Bile Exclusion Research Articles

Gastrointestinal phase ofListeria monocytogenesinfection

For many years the Gram-positive facultatively intracellular food-borne pathogen Listeria monocytogenes has been used as a model organism for the study of intracellular parasitism. Whilst the basic mechanisms of cellular pathogenesis have been elucidated by a series of elegant studies, recent research has begun to focus upon the gastrointestinal (GI) phase of L. monocytogenes infection. Epidemiological studies of outbreaks of human disease now demonstrate that the pathogen can cause gastroenteritis in the absence of invasive disease and associated mortality. Furthermore, the pathogen has the ability to colonize the gallbladder in infected mice and this may function as a significant focus of infection during pathogenesis. The ability of the pathogen to survive within the various microenvironments of the GI tract is essential for the causation of food-borne infection. Survival of the pathogen within gastric acid requires the glutamate decarboxylase (GAD) system. In addition, the carnitine uptake system, OpuC is essential for adaptation within the murine GI tract and subsequent invasive disease. Survival of bile salts both in vitro and in vivo requires a number of mechanisms including bile salt hydrolase (Bsh) and the recently described bile exclusion system, BilE (previously OpuB). Finally a number of these systems (including Bsh, OpuC and BilE) are regulated by both the alternative stress sigma factor, Sigma B and by the regulator of virulence gene expression, PrfA.

A PrfA‐regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851-853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two-gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus sigmaA- and sigmaB-dependent promoter-binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of sigmaB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five-log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by sigmaB and PrfA, represents a new and important virulence factor in L. monocytogenes.

Pathophysiological role of cholecystokinin in humans.

Cholecystokinin (CCK) is a major gastrointestinal hormone that plays an important role in stimulation of pancreatic secretion and gall-bladder contraction, regulation of gastrointestinal motility and induction of satiety. Ingestion of fat and protein induces significant increases in plasma CCK. Intraluminal mediators of CCK secretion, luminal CCK releasing factor and diazepam-binding inhibitor, were purified from rat intestinal secretion. These CCK-releasing factors (RF) are secreted tonically by the small intestine and stimulate CCK release. Another kind of CCK-RF named 'monitor peptide' was purified from the rat pancreatic juice that stimulates CCK secretion when introduced into rat intestine. Bile exclusion from the duodenum causes an increase in basal CCK and enhances stimulated plasma CCK release, and bile salt replacement reverses these effects. Thus, the CCK-RF are spontaneously secreted into the intestinal lumen in humans, while the CCK-producing cells are under constant suppression by intraduodenal bile acids. In acute pancreatitis, plasma CCK levels are high in patients with gallstone pancreatitis, but not in patients with pancreatitis from other causes, such as alcoholic and idiopathic pancreatitis. A transient disturbance of bile flow into the duodenum by stones or oedema of the pancreas together with impairment of pancreatic exocrine function might cause the increase in plasma CCK release in gallstone pancreatitis. Patients with chronic pancreatitis with mild to moderate impairment of exocrine function and abdominal pain, had significantly higher plasma CCK concentrations, whereas patients with pancreatic insufficiency had a significantly lower plasma CCK response to a test meal than the healthy subjects. The increased CCK may further aggravate pancreatitis and worsen the prognosis of pancreatitis by stimulating the injured pancreas, resulting in the vicious circle via endogenous CCK release. The CCK-A receptor antagonist might be therapeutically useful in acute pancreatitis by stopping the vicious circle.

Increased Circulating Cholecystokinin in Obstruction-Induced Acute Pancreatitis: I. Bile Duct Obstruction with and without Pancreatic Duct Obstruction

Bile exclusion from the gut exacerbates pancreatic duct obstraction-induced acute pancreatitis. We hypothesized that obstruction-induced acute pancreatitis involves an increase in circulating cholecystokinin (CCK), as bile and pancreatic juice exclusion from the gut stimulates duodenal CCK release. We studied 54 rats after the following operations: (1) sham operation ( n = 18). (2) hepatic bile duct obstruction alone ( n = 18), (3) hepatic bile duct and common bile-pancreatic duct obstruction ( n = 18). Rats recovered and were killed in subgroups of six rats each at 3.6. and 18 hr after operation; blood was collected for measurement of plasma CCK and amylase concentrations. Each pancreas was excised, weighed, and processed for histological examination; an acute pancreatitis score was determined. Combined bile and pancreatic duct obstruction induced acute pancreatitis and was associated with a marked increase of circulating CCK concentration. Bile duct obstruction alone did not induce acute pancreatitis but was associated with an increase of circulating CCK of lower magnitude. The time course of circulating CCK increase showed an early peak. These findings support our hypothesis and suggest that CCK plays a role in the pathogenesis of obstruction-induced acute pancreatitis.

Increased Circulating Cholecystokinin in Obstruction-Induced Acute Pancreatitis: II. Pancreatic Duct Obstruction with and without Bile Duct Obstruction

Pancreatic exocrine stimulation by cholecystokinin (CCK) has been implicated in pathogenesis of experimental acute pancreatitis. Bile exclusion from the gut stimulates duodenal CCK release and exacerbates obstruction-induced acute pancreatitis. Pancreatic and bile duct obstruction increases circulating CCK concentration. We hypothesized that acute pancreatitis induced by pancreatic and bile duct obstruction would be ameliorated when bile was returned to the duodenum. As many small pancreatic ducts drain into the bile duct in rats, preservation of bile flow required the use of a bile shunt. We studied acute pancreatitis and the time course of circulating CCK increase in three groups of rats after: (1) sham operation (dissection, no obstruction), (2) bile and pancreatic duct obstruction, and (3) bile and pancreatic duct obstruction with bile shunt. The rats were killed at 3-, 6-, and 18-hr intervals after operation. Their blood was collected for measurement of CCK, amylase, and bilirubin concentrations. The pancreata were excised, weighed, and processed for histological examination. The shunting of bile back to the duodenum ameliorated the acute pancreatitis along with a simultaneous limitation of the rise in CCK concentration. This suggests that bile duct obstruction, an other form of bile exclusion, exacerbates pancreatic duct obstruction-induced acute pancreatitis. The elevation in CCK concentration showed an early peak indicating that the potential role of CCK in the pathogenesis of obstruction-induced acute pancreatitis is predominantly in the early phase of its development.

Bile Exclusion from the Gut Exacerbates Acute Pancreatitis Caused by Pancreatic Duct Obstruction in Rats

Acute pancreatic duct obstruction causes hyperamylasemia and mild pancreatic inflammation. We hypothesized that bile exclusion from the gut, which stimulates pancreatic secretion, exacerbates acute pancreatitis caused by pancreatic duct obstruction. Rats were surgically prepared with gastric, duodenal, bile, and pancreatic fistula catheters and a jugular vein catheter. After a 4-day recovery, groups of rats (a) served as controls, (b) had complete pancreatic duct obstruction for 6 h, or (c) had bile excluded from the gut for 24 h and then, during the final 6 h, complete pancreatic duct obstruction. Plasma amylase was measured, and all rats were euthanized at the end of experiments. Each pancreas was excised and weighed, and portions were fixed in formalin and glutaraldehyde. In blind fashion, each pancreas was examined under light microscopy and assigned a pancreatitis score based on presence of edema and severity of acinar cell changes and inflammation. Acute pancreatic duct obstruction was associated with increased pancreas weight, hyperamylasemia, and elevated pancreatitis score; moderate acinar cell vacuoles, which were observed in the cytoplasm near the basolateral membrane, and loss of microvilli were noted with electron microscopy. Bile exclusion from the gut exacerbated the acute pancreatitis caused by pancreatic duct obstruction; acinar cell distortion and destruction, as well as marked inflammation, were seen microscopically. These observations suggest that the absence of intestinal bile contributes to the pathogenesis of acute pancreatitis associated with pancreatic duct obstruction.

In vitro dissolution of cholesterol gallstones: A study of factors influencing rate and a comparison of solvents

Models of the common bile duct and gallbladder were constructed to study conditions that affect the rate of cholesterol gallstone dissolution by monooctanoin and other potential solvents. In the bile duct model, the rate of monooctanoin infusion was not an important factor in accelerating dissolution time. In contrast, the exclusion of bile from interfering with solvent-stone contact or the enhancement of solvent-stone contact by stirring significantly accelerated stone dissolution. The combination of both bile exclusion and stirring increased the dissolution rate of gallstones by monooctanoin 15-fold. When compared with two other ethers and with monooctanoin, methyl tert-butyl ether was found to be the most potent gallstone solvent. Methyl tert-butyl ether completely dissolved 219-mg cholesterol stones within 60 min. In the gallbladder model, in the absence of stirring both methyl tert-butyl ether and monooctanoin floated on bile, whereas the gallstones sank resulting in minimal stone-solvent contact. To increase the stone-solvent contact, we used a pump to create sufficient turbulence to mix the solvent with bile. Pump stirring of monooctanoin in the presence of bile achieved rates of stone dissolution approaching that of stirred monooctanoin without bile. Stirring of methyl tert-butyl ether and bile, however, did not achieve sufficient solvent-stone contact to appreciably accelerate dissolution in the presence of 50% bile. Stone-solvent contact was a critical factor in determining the rate of gallstone dissolution in both gallbladder and common bile duct models. Efforts to enhance contact include bile exclusion and intraluminal stirring—both of which are clinically applicable. Methyl tert-butyl ether is a potent new cholesterol gallstone solvent with excellent potential for use in humans. Even with this potent agent, however, rapid gallstone dissolution is likely to require removal of most of the bile from the dissolution medium.

Effect of bile on vitamin B12 absorption.

The standard double-isotope Schilling test was used to study vitamin B12 absorption in seven patients with obstructive jaundice and 10 with T-tube bile duct drainage after cholecystectomy and bile duct exploration. In three and five of these patients respectively absorption was impaired. In the second group six patients were restudied after removal of the T tube, and in each case absorption was improved. Similar results were obtained after bile duct ligation in rats. Bile exclusion produced a 50-60% reduction in renal and hepatic uptake of vitamin B12 from the intestinal lumen. The malabsorption was corrected by replacing bile. These studies suggest that bile plays a part in the normal absorption of vitamin B12.

Effect of Exclusion of Bile from the Intestine on Gastric Acid Secretion

Summary Decreased acid secretion from Heidenhain pouches in response to food was found in cholecystectomized dogs with bile excluded from the intestine by cannulation of the common bile duct through a duodenal fistula when the animals were fed 3 hr after the onset of bile exclusion. The secretory response with exclusion of bile for 1 hr before feeding was not different from that without exclusion of bile. Secretin inhibited gastric secretion stimulated by a meal to the same degree when bile was excluded from the intestine as when it was not. These studies show that the presence of bile in the intestine is a factor which contributes to the regulation of gastric secretion and that inhibition of gastric secretion by secretin is unaltered by removal of this factor.