Shc is a substrate of the rat intestinal epidermal growth factor receptor tyrosine kinase

Epidermal growth factor (EGF) has been shown to induce intestinal proliferation and maturation; however, little information is available regarding substrates of the intestinal EGF receptor tyrosine kinase. The purpose of this study was to determine if src homologous collagen-like protein (Shc) was an in vivo substrate of the intestinal EGF receptor. Ten-day-old rats were treated with EGF or were breast-fed. In some experiments, IEC-6 cells were treated with EGF. Intestinal tissue and cell fractions were studied by immunodetection to compare the tyrosine phosphorylation state and the subcellular localization of intestinal proteins. The total tyrosine phosphorylation state of intestinal proteins was increased threefold by EGF. Tyrosine phosphorylation of the EGF receptor and Shc were rapidly increased by EGF. The association of Grb2 with Shc increased fourfold and fivefold. Plasma membrane translocation of Shc and associated phosphotyrosyl proteins was increased within 30 seconds of EGF treatment. Shc is a substrate of the intestinal EGF receptor in vivo. EGF-induced association of Shc with the adapter protein Grb2 may have implications for activation of the p21^ras signaling pathway in the intestine. The EGF-induced membrane association of Shc with two other phosphotyrosyl proteins suggests involvement of Shc in additional aspects of EGF-receptor signaling in the intestine.     

Antioxidant activity of silybin in vivo during long-term iron overload in rats

Hepatic iron toxicity may be mediated by free radical species and lipid peroxidation of biological membranes. The antioxidant property of silybin, a main constituent of natural flavonoids, was investigated in vivo during experimental iron overload. Rats were fed a 2.5% carbonyl-iron diet and 100 mg · kg body wt⁻¹ · day⁻¹ silybin for 4 months and were assayed for accumulation of hepatic lipid peroxidation by-products by immunocytochemistry, mitochondrial energy-dependent functions, and mitochondrial malondialdehyde content. Iron overload caused a dramatic accumulation of malondialdehyde-protein adducts into iron-filled periportal hepatocytes that was decreased appreciably by silybin treatment. The same beneficial effect of silybin was found on the iron-induced accumulation of malondialdehyde in mitochondria. As to the liver functional efficiency, mitochondrial energy wasting and tissue adenosine triphosphate depletion induced by iron overload were successfully counteracted by silybin. Oral administration of silybin protects against iron-induced hepatic toxicity in vivo. This effect seems to be caused by the prominent antioxidant activity of this compound.     

Cost-effectiveness model for colon cancer screening

The relative efficacy and effectiveness of different colon screening programs has not been assessed. The purpose of this analysis was to provide a model for comparing several colon screening programs and to determine the key variables that impact program effectiveness. Five screening programs were compared: annual fecal occult blood test (FOBT) alone, flexible sigmoidoscopy, flexible sigmoidoscopy and FOBT combined, one-time colonoscopy, and air-contrast barium enema. Key variables were adjusted for sensitivity analyses. Cost-effectiveness was defined as the cost per cancer death prevented. FOBT alone prevents fewer cancer deaths than the other programs. The addition of flexible sigmoidoscopy to the FOBT increases the rate of cancer prevention. One-time colonoscopy has the greatest impact on colorectal cancer mortality, largely because of assumptions that cancer would be prevented in most patients who undergo polypectomy. FOBT alone is the most cost-effective of the programs, but the cost is sensitive to several key variables. The model shows key variables that impact the cost-effectiveness of colon screening programs. Compliance is an important determinant of effectiveness of all of the screening programs. Future study should be focused on methods of patient education that improve patient compliance with screening.     

Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B

To test whether the dominant cytoplasmic expression of hepatitis B core antigen (HBcAg) in active chronic hepatitis B is secondary to liver damage and regeneration, the relationship between subcellular localization of HBcAg, liver inflammatory activity, and hepatocyte regeneration in chronic hepatitis B was studied. Correlation of the clinical and laboratory data with the topographical distribution of HBcAg was studied in 30 patients. The subcellular localization of HBcAg in relation to hepatocyte cell cycles was studied by double immunostaining of HBcAg and proliferating cell nuclear antigen. Patients with predominant cytoplasmic HBcAg had significantly higher levels of biochemical and histological activities and proliferating cell nuclear antigen expression than patients with predominant nuclear HBcAg. The levels of proliferating cell nuclear antigen expression correlated positively with biochemical and histological activities and degrees of cytoplasmic HBcAg expression but negatively with degrees of nuclear HBcAg expression. Proliferating cell nuclear antigen expression was shown in 49% of hepatocytes with cytoplasmic HBcAg but in only 2% of hepatocytes with nuclear HBcAg. These findings suggested that, following liver damage, the regeneration of surviving hepatocytes might cause the shift of intracellular HBcAg from nucleus to cytoplasm. As a result, the extent of nuclear HBcAg expression reduces with concomitant increase in cytoplasmic HBcAg expression.     

The site-specific delivery of ursodeoxycholic acid to the rat colon by sulfate conjugation

Because ursodeoxycholate has been shown to act as a tumor-suppressive agent in the colon, the absorption and metabolism of its sulfate conjugates were examined in rats to show that sulfation would facilitate the site-specific delivery of ursodeoxycholate to the colon. Bile acids were measured in intestinal contents, feces, urine, plasma, and liver tissue after oral administration of ursodeoxycholate and its C-3, C-7, and C-3,7 sulfate derivatives. Ursodeoxycholate was found in the jejunum after administration of all bile acids, but the mass was greatest for ursodeoxycholic acid administration. In the colon, lithocholic acid, normally found in negligible amounts, became the major bile acid after ursodeoxycholate administration. In contrast, reductions in mass and proportions of lithocholate and deoxycholate occurred after administering the C-7 sulfates. The fecal lithocholate/deoxycholate ratio, a risk marker for colon cancer, increased markedly after administration of ursodeoxycholate and its C-3 sulfate, but did not change after administering the C-7 sulfates. Unlike ursodeoxycholate or its C-3 sulfate, which increased liver concentrations of lithocholate and ursodeoxycholate, the C-7 sulfates had the opposite effect, which was consistent with poor absorption. Sulfation of ursodeoxycholate, specifically at the C-7 position, protects the molecule from bacterial degradation and inhibits its intestinal absorption, thereby facilitating delivery to the colon.     

An intravenous loading dose of azathioprine decreases the time to response in patients with Crohn's disease

Azathioprine, an effective therapy for Crohn's disease, is limited by a prolonged time to response. The aim of this study was to determine the safety and utility of a loading dose of azathioprine to decrease the time to response in patients with Crohn's disease. Twelve patients were studied: 6 with 13 fistulae and 6 with inflammatory disease. All patients received an intravenous infusion of azathioprine (50 mg/h for 36 hours). Response was determined by physical and radiographic examination for fistulae and by the Crohn's Disease Activity Index for inflammatory disease. Erythrocyte concentrations of azathioprine metabolites were measured by chromatography. Seven of 13 fistulae closed by week 4, and three had a temporary decrease in drainage. One fistula improved at week 16. Two fistulae failed to improve. Four of 6 patients with inflammatory disease achieved remission, and 1 improved temporarily. Improvement was rapid (≤4 weeks). Peak concentrations of azathioprine metabolites occurred within 3 days. Clinical response did not correlate with azathioprine metabolite concentrations at the azathioprine dose studied. No adverse events occurred. An 1800-mg intravenous loading dose of azathioprine is safe and may decrease the time to response to ≤4 weeks in patients with Crohn's disease. Correlation between clinical response and azathioprine metabolite concentrations at larger azathioprine doses should be determined.     

The effects of and interactions between fermentable dietary fiber and lipid in germfree and conventional mice

Dietary fiber can stimulate intestinal epithelial cell proliferation. The aim of this study was to resolve the different roles of fermentation and intraluminal viscosity on this trophic action and to investigate reported interactions between fiber and dietary fat. Conventional and germfree mice were fed guar gum in combination with low- or high-lipid diets for 2 weeks, and crypt cell production rates were determined. Guar gum significantly stimulated proliferation in the small intestine, especially when combined with fat. Lipid itself also stimulated proliferation in the small intestine and had a direct trophic effect in the cecum and colon of the germfree mice. Fiber markedly stimulated proliferation in the cecum and colon but only in the conventional group. Interactions between lipid and bacteria and between guar gum and bacteria were also observed in the small intestine. Guar gum has a trophic effect in the small bowel, probably related to viscosity, in addition to its fermentation-related actions in the colon. Positive interaction with lipid may be associated with delayed absorption. Lipid also has its own direct actions on small bowel mucosal proliferation, which are attenuated by the presence of bacteria.     

Role of hepatocytes in direct clearance of lipopolysaccharide in rats

The liver is the clearance organ for lipopolysaccharide (LPS). The aim of this study was to investigate the biliary excretion of LPS using fluorescein isothiocyanate (FITC)-labeled LPS. After FITC-LPS was injected intravenously into rats, the cellular localization of fluorescence in the liver was examined and the biliary excretion of fluorescence was measured. The effects of gadolinium chloride, a blocker of Kupffer cells, and colchicine, an inhibitor of microtubules, on the biliary excretion of fluorescence was investigated, and bile was analyzed using high-performance liquid chromatography. Laser scanning confocal microscopy showed that fluorescence was taken up by hepatocytes 5 minutes after injection of FITC-LPS into the portal vein. When FITC-LPS was injected into the portal vein, fluorescence was rapidly secreted into bile, peaking at 20 minutes, and 25.1% of the injected dose appeared in bile within 60 minutes. When the same dose of FITC-LPS was injected into the tail vein, 15.8% appeared in bile within 60 minutes. Chromatography showed that FITC-LPS was excreted into bile in an unchanged form over a period of 20 minutes after injection. Colchicine significantly reduced the biliary excretion of fluorescence, but gadolinium chloride had no effect. LPS was directly and effectively processed by hepatocytes and secreted into the bile canalicular system via a microtubule-dependent vesicular pathway.     

A 7-year experience of severe acetaminophen-induced hepatotoxicity (1987–1993)

Five hundred sixty patients admitted between January 1, 1987, and December 31, 1993, with severe acetaminophen-induced hepatotoxicity were studied. The aim of this study was to identify why severe acetaminophen-induced hepatotoxicity still occurs and to determine how known risk factors and advances in management have affected the pattern of illness and outcome. This was a retrospective study of the etiologic factors and the clinical course of all acetaminophen-related admissions. The number of admissions increased from 58 in 1987 to 123 in 1993. During the corresponding period, overall survival improved from just <50% to 78%. The percentage of admissions treated with N-acetylcysteine increased from 40% in 1987 to 83% in 1993. The frequency with which grade III or IV encephalopathy developed decreased from 62% in 1987 to 40% in 1993, and the percentage of these patients who developed cerebral edema decreased from 61% to 45% during the same period. There was an increase in both the number of patients transplanted and the survival of those managed medically. Severe acetaminophen-induced hepatotoxicity remains a serious condition, but the increasing use of N-acetylcysteine, advances in medical management, and the increasing availability of transplantation have resulted in a significant improvement in survival rates.     

Interruption of a transforming growth factor α autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides

We have previously shown that Caco-2 cell proliferation is driven by basolateral membrane epidermal growth factor receptors. The aim of this study was to investigate whether autocrine production of transforming growth factor α (TGF-α) activates these receptors and stimulates proliferation using antisense oligodeoxynucleotides. Caco-2 cells grown on microporous membranes or Jurkat cells were exposed to conventional or 5′ cholesterol-modified oligodeoxynucleotides synthesized with random, antisense, or missense base sequences. Indices of proliferation were measured, including [³H]thymidine or [³H]uridine uptake for studies of short-term stimulation and the methylthiotetrazole assay as an index of cell number increase over longer periods. Secretion of TGF-α by cells was detected using a soft agar bioassay. Incubation with antisense oligodeoxynucleotides inhibited TGF-α secretion compared with controls. Random and missense oligodeoxynucleotides had no effect on proliferation. The TGF-α antisense oligodeoxynucleotides markedly inhibited proliferation, an effect that was abolished by adding TGF-α to the medium. Oligonucleotides had no effect on Jurkat cells, a lymphocytic cell line lacking epidermal growth factor receptors. Cholesterol-modified oligodeoxynucleotides were more effective and specific than unmodified oligodeoxynucleotides. Caco-2 cell proliferation is driven by autocrine stimulation of epidermal growth factor receptors by TGF-α. This mechanism may be effectively inhibited by antisense oligodeoxynucleotides, particularly those modified by the 5′ attachment of cholesterol.     

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene

Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.     

Microsatellite instability in human colonic cancer is not a useful clinical indicator of familial colorectal cancer

Microsatellite instability is a property of most tumors occurring in the context of hereditary nonpolyposis colon cancer. Instability also occurs in 10%–15% of apparently sporadic colorectal cancers, and it has been hypothesized that this instability may indicate a genetic predisposition to colonic cancer. This study evaluated whether there is a clinically useful association between colon cancer instability and a family history of cancer. Colon cancer cases (n = 188) from a population-based study were evaluated for microsatellite instability with 10 polymerase chain reaction primer sets. Instability results were compared with family history and other clinical and biological characteristics. Microsatellite instability was found in 16.5% of tumors. It was predominantly a feature of right-sided tumors (P = 0.003) and was associated with the youngest and oldest ages at diagnosis (P = 0.01). Instability was not associated with family history of cancer, sex of the individual, or the glutathione-S-transferase mu 1 null genotype. Although some very small, and as yet undefined, proportion of colon cancer may be caused by inherited mutations leading to microsatellite instability, tumoral instability by itself is not a marker for familiality and should not be considered as evidence for an inherited syndrome.     

Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor

Considerable indirect evidence suggests that cytokine tumor necrosis factor α contributes to the hepatocellular damage caused by toxic liver injury. The effects of tumor necrosis factor α neutralization on liver cell injury were determined in an in vivo model of toxic liver injury. The in vivo effects of tumor necrosis factor α were examined in carbon tetrachloride liver injury through the administration of a soluble tumor necrosis factor receptor to neutralize the effects of this cytokine. Soluble tumor necrosis factor receptor treatment decreased the degree of liver injury as measured by reduced levels of serum liver enzymes and improved histology. Soluble tumor necrosis factor receptor administration also lowered the mortality from a lethal dose of carbon tetrachloride from 60% to 16%. Tumor necrosis factor α neutralization had no detrimental effect on liver regeneration as determined by the timing of histone gene expression and postinjury liver weight. These data provide direct evidence for a role of tumor necrosis factor α in toxin-induced liver cell injury. In addition, these investigations suggest that soluble tumor necrosis factor receptor therapy may be of benefit in the treatment of human liver disease.     

Esophageal body and lower esophageal sphincter function in healthy premature infants

Gastroesophageal reflux is a common problem in premature infants. The aim of this study was to use a novel manometric technique to measure esophageal body and lower esophageal sphincter pressures in premature infants. Micromanometric feeding assemblies (OD, ≤2 mm) incorporating 4–9 manometric channels were used in 49 studies of 27 premature neonates. Esophageal body motility was recorded at three sites for 20 minutes after feeding. Twenty attempts (one per minute) were made to stimulate swallowing via facial stimulation (Santmyer reflex). In 32 studies, lower esophageal sphincter pressures were recorded (sleeve) for 15 minutes before and after feeding. Peristaltic motor patterns were less common than nonperistaltic motor patterns (26.6% vs. 73.4%; P < 0.0001) that comprised 31.1% synchronous, 34.6% incomplete, and 6.3% retrograde pressure waves. Reflex swallowing was elicited more frequently in neonates older than 34 weeks postconceptional age than in younger infants (33.4% vs. 20.4%; P < 0.05). Mean lower esophageal sphincter pressure was 20.5 ± 1.7 mm Hg before and 13.7 ± 1.3 mm Hg after feeding (P < 0.0005). Premature infants show nonperistaltic esophageal motility that may contribute to poor clearance of refluxed material. In contrast, the lower esophageal sphincter mechanisms seem well developed.     

Platelet-activating factor acetylhydrolases in Caco-2 cells and epithelium of normal and ulcerative colitis patients

Platelet-activating factor (PAF) is a potent inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease and necrotizing enterocolitis. Metabolism by platelet-activating factor acetylhydrolase (PAF-AH) is the major pathway for platelet-activating factor degradation. The aim of this study was to investigate the possible role of intestinal epithelium as a source of PAF-AH. Intracellular and secreted PAF-AHs were characterized in human colonic epithelial cells isolated from histologically normal mucosa and inflamed mucosa from patients with ulcerative colitis and in the human intestinal epithelial cell line Caco-2 by measuring the metabolism of [³H]-PAF to [³H]lysoPAF. Human colonic epithelial cells and Caco-2 cells synthesize and secrete PAF-AH as shown by in vitro hydrolysis of [³H]PAF to [³H]-lysoPAF in cell lysates and conditioned media. Both intracellular and secreted PAF-AHs are calcium-independent and substrate-specific for phospholipids similar to PAF. Epithelial cells from involved areas of resections for ulcerative colitis had increased levels of secreted PAF-AH and decreased levels of intracellular PAF-AH compared with epithelial cells from histologically normal areas. Human colonic epithelial cells and Caco-2 cells produce intracellular and secreted PAF-AHs, which are distinct proteins. This is the first demonstration of PAF-AH production by epithelial cells.     

Medical costs in community subjects with irritable bowel syndrome

Costs of management of irritable bowel syndrome (IBS) are unknown. The direct medical charges in community subjects with IBS were estimated. An age- and sex-stratified random sample of residents of Olmsted County, Minnesota, ranging in age from 20 to 95 years, was mailed a valid self-report questionnaire. Subjects were categorized as having IBS, having some symptoms but inadequate criteria for IBS, and controls. All charges (in 1992 U.S. dollars) for health services rendered in the year before completing the survey were obtained (except outpatient medications). A total of 88% of subjects with IBS, 86% of subjects with some symptoms of IBS, and 83% of controls incurred direct medical charges during the study year. The odds of incurring charges were 1.6 times greater in subjects with IBS relative to those without symptoms (P < 0.01) adjusting for age, sex, education, marital status, and employment. Overall median charges incurred by subjects with IBS were $742 compared with $429 for controls and $614 for subjects with some symptoms. Among those subjects with nonzero charges, there were significant positive associations with age, higher education, and symptom groups (all P < 0.01) but not sex. The economic impact of IBS is significant. A better understanding of the determinants of these costs is needed so that cost-saving strategies can be implemented.     

Decreased activity of intestinal and urinary intrinsic factor in Gräsbeck-Imerslund disease

The pathogenesis of inherited intestinal cobalamin malabsorption (Gräsbeck-Imerslund disease) remains unknown. The authors studied whether the disease corresponds to a defective expression and/or function of the intrinsic factor-cobalamin receptor in the ileum. Intrinsic factor-cobalamin receptor activity was measured using radioisotope assay and gel-filtration exclusion chromatography in ileal biopsy specimens and urine concentrates from 4 patients with Gräsbeck-Imerslund disease and 5 controls. Receptor activity was 164 ± 13 fmol/mg of protein in control biopsy specimens and <2.6 fmol/mg protein in specimens from patients. The association constant was estimated to be 3.8 ± 0.4 (nmol/L)⁻¹ in controls. A dramatic decrease in receptor activity was also observed in urine concentrate from patients with an association constant of 1.9 and 3.3 (nmol/L)⁻¹. Isoelectrofocusing of the cross-linked intrinsic factor-cobalamin receptor complex showed an isoelectric point at 4.8 in a patient as well as in control samples. It is concluded that Gräsbeck-Imerslund disease is related to decreased intrinsic factor-receptor activity in intestinal mucosa; the receptor assay in urine can be helpful for diagnosis.     

Effect of germfree state on the capacities of isolated rat colonocytes to metabolize n-Butyrate, glucose, and glutamine

Among substrates available to the colonic mucosa, n-butyrate from bacterial origin represents a major fuel. The present work investigated possible modifications of energy substrate metabolism in colonocytes isolated from germfree rats. Colonocytes isolated from germfree vs. conventional rats were incubated (30 minutes at 37°C) in the presence of ¹⁴C-labeled n-butyrate (10 mmol/L), glucose (5 mmol/L), or glutamine (5 mmol/L). ¹⁴CO₂ and metabolites generated were measured. Possible regulatory steps were also investigated. Glucose use rate was 25% lower in germfree rat colonocytes due to a reduced glycolytic capacity in these cells. Differences in 6-phosphofructo-1-kinase activity could account for this decrease. In contrast, glutamine use rate was 45% higher, and this was correlated with a higher maximum velocity of glutaminase in these cells. Nevertheless, the capacities to oxidize glucose and glutamine remained unchanged. Although the capacity to use n-butyrate was maintained in colonocytes of germfree rats, the ketogenic capacity was lower, whereas the capacity to oxidize n-butyrate was higher. The mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein was identified in the colonic mucosa. Moreover, the messenger RNA and amount of protein were 75% lower in the germfree state. The absence of an intestinal microflora induces specific changes in the metabolic capacities of colonocytes.     

Hansenula anomala killer toxin induces secretion and severe acute injury in the rat intestine

The yeast Hansenula anomala has been associated with gastrointestinal symptomatology and damage to the intestinal wall in humans. In vitro and in vivo, H. anomala secretes a toxin, killer toxin, which is lethal to other microorganisms. In view of the very high rate of killer phenotype expression recorded for H. anomala strains in nature, this study aimed to investigate the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis. Effects of active and heat-inactivated H. anomala killer toxin on intestinal fluid homeostasis and electrolyte balance were investigated in rat small intestine using a standard intestinal perfusion technique. Sections of the perfused jejunum tracts were examined histologically. H. anomala killer toxin induced a significant secretion of water and electrolytes. No significant change was observed when either heat-inactivated H. anomala killer toxin or control growth medium were tested. Histological analysis showed ischemic degeneration of villi and sloughing of surface epithelium in 50% of active H. anomala killer toxin-perfused jejuna. This paper presents original observations compatible with the hypothesis that H. anomala killer toxin plays a role in the pathogenesis of H. anomala-induced enteritis.