight were evaluated. At the end of pregnancy, a 10±3% increase in SBP was found in P-INS rats, contrasting with a fall of -15±4% in P rats (P < 0.01). In the l-ARG group at the end of pregnancy, SBP values had fallen by -14±2%, to values comparable with those of P rats. The increase in UNOx excretion was 175±38% in P rats, 106±12% in l-ARG rats and 41±8% in P-INS rats (P < 0.01 compared with P and l-ARG groups). No differences were found in the urinary excretion of noradrenaline or in the plasma levels of endothelin-1 between the pregnant groups. Fetal number was similar in all groups, but fetal body weight was lower for P-INS rats compared with P and l-ARG rats. Thus the blood pressure response to l-arginine strongly suggests that a decrease in NO availability may be the main pathogenic mechanism involved in the development of hypertension in this model.

Objective In active ulcerative colitis, colonic nitric oxide (NO) generation is enhanced and probably has an important role in its pathogenesis. We tested the reliability of an NO electrode in monitoring colonic NO levels in ulcerative colitis patients and control subjects and its possible usage as a marker of disease activity. Methods Colonie NO level was determined by the NO detection system model NO-501 (InterMedical, Nagoya, Japan). The working electrode was inserted into a 7-mm diameter polyvinyl tube and introduced at a distance 6 cm from the anus. In each subject sigmoidoscopy was performed and mucosal biopsies were obtained. NO synthase (NOS) activity was determined by monitoring the conversion of 3 H-arginine to citrulline. Results Colonie NO level is significantly increased in patients with active ulcerative or Crohn's colitismore than 2-fold higher than in control subjects. There was good correlation between colonic NO level and NOS activity and the clinical and endoscopic indices of disease activity. Conclusion: Direct determination of colonic NO level is convenient, and reliable, and may help to monitor disease activity in ulcerative colitis.

Substance P (SP), vasoactive intestinal polypeptide (VIP), and somatostatin content in rectal mucosa were determined by radioimmunoassay (RIA) in 38 diabetic patients (12 with normal bowel function, 13 with diabetic diarrhea, and 13 with constipation) and in 10 nondiabetic controls with normal bowel function. SP content (picograms per milligram) in the rectal mucosa of diabetics with normal bowel function was significantly higher than that of nondiabetic controls (P.05). SP content in the rectal mucosa of diabetics with diabetic diarrhea and constipation was significantly lower than in diabetics with normal bowel habits and nondiabetic controls (P.05). No differences were found in the rectal mucosa content of VIP and somatostatin between the different groups of diabetics and controls. Diabetic diarrhea is a condition with an intermittent nature and frequently alternates with constipation. Our findings showing low levels of rectal mucosa SP in both conditions suggest a possible common role of SP in the pathogenesis of diabetic diarrhea and constipation.