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.     

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.     

Histamine inhibits prostaglandin E2-stimulated rabbit duodenal bicarbonate secretion via H2 receptors and enteric nerves

The gastroduodenal epithelium is protected from acid peptic damage by an adherent mucus-bicarbonate layer. Bicarbonate is secreted by the surface epithelial cells into this mucus layer. Patients with duodenal ulcer disease have impaired proximal duodenal bicarbonate secretion. Mast cells, present in large numbers in the duodenal mucosa, release a number of inflammatory mediators, including histamine. Release of such mast cell mediators has been implicated in ulcer disease. In this study, the ability of histamine to regulate bicarbonate secretion was examined. Bicarbonate secretion by rabbit proximal duodenal mucosa was examined in vitro, and the effects of histamine, its agonists, and its antagonists were studied. Histamine essentially eliminated prostaglandin E₂-stimulated duodenal mucosal bicarbonate secretion, an effect reversed both by the neurotoxin, tetrodotoxin, and the histamine H₂-receptor antagonist, cimetidine, as well as reproduced by the H₂-receptor agonist, dimaprit. In addition to the stimulatory action of histamine on gastric acid secretion, histamine expresses an additional antidefensive action by inhibiting prostaglandin E₂-stimulated duodenal epithelial bicarbonate secretion. This effect of histamine is likely mediated via H₂ receptors located on enteric nerves.     

Involvement of eicosanoids and macrophage-like cells in cytokine-mediated changes in rat myenteric nerves

Proinflammatory cytokines alter function in enteric nerves, but little is known about underlying mechanisms. This study was designed to investigate the roles of prostanoids and of macrophage-like cells in cytokine-induced suppression of [³H]norepinephrine release from rat myenteric plexus. The release of ³H from jejunal longitudinal muscle-myenteric plexus preparations that had been loaded with [³H]norepinephrine was measured. Measurements of ³H release as well as concentrations of prostaglandin E₂ and leukotriene were made in preparations exposed to interleukin 1β plus interleukin 6 and in the presence or absence of piroxicam, 5-lipoxygenase inhibitor MK886, cycloheximide, or cyclosporin A. An ultrastructural analysis was also performed to investigate the presence of macrophage-like cells in the myenteric plexus. Interleukin 1β plus interleukin 6 suppressed ³H release and caused an increase in tissue prostaglandin E₂ butnot leukotriene E₄. Piroxicam and cycloheximide but not MK886 attenuated the cytokine-induced increase in prostaglandin E₂ and the suppression of [³H]norepinephrine release. Ultrastructural analysis showed macrophage-like cells in the plexus, and the cytokine effects were inhibited by cyclosporin A. Prostanoids but not leukotrienes mediate the cytokine-induced suppression of norepinephrine release, and the results of this study suggest that macrophage-like cells are also involved.     

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells

The type and function of 5-hydroxytryptamine (5-HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. Contraction, relaxation, inositol 1,4,5-triphosphate (IP₃) and adenosine 3′,5′-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. 5-HT binding was completely inhibited by 5-HT and partially by 5-HT_2A (ketanserin), 5-HT₄ (SDZ-205,557), and 5-HT_1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP₃, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT_2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT_2A receptors mediating contraction and 5-HT₄ receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT₄ receptors are closely similar or identical to 5-HT_1p receptors.     

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.     

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.     

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.     

Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats

Recent evidence suggests an active role of endothelial cells and inflammatory cells in radiation-induced vascular dysfunction and organ damage. The aim of this study was to characterize the endothelial cell-leukocyte interactions, their molecular mechanisms, and the associated microvascular dysfunction in postcapillary venules exposed to ionizing radiation. Leukocyte rolling, adherence, and emigration and leakage of fluorescein isothiocyanate albumin in rat mesenteric venules were measured in control conditions and at 2, 4, and 6 hours after abdominal irradiation. Some animals were treated with monoclonal antibodies against leukocyte (CD18) or endothelial cell (intercellular adhesion molecule 1, P-selectin) adhesion molecules before radiation and 5 hours thereafter. In comparison with controls, irradiated animals had a marked increase in the number of rolling leukocytes at 2 hours after radiation. In animals studied 6 hours after radiation, a significant increment in the number of adherent and emigrated leukocytes was observed. This was associated with an increased permeability to fluorescein isothiocyanate albumin. Treatment with antibodies against either CD18 or intercellular adhesion molecule 1, but not P-selectin, significantly attenuated leukocyte adherence, emigration, and the increase in permeability induced by radiation. Radiation-induced leukocyte adherence and emigration involves an interaction between CD11/CD18 on leukocytes and intercellular adhesion molecule 1 on vascular endothelium. These interactions are implicated in the early increase in vascular permeability after irradiation.     

Desensitization of platelet-activating factor receptors, induced by inflammation in guinea pig ileal smooth muscle cells

Platelet-activating factor (PAF) is involved in the pathophysiology of motility changes induced by intestinal inflammation. The aim of this study was to evaluate a possible desensitization of PAF receptors in guinea pig intestinal smooth muscle cells after experimental inflammation induced by trinitrobenzenesulfonic acid (TNB). Saline or TNB (80 mg/kg) was injected in the intestinal lumen, and the animals were killed 6 days later. Smooth muscle cells from the circular layer were isolated, and cell contraction induced by PAF was measured. In cells from saline-treated animals, PAF induced a maximal cell contraction at 10 nmol/L and half-maximal contraction (EC₅₀) was 9 ± 0.2 pmol/L. After TNB injection, the maximal contraction induced by PAF was observed at 1000 nmol/L and EC₅₀ was 300 ± 70 pmol/L, indicating a 2 logmol/L right shift of the concentration-response curve of PAF. When animals were treated with the PAF antagonist, 20 mg/kg BN52021, or with 2 mg/kg indomethacin for the 6 days after TNB instillation, the right-sided shift of the concentration-response curve of PAF did not occur. Desensitization of PAF receptors occurring in intestinal smooth muscle cells after TNB instillation could be mediated in vivo by PAF itself via a prostaglandin-dependent pathway.     

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.     

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.     

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.     

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.     

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.     

Human cholangiocarcinomas express somatostatin receptors and respond to somatostatin with growth inhibition

Cholangiocarcinoma, a malignancy of biliary epithelia, is usually fatal because of absence of tests for early detection and lack of effective therapy. Somatostatin (SS) receptors are expressed in several malignancies and in rodent biliary epithelia. We tested the hypothesis that SS receptors are present in cholangiocarcinomas. We examined tissue from seven surgically resected human cholangiocarcinomas and a human bile duct cancer cell line for the messenger RNA for one subtype of SS receptors (SSTR2) and studied binding and growth-active properties of SS and its analogues. SSTR2 messenger RNA was expressed in all seven human cholangiocarcinoma specimens. Experiments with the human cholangiocarcinoma cell line showed specific, saturable binding of an SS analogue (MK-678) with high affinity for SSTR2 on cholangiocarcinoma membranes; inhibition in vitro of tumor cell proliferation by SS-14 and its analogue, octreotide; and inhibition in vivo of tumor growth in athymic mice implanted with human cholangiocarcinoma cells and treated with lanreotide, another SS analogue. Experiments to elucidate a possible mechanism of growth inhibition by SS showed it was not through changes in cellular cyclic adenosine monophosphate or calcium levels. Using gamma camera imaging with an ¹¹¹In-SS analogue, we localized a histologically proven cholangiocarcinoma in a patient. These results suggest that SS analogues may be useful for diagnostic localization and treatment of biliary tract malignancies.     

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.     

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.