Colonic delivery of dexamethasone from a prodrug accelerates healing of colitis in rats without adrenal suppression

Dexamethasone-β-d-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohn's colitis. The aim of this study was to evaluate colonic delivery and efficacy of this prodrug in the rat. Distribution of dexamethasone in luminal contents and tissues of the gastrointestinal tract and in plasma was measured after oral administration of dexamethasone-β-d-glucuronide or free dexamethasone. Efficacy of the prodrug and free drug was tested in an acetic acid-induced rat colitis model. Healing of induced colitis was assessed by measuring net intestinal fluid absorption, colonic surface area of ulceration, histology, and myeloperoxidase activity. Glucocorticosteroid toxicity was evaluated with serum corticosterone and plasma adrenocorticotropic hormone levels. The drug delivery index (a measure of relative targeting efficiency) was 6.7 and 8.6 in the cecal and colonic mucosa, respectively. The prodrug was significantly more potent than free drug in improving net colonic fluid absorption while significantly reducing surface area of ulceration and histological grade in colitic rats. Treatment with free dexamethasone significantly reduced serum corticosterone levels to subnormal levels, and treatment with the prodrug maintained serum corticosterone and plasma adrenocorticotropic hormone levels near control levels. The prodrug dexamethasone-β-d-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Absence of human papillomavirus DNA detected by polymerase chain reaction in French patients with esophageal carcinoma

Recent studies have suggested that esophageal human papillomavirus infection could be a risk factor for esophageal squamous cell carcinoma. The aim of this study was to evaluate the prevalence of human papillomavirus DNA sequences in the esophagus of French patients with esophageal squamous cell carcinoma. Multiplex polymerase chain reactions with consensus primers directed to the L1 gene or specific primers for human papillomavirus types 6, 11, 16, 18, 31, and 33 directed to E6 gene (40 cycles followed by restriction mapping of the amplified products) were used to determine the presence of human papillomavirus DNA sequences in esophageal squamous cell carcinoma (n = 75), normal adjacent mucosa (n = 49), and metastatic lymphadenopathies (n = 5). As an internal control, a target located in the embryonic myosin heavy-chain gene was used in each reaction. Human papillomavirus DNA sequences could not be detected in any of the tumoral samples, the normal adjacent mucosa, or the metastatic lymphadenopathies. Human papillomavirus seems not to be implicated in esophageal carcinogenesis, at least in French patients, because the viral genomes are not associated with esophageal squamous cell carcinomas.     

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.     

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.     

Beneficial effect of fructose-1,6-bisphosphate on mitochondrial function during ischemia-reperfusion of rat liver

Several groups have reported that administration of fructose-1,6-bisphosphate (FBP) reduces ischemic injury. The aim of this study was to determine the protective effect of FBP on the impairment of mitochondrial oxidative phosphorylation by ischemia-reperfusion injury in the rat liver. The respiratory control ratio (RCR) and the adenine nucleotide content of mitochondria isolated from ischemic and reperfused livers with or without FBP treatment were measured. In FBP-treated livers, the cellular adenosine triphosphate level was restored to more than 50% of normal after 120 minutes of reperfusion following 120 minutes of ischemia, whereas that of control livers only reached 15% of normal. The RCR and the adenine nucleotide content of mitochondria isolated from FBP-treated livers were significantly higher than those of mitochondria from control livers after ischemia and reperfusion. FBP strongly suppressed the formation of lipid peroxides during reperfusion. In vitamin E-deficient rats, the RCR decreased markedly during reperfusion, but FBP protected the mitochondria against reperfusion injury. FBP has a protective effect against ischemia-reperfusion injury on the liver and especially preserves the oxidative phosphorylation capacity of hepatic mitochondria.     

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.     

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.     

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.     

Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites

Cirrhotic patients are predisposed to develop spontaneous bacteremias and/or peritonitis, mainly caused by enteric bacteria. The aim of this study was to investigate if bacterial translocation, which is the passage of bacteria from the intestinal lumen to regional lymph nodes and/or the systemic circulation, is increased in a rat model of cirrhosis. Rats were studied after 12–16 weeks of CCl₄ inhalation, when samples of mesenteric lymph nodes, blood, liver, and spleen for standard bacteriologic cultures and a fragment of colon and liver for histology were obtained. Immunostaining of the cecum was performed using a polyclonal anti-Escherichia coli antibody. A significantly greater proportion of rats with cirrhosis and ascites (5 of 9; 56%) had positive mesenteric lymph node cultures compared with cirrhotics without ascites (0 of 9) and normal controls (0 of 12) (P < 0.01). In one cirrhotic rat, E. coli was isolated from both mesenteric lymph nodes and ascites. Rats with cirrhosis and ascites had significantly greater cecal submucosal edema and inflammation than rats with no ascites and controls. Immunoreactivity with E. coli was present in the cecal wall in 3 of 5 animals with E. coli translocation to mesenteric lymph nodes. In cirrhotic rats, bacterial translocation is increased after the development of ascites and may be a major factor in the development of spontaneous infections in cirrhosis.     

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.     

Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome

The process and the site of hepatitis C virus (HCV) particle formation in cells after infection remain unknown. The aim of this study was to create an in vitro model for the study of HCV particle formation. HeLa G cells were transfected with the full-length sequence of the HCV genome. Viral protein expression was analyzed using immunoblotting. The cells were examined using immunoelectron and conventional electron microscopy. Core, E2, NS3, NS5a, and NS5b proteins were identified using immunoblotting. Immunoelectron microscopy showed that the core antigen was located along the membrane of the endoplasmic reticulum (ER) and occasionally in its cisternae. Core antigen-positive particles of 30 nm in diameter were found in the cytosol and in the cisternae of the ER. The particles in the cisternae were coated with an outer membrane that was connected to the ER membrane. Conventional electron microscopy revealed particles of 45 nm in diameter with electrondense cores in the cisternae of the ER. The outer membrane of the particles was occasionally connected to the ER membrane. The findings suggest that HCV core proteins are synthesized and assembled into particles in the cytosol and that they bud into the cisternae of the ER to form coated particles.