Steroid-sparing properties of sargramostim in patients with corticosteroid-dependent Crohn's disease: a randomised, double-blind, placebo-controlled, phase 2 study.

OBJECTIVE: Although treatment with corticosteroids induces remission in Crohn's disease, prolonged exposure to corticosteroids is undesirable. This randomised clinical trial evaluated the efficacy of recombinant human granulocyte-macrophage colony-stimulating factor (sargramostim), an activator of innate immunity, in corticosteroid-dependent patients with Crohn's disease. DESIGN: Patients were randomised in a 2:1 ratio, to sargramostim 6 microg/kg subcutaneously once daily or placebo for up to 22 weeks. The study consisted of (1) an adjunctive phase (weeks 1-4) in which patients received study drug plus corticosteroid therapy; (2) a forced corticosteroid tapering phase (weeks 4-14); and (3) an observation phase (4 weeks) in which patients received study drug plus prednisone < or =7.5 mg. The primary endpoint was corticosteroid-free remission (Crohn's Disease Activity Index (CDAI) < or =150) 4 weeks after corticosteroid elimination. Secondary endpoints were corticosteroid-free response (CDAI decreased by > or =100) and induction of remission in patients who reduced the dose of corticosteroid to 2.5-7.5 mg. RESULTS: Eighty-seven patients were randomised to sargramostim and 42 to placebo. Significantly more sargramostim-treated patients than placebo patients achieved corticosteroid-free remission (18.6% vs 4.9%; p = 0.03). Similar differences were seen for corticosteroid-free response and in patients who tapered corticosteroids to 2.5-7.5 mg/day. Sargramostim treatment was also associated with significant improvements in health-related quality of life. Patients who received sargramostim were more likely to experience musculoskeletal pain, injection site reactions and dyspnoea. CONCLUSIONS: Sargramostim was more effective than placebo for inducing corticosteroid-free remission in patients with Crohn's disease with corticosteroid dependence. Sargramostim may provide significant benefit in this population if these findings are confirmed.

Repifermin (keratinocyte growth factor-2) for the treatment of active ulcerative colitis: a randomized, double-blind, placebo-controlled, dose-escalation trial.

BACKGROUND: Repifermin (keratinocyte growth factor-2) has been shown to reduce inflammation in animal models of colitis. AIM: To evaluate repifermin for the treatment of active ulcerative colitis. METHODS: Eighty-eight patients with active ulcerative colitis were enrolled in a 6-week, double-blind trial. Patients were randomized to receive treatment for five consecutive days with intravenous repifermin at a dose of 1, 5, 10, 25 or 50 microg/kg, or placebo. The primary objective of the study was to evaluate the safety of repifermin. The primary efficacy outcome was clinical remission at week 4, defined as a score of zero on the endoscopic appearance and stool blood components of the Mayo score and a score of zero or unity on the stool frequency and physician's global assessment components. RESULTS: At week 4, the rates of clinical remission in the 1, 5, 10, 25 and 50 microg/kg repifermin groups were 19%, 9%, 0%, 0% and 0%, respectively, and 11% for the placebo group (P = 0.32 for repifermin vs. placebo). The frequencies of commonly occurring adverse events and severe adverse events were similar in both groups. CONCLUSIONS: Intravenous repifermin at a dose of 1-50 microg/kg was very well tolerated, but there was no evidence that repifermin was effective for the treatment of active ulcerative colitis at these doses. An additional study to determine the efficacy of repifermin at doses of > 50 microg/kg or for a longer treatment duration may be warranted, as the maximally tolerated dose was not reached in the present study.

The role of leaf nitrogen content in determining turtlegrass (Thalassia testudinum) grazing by a generalized herbivore in the northeastern Gulf of Mexico.

In shallow marine environments the variability in grazing on seagrasses has been hypothesized to be controlled, in part, by the nutritive quality (i.e., nitrogen content) of their leaves. The few existing studies of the relationship between leaf nitrogen content and seagrass grazing have all found a positive relationship between leaf nitrogen content and preference by selective vertebrate grazers (i.e., the bucktooth parrotfish, green sea turtles, and dugongs). However, most marine herbivores (both vertebrate and invertebrate) are thought to be extreme generalists with broad diets of variable nutritive quality (e.g., detritus, living plants, and animals), suggesting the currently held view on the role leaf nutrient content in explaining the variability of seagrass grazing is an oversimplification.In this study, we evaluated how leaf nitrogen content influenced grazing on turtlegrass by a generalist invertebrate herbivore (the pink sea urchin Lytechinus variegatus) in the northeastern Gulf of Mexico. Using a short-term laboratory test and a longer-term field experiment, we tested the hypothesis that leaf nitrogen content controls sea-urchin grazing on seagrass leaves. We hypothesized that if poor nutritive value of seagrasses is responsible for reduced rates of feeding, then increasing leaf nitrogen concentrations should lead to increased rates of seagrass consumption by sea urchins.In the field experiment, we significantly enriched seagrass leaf nitrogen concentrations (some 10-20% depending on month) in experimental plots with a commercial fertilizer and we manipulated grazing intensity by enclosing adult sea urchins at densities that bracketed the range of average densities observed in the region (i.e., 0, 10 and 20 individuals/m(2)). Comparisons of changes in aboveground seagrass production and biomass showed no evidence that sea urchins grazed significantly more in treatments where leaf nitrogen was enriched. Because the statistical power of our test to detect such differences was low and aboveground seagrass production varied significantly among treatments, we also used a mass balance equation to estimate sea urchin consumption of nitrogen-enriched and unenriched leaves. This showed that sea urchins compensated for low nitrogen levels in our unenriched treatments by eating more leaves than in treatments where leaf nitrogen was elevated. Using a laboratory test, we also found that sea urchins ate less nitrogen-enriched seagrass than unenriched seagrass. In combination, these results show that, in contrast to findings reported for vertebrate herbivores, sea urchins feed at higher rates when offered seagrass leaves of lower leaf nitrogen content, and that low levels of leaf nitrogen are not always an effective defense against herbivores.

Mucosal injury and inflammatory cells in response to brief ischaemia and reperfusion in the equine large colon.

REASON FOR PERFORMING STUDY: Intestinal ischaemia and reperfusion (I/R) can activate inflammatory cells in the equine colon, although effects on different types of inflammatory cells have received little attention. OBJECTIVES: To assess early mucosal injury, the reaction of mucosal neutrophils, eosinophils, mast cells and macrophages, and cyclooxygenase (COX)-1 and -2 expression in response to I/R in the equine large colon. METHODS: Large colon ischaemia was induced for 1 h (1hI) followed by 4 h of reperfusion in 6 horses, and mucosal biopsies were sampled before and after ischaemia, and after 1, 2 and 4 h of reperfusion. Semithin sections (500 nm) of epon-embedded biopsies were stained with toluidine blue for histomorphometric evaluation. The number and distribution of mucosal macrophages (CD163), neutrophils (calprotectin), eosinophils (LUNA) and mast cells (toluidine blue) were determined, and mucosal COX-1 and -2 expression was identified. RESULTS: Ischaemia caused epithelial cell and nuclear swelling (mean ± s.e. nuclear width; control: 2.7 ± 0.2 µm vs. 1hI: 4.2 ± 0.2 µm; P<0.01), subepithelial oedema (control: 0.2 ± 0.1 µm vs. 1hI: 3.2 ± 0.2 µm; P<0.01) and increased epithelial apoptosis (control: 14.3 ± 4.1 apoptotic cells/mm mucosa vs. 1hI: 60.4 ± 14.0 apoptotic cells/mm mucosa; P<0.01). COX-2 expression (P<0.01) was evident after ischaemia. Reperfusion caused paracellular fluid accumulation (control: 0.9 ± 0.1 µm vs. 1hI: 0.6 ± 0.6 µm vs. 1hI + 4hR: 1.6 ± 0.2 µm; P<0.05). Epithelial repair started at 1 h of reperfusion (P<0.001), followed by migration of neutrophils into the mucosa after 2 h (control: 72.3 ± 18.4 cells/mm(2) mucosa vs. 1hI + 2hR: 1149.9 ± 220.6 cells/mm(2) mucosa; P<0.01). Mucosal eosinophils, mast cells and macrophages did not increase in numbers but were activated. CONCLUSIONS: Epithelial injury and COX-2 expression caused by short-term hypoxia were followed by intense inflammation associated with epithelial repair during reperfusion. POTENTIAL RELEVANCE: Equine colonic mucosa subjected to a brief period of ischaemia can repair during reperfusion, despite increased mucosal inflammation.

Daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), for the treatment of moderately to severely active ulcerative colitis: a randomised, double blind, placebo controlled, dose ranging trial.

BACKGROUND: An uncontrolled pilot study demonstrated that daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), might be effective for the treatment of active ulcerative colitis. METHODS: A randomised, double blind, placebo controlled trial was conducted to evaluate the efficacy of daclizumab induction therapy in patients with active ulcerative colitis. A total of 159 patients with moderate ulcerative colitis were randomised to receive induction therapy with daclizumab 1 mg/kg intravenously at weeks 0 and 4, or 2 mg/kg intravenously at weeks 0, 2, 4, and 6, or placebo. The primary end point was induction of remission at week 8. Remission was defined as a Mayo score of 0 on both endoscopy and rectal bleeding components and a score of 0 or 1 on stool frequency and physician's global assessment components. Response was defined as a decrease from baseline in the Mayo score of at least 3 points. RESULTS: Two per cent of patients receiving daclizumab 1 mg/kg (p = 0.11 v placebo) and 7% of patients receiving 2 mg/kg (p = 0.73) were in remission at week 8, compared with 10% of those who received placebo. Response occurred at week 8 in 25% of patients receiving daclizumab 1 mg/kg (p = 0.04) and in 33% of patients receiving 2 mg/kg (p = 0.30) versus 44% of those receiving placebo. Daclizumab was well tolerated. The most frequently reported adverse events in daclizumab treated patients compared with placebo treated patients were nasopharyngitis (14.6%) and pyrexia (10.7%). CONCLUSION: Patients with moderate ulcerative colitis who are treated with daclizumab are not more likely to be in remission or response at eight weeks than patients treated with placebo.

Mesalamine induces manganese superoxide dismutase in rat intestinal epithelial cell lines and in vivo.

Mesalamine (5-ASA) is effective in the treatment of inflammatory bowel diseases. However, the mechanisms of action of 5-ASA remain unclear. IEC-6 and IRD-98, nontransformed rat small intestinal epithelial cell lines, were used to examine the effect of 5-ASA on the expression of manganese superoxide dismutase (MnSOD). Rats were given 5-ASA enemas to determine the effect on colonic MnSOD expression. Treatment with 5-ASA at 0.02 or 2 mg/ml induced MnSOD mRNA levels 2.67-fold or 5.66-fold, respectively. Inhibition of 5-lipoxygenase activating protein with MK-886 or cyclooxygenase with indomethacin did not influence the level of MnSOD mRNA. Nuclear run-on experiments demonstrated an increase in de novo transcription following treatment with 5-ASA. MnSOD protein levels were induced 2-fold at 24 h and 4.23-fold at 48 h following treatment with 1 mg/ml 5-ASA. 5-ASA increased MnSOD 1.7-fold in vivo. Pretreatment with 5-ASA significantly protected IRD-98 cells from tumor necrosis factor-alpha cytotoxicity. This is the first example of transcriptional gene regulation by 5-ASA. The induction of MnSOD by 5-ASA may contribute to the therapeutic mechanism of 5-ASA.

Glucocorticoids repress basal and stimulated manganese superoxide dismutase levels in rat intestinal epithelial cells.

BACKGROUND/AIMS: Elevated expression of manganese superoxide dismutase (SOD) has been shown to mitigate the toxic effects of cytokine and free radical production. Because of the multiple anti-inflammatory effects of glucocorticoids, we hypothesized that the MnSOD gene may be under glucocorticoid regulation. METHODS: IEC-6 cells were treated with 0.5 mumol/L dexamethasone (DEX), 50 mumol/L cycloheximide, 4 mumol/L actinomycin D, 0.5 microgram/mL lipopolysaccharide, or 10 ng/mL tumor necrosis factor alpha. MnSOD messenger RNA was evaluated by Northern analysis. MnSOD protein levels were evaluated by Western analysis. RESULTS: IEC-6 treatment with DEX reduced MnSOD messenger RNA by 77% at 8 hours. Treatment with DEX plus cycloheximide or actinomycin showed a requirement for protein synthesis and implicated transcriptional regulation of MnSOD messenger RNA by DEX. DEX cotreatment inhibited the induction of MnSOD messenger RNA by lipopolysaccharide or tumor necrosis factor alpha. Twenty-four hours of DEX exposure reduced basal MnSOD protein levels by 43%, whereas 24-hour treatment with lipopolysaccharide or tumor necrosis factor alpha resulted in a 3.5-fold and 2.4-fold increase in MnSOD protein levels, respectively, that was blocked by DEX. CONCLUSIONS: DEX represses both basal and stimulated MnSOD messenger RNA and protein levels. Repression of MnSOD seems detrimental; however, this may not be the case because DEX also inhibits oxygen free radical production as well as cytokine release and action.

Acute-phase induction of manganese superoxide dismutase in intestinal epithelial cell lines.

Cellular protection from immune-generated oxygen free radicals is initiated by the reduction of oxygen radicals by manganese superoxide dismutase (MnSOD) and copper/zinc superoxide dismutase (Cu/ZnSOD). Using rat adult (IEC-6) and fetal (IRD-98) intestinal epithelial cell lines, factors involved in the regulation of the SODs at the messenger RNA (mRNA) level were examined. Exposure of IEC-6 and IRD-98 to Escherichia coli lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha) results in a marked increase in MnSOD mRNA as early as at 1 hour. Cotreatment of cells exposed to LPS or TNF-alpha with actinomycin D or cycloheximide showed that de novo transcription but not protein synthesis is required for the LPS- and TNF-alpha-dependent induction in MnSOD mRNA. Treatment with interleukin 1 beta results in a 12-fold increase in MnSOD mRNA, but no change was observed with interleukin 6 or interferon alpha. No change was observed in the level of Cu/ZnSOD mRNA under any condition tested. The results indicate that MnSOD functions as a cytokine-regulated acute phase protein involved in cellular protection from free radical-mediated damage.

Osteopenia and osteoporosis in gastrointestinal diseases: diagnosis and treatment.

An increased awareness of the higher incidence of osteopenia and osteoporosis associated with a number of gastrointestinal disease states has occurred over the last few years. High rates of bone loss have been reported in luminal diseases such as inflammatory bowel disease and celiac disease as well as in cholestatic liver diseases and in the post-liver transplant setting. The post-gastrectomy state and chronic pancreatitis are also associated with decreased bone density. Publications over the last year have provided a better understanding of the true incidence of osteoporosis and fracture risk in these gastrointestinal disease states. Dual-energy x-ray absorptiometry remains the diagnostic procedure of choice. Biochemical markers of bone resorption have a role in identifying those patients with ongoing bone loss and monitoring their response to therapy. Identification of patients at risk and initiation of measures to prevent bone loss form the optimal therapeutic strategy. This article reviews advancements in the understanding of the development and activation of osteoblasts and osteoclasts. It also reviews the recent data concerning the diagnosis and treatment of bone loss associated with various gastrointestinal disease states.

Prevention and treatment of osteoporosis in patients with inflammatory bowel disease.

Osteopenia or osteoporosis is common in patients with inflammatory bowel disease. The use of corticosteroids contributes to the decline in bone loss; however, osteoporosis may develop in patients with inflammatory bowel disease independent of corticosteroid use. Risk factors for the development of low bone mass in patients with inflammatory bowel disease include the general risk factors for osteoporosis as well as additional factors such as the presence of chronic inflammation, use of corticosteroids and other pharmaceuticals, and nutritional deficiencies as the result of small bowel disease or small bowel resections. Despite the high prevalence, few patients are entered into prophylactic regimens to prevent corticosteroid-induced bone loss. The American College of Rheumatology has recently published recommendations for the prevention and treatment of corticosteroid-induced osteoporosis. In this article, we highlight the special risks for osteoporosis in patients with IBD and adapt the recommendations for prevention and treatment of osteoporosis to this clinical setting.

Clearance of furosemide by the gastrointestinal tract.

Approximately 45% of i.v. administered furosemide is eliminated by nonrenal clearance mechanisms. Indirect evidence suggests this might represent intestinal secretion. Therefore, we examined whether the intestinal tract serves as a drug-eliminating organ in man. Intestinal perfusion studies were performed in six healthy volunteers during i.v. furosemide administration (mean serum concentration, 3.74 +/- 0.64 microgram/ml). Subjects were intubated with a multilumen tube which allowed examination of transmucosal water, solute and furosemide movement at separate levels of the gastrointestinal tract. A poorly absorbable electrolyte-mannitol solution was infused in the jejunum (15 ml/min), with polyethylene glycol as a nonabsorbable marker. Furosemide elimination occurred at an equally low rate in all areas of the intestinal tract. Furosemide clearance for the total gastrointestinal tract was 2.1 +/- 0.4 ml/min (mean +/- S.E.M.) compared to a renal clearance of 93.1 +/- 4.6 ml/min. Thus, gastrointestinal elimination amounted to only 2% of renal elimination. The luminal concentration of furosemide in the intestinal tract did not exceed a mean of 0.5 microgram/ml. When the experiments were repeated after administration of probenecid, gut clearance was unchanged but renal clearance was reduced by 70%. In the ileum, furosemide enhanced bicarbonate secretion and induced chloride absorption. We conclude that the intestinal tract contributes only minimally to furosemide elimination in man. From concentration gradients between lumen and plasma and from the fact that probenecid had no effect on elimination rate, it appears likely that active secretion into the intestinal lumen does not occur and that all furosemide appearance in the gut results from passive diffusion.

Colitis and interleukin 1beta up-regulate inducible nitric oxide synthase and superoxide dismutase in rat myenteric neurons.

BACKGROUND & AIMS: Manganese superoxide dismutase (MnSOD) is rapidly induced in myenteric plexus neurons (MPNs) in acute colitis and may protect cells from nitric oxide toxicity. Inducible nitric oxide synthase (iNOS) regulation was examined in acute colitis, and MnSOD and iNOS were examined in primary cultures of MPNs. METHODS: Acute colitis in rats was induced with 5% acetic acid. iNOS messenger RNA (mRNA) was analyzed by Northern analysis, and reduced nicotinamide adenine dinucleotide phosphate diaphorase was used to identify potential NO synthase activity. MnSOD and iNOS mRNA levels were evaluated in cultured MPNs after treatment with tumor necrosis factor alpha, interleukin (IL) 1beta, or IL-6. iNOS and MnSOD protein expression in control and IL-1beta-treated neurons was evaluated by immunofluorescence microscopy. RESULTS: iNOS mRNA was detected in the mucosal and muscularis layers after the initiation of colitis. Reduced nicotinamide adenine dinucleotide phosphate diaphorase localized NO synthase activity to MPNs in controls and in epithelial cells and MPNs in the inflamed colon. In MPN cultures, tumor necrosis factor alpha and IL-1beta treatment resulted in induction of MnSOD, but only IL-1beta induced iNOS. Immunolocalization confirmed that the neurons were the primary source of iNOS and MnSOD. CONCLUSIONS: Induction of MnSOD and iNOS are coordinated and may limit NO cytotoxicity. The function of iNOS in gut neurons remains to be delineated.

Regulation of superoxide dismutase in primary cultures of rat colonic smooth muscle cells.

We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuronal, and smooth muscle cells (SMC) after acetic acid-induced colitis. To examine the regulation of MnSOD in the SMC more specifically, primary cultures of colonic SMC were developed by enzymatic digestion of the circular muscle layer from an adult rat. SMC were treated for 2-72 h with 0.5 microgram/ml Escherichia coli endotoxin [lipopolysaccharide (LPS)], 10 ng/ml tumor necrosis factor (TNF)-alpha, or 2 ng/ml interleukin-1 beta (IL-1 beta). Cotreatments were performed with IL-1 beta and 4 microM actinomycin or 50 microM cycloheximide. Northern analysis demonstrated 23-fold, 8-fold, and 6-fold inductions of MnSOD mRNA by IL-1 beta, LPS, and TNF-alpha, respectively. Induction of MnSOD by IL-1 beta was eliminated by actinomycin but not by cycloheximide, implicating a requirement for de novo transcription. Western analysis resulted in a 23.7-fold induction of MnSOD protein after 48-h treatment with IL-1 beta. Induction of MnSOD by IL-1 beta and other inflammatory mediators may serve as a protective mechanism to reduce oxygen free radical- and nitric oxide-mediated cell damage during inflammation.

Induction and immunolocalization of manganese superoxide dismutase in acute acetic acid-induced colitis in the rat.

BACKGROUND & AIMS: Oxygen radicals and reactive oxygen species play an important role in inflammatory episodes in the bowel. Nonetheless, little is known about the regulation of colonic superoxide dismutases and key antioxidant enzymes with cytoprotective and radical detoxifying properties. The aim of this study was to examine the regulation of manganese superoxide dismutase (SOD) in acute acetic acid-induced colitis. METHODS: Colitis was induced in adult rats by the rectal administration of 5% acetic acid. Total RNA and protein were isolated from the inflamed colon from 1 to 24 hours after the induction of colitis. MnSOD messenger RNA and protein levels were evaluated by Northern and Western analyses, respectively. MnSOD protein was localized in cross sections of the colon by immunocytochemistry. RESULTS: MnSOD messenger RNA levels showed a rapid 14-96-fold induction in response to acetic acid administration. Western analysis showed a 22-49-fold induction in MnSOD protein levels. Immunocytochemistry showed induction of MnSOD protein, specifically in smooth muscle cells, epithelial cells at the base of the glands, and myenteric plexus neurons. CONCLUSIONS: MnSOD messenger RNA and protein levels are rapidly induced following the inflammatory insult, implicating a role for MnSOD in the acute phase of colonic inflammation. We suggest that induction of MnSOD in specific cell types may have a cytoprotective function.