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Background & Aims Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. Methods Germ-free, wild-type, and interleukin ( Il ) 10 −/− mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10 −/− mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium . Results In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2–related factor 2–, IL-10–, and Toll-like receptor–dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10 −/− mice. Administration of CoPP to Il10 −/− mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10 −/− mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium –induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli , E faecalis , and S typhimurium , whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. Conclusions Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.

Regression of low-grade B cell gastric lymphoma of mucosa-associated lymphoid tissue after eradication of Helicobacter pylori with antibiotic therapy was recently shown in a small number of patients with low-volume tumors. A patient with a > 10 cm nodular gastric mucosa-associated lymphoid tissue lymphoma that caused hematemesis and weight loss is described. Antibiotic therapy of H. pylori resulted in full clinical recovery and resolution of the mass lesion and morphological features of lymphoma on routine histological examination. However, monotypic immunostaining of plasma cells persisted in a separate and grossly normal-appearing region of the stomach. Antibiotic therapy may be of benefit in patients with mucosa-associated lymphoid tissue lymphoma with mass lesions and significant signs and symptoms, but periodic search for residual lymphoma is needed.

Background: Interaction of the enteric nerves in regulating mammalian duodenal mucosal bicarbonate secretion is not well understood. The purpose of the present experiments was to evaluate the role of the enteric nervous system on bicarbonate secretion from rabbit duodenal mucosa in vitro. Methods: Proximal duodenum from male New Zealand White rabbits was stripped of seromuscular layers, mounted in Ussing chambers, and studied under short-circuited conditions. Effects of electrical field stimulation, vasoactive intestinal polypeptide (VIP), carbachol, prostaglandin E2 (PGE2), dibutyryl-cyclic adenosine monophosphate (db-cAMP), and the neurotoxin tetrodotoxin (TTX) and muscarinic blockade by atropine were studied. Results: Electrical field stimulation significantly (P < 0.01) stimulated bicarbonate secretion, short-circuit current (Isc), and electrical potential difference (PD) that was sensitive to both TTX and atropine. VIP-stimulated bicarbonate secretion was significantly inhibited by TTX (−73%), yet Isc and PD remained unchanged. Atropine decreased VIP-induced bicarbonate secretion (−69%) and Isc (−43%). Carbachol-stimulated bicarbonate secretion, Isc, and PD were abolished by atropine, whereas TTX was without affect. Neither TTX nor atropine had a significant effect on PGE2 or db-cAMP-stimulated bicarbonate secretion. Conclusions: These results suggest that (1) enteric nerve stimulation activates an acetylcholine receptor that in turn stimulates duodenal epithelial bicarbonate secretion; (2) VIP stimulates bicarbonate secretion, in large part, via the enteric nervous system; and (3) PGE2 and cAMP stimulate bicarbonate secretion independent of the enteric nervous system.

Sugar absorption by the biliary ductular epithelium under steady-state conditions was examined using isolated perfused rat liver. The test sugar and mannitol (as a putative marker of paracellular entry) were added to the glucose-free recirculating perfusate each at a concentration of 5 mmol/L, and apparent active biliary ductular absorption equated with the change in concentration of the test sugar relative to that of mannitol. A metabolizable hexose (D-glucose), pentose (D-xylose), and three nonmetabolizable hexoses (alpha-methyl-glucoside, 3-o-methyl-glucose, and L-glucose) were used. All five monosaccharides were well absorbed at constant rates for 2 hours with apparent rates of absorption (mumol.kg body weight-1.min-1, mean +/- SE) of D-glucose, 0.24 +/- 0.01; L-glucose, 0.20 +/- 0.02; 3-o-methyl-glucose, 0.19 +/- 0.02; alpha-methyl-glucoside, 0.16 +/- 0.03; and D-xylose, 0.10 +/- 0.04. The addition of phloridzin to the perfusate inhibited D-glucose absorption in part but did not inhibit L-glucose absorption. When perfusate Na+ was replaced by N-methylglucamine, the bile-plasma ratio of mannitol remained unchanged, as did the apparent absorption rate of D-glucose and 3-o-methyl-glucose. In contrast, absorption of L-glucose and alpha-methyl-D-glucoside gradually ceased. The addition of 15 mmol/L glucose to the perfusate caused decreased bile flow and increased taurocholate concentration in bile, suggesting that glucose absorption by the biliary ductules induced water reabsorption. It is concluded that sugars are absorbed by the biliary ductular system by Na(+)-dependent and Na(+)-independent transport systems, the substrate affinities of which differ from those reported for apical membrane hexose transport systems in renal tubular and intestinal epithelia. Ductular absorption of solutes such as glucose that enter bile passively may have biological use, because ductular absorption decreases the concentration of substrates for bacterial growth in gallbladder bile. On the other hand, ductular absorption of solutes induces reabsorption of biliary water, resulting in decreased bile flow; this might contribute to cholestasis during prolonged hyperalimentation with solutions containing glucose.

Active transport of conjugated bile acids by the distal ileum is required for efficient enterohepatic cycling of bile acids. Experiments were performed in the rat to obtain accurate values for Tmax and Michaelis constant (Km) of the absorptive area of the rat ileum and to define the structural specificity of the transport system. The distal fifth (20 cm) of the small intestine from an anesthetized animal with a biliary fistula was perfused using solutions of 10 taurine-conjugated bile acids; a flow rate was used that was sufficiently high such that unstirred water layer effects were negligible and the intraluminal concentration remained unchanged throughout the perfused segment. The absorption rate was equated with the rate of hepatic bile acid secretion. Values of Tmax (mumol/min.kg) were markedly influenced by bile acid structure: cholyltaurine, 12.9; ursocholyltaurine, 9.6; ursodeoxycholyl taurine, 5.0; and lagodeoxycholyl-(3 alpha,12 beta-dihydroxy-cholanoic acid)-taurine, 1.2. Decreasing the length of the side chain of ursodeoxycholate conjugates from 8 to 6 carbon atoms was associated with a modest increase in Tmax values from 5.0 to 9.1 mumols/min.kg. Values of Km correlated with Tmax values and ranged from 0.5 to 5 mmol/L, being highest for those bile acids that were best transported. The Tmax for cholyltaurine transport was not reached when the intraluminal concentration was as high as its critical micellization concentration, precluding the definition of its Tmax; however, for ursocholyltaurine, with a critical micellization concentration of 40 mmol/L, saturation of transport was clearly shown. Kinetic parameters could not be obtained for two common dihydroxy conjugates (chenodeoxycholyltaurine and deoxycholyltaurine) because at a transport rate of 2 mumols/min.kg systemic toxicity and death occurred. These studies define the maximal transport capacity of the rat ileum for taurine-conjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituents and side chain length influence the transport rate of taurine-conjugated bile acids.

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5- bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision. (Gastroenterology 1996 Dec;111(6):1493-500)

Fatty acids and bile acids are tumor promoters of experimental colon cancer in rats. Calcium can inhibit their effects. After intestinal bypass (IB), fecal bile acid and lipid levels increase markedly. In rats, IB increases colonic cell proliferation and carcinogen-induced colon tumor incidence. Whether fecal bile acids and lipids influence rectal epithelial proliferation in humans is uncertain. This study compared rectal epithelial proliferation in IB subjects and in controls matched for age, sex, and body mass index and investigated the effects of calcium carbonate supplementation (2400 or 3600 mg Ca2+/day for 12 weeks) on proliferation indices in IB subjects.Epithelial proliferation was studied by in vitro incubation of rectal biopsy specimens with [3H] thymidine. Twenty-four-hour stool collections were assayed for bile acids, lipids, and calcium.Whole crypt labeling index (LI) and upper crypt LI were increased in IB subjects compared with controls (P0.005). Calcium reduced whole crypt LI by 38% (P0.001) and upper crypt LI by 56% (P0.05). Levels of fecal bile acids (4.5 mmol/day) and lipids (131.9 g/day) were markedly elevated in IB subjects (P0.005).IB induces rectal hyperproliferation and expansion of the proliferative zone in association with excessive output of fecal bile acids and lipids. Oral calcium reverses the proliferative changes.

Background: Colorectal cancer (CC) discovered within 3 years after a normal or clearing colonoscopy (C) is considered to be the result of a missed polyp or cancer at the time of the preceding C. Multiple studies comparing sequential images of colon lesions over time have calculated lesion volume doubling rates varying from 4 months for very large lesions (~35 mm) to more than 2 years for medium size polyps (~10 mm). Aim: To estimate the size of CC considered missed (i.e., diagnosed within 3 years of a prior C) using the tumor size at the time of CC diagnosis.Methods: A large dataset was created containing (1) patients who had undergone C at Mayo Clinic Rochester (MCR) between 1992 and 2002 (138,318 Cs in 98,960 patients), and (2) patients who had a tissue diagnosis of CC between 1992 and 2004 (N=10,136). We defined a missed CC (CCdespiteC) as a CC occurring from 90 to 1095 days after C. The estimated size of the tumor at the time of the C preceding the date of CC diagnosis was estimated using the exponential growth function y=xeβt where t is the interval between date of the preceding C and the date of CC diagnosis; y and x are the tumor size (diameter of the greatest dimension) at the time of the CC diagnosis and the preceding C respectively. Despite the fact that most lesions missed during preceding C were expected to be medium sized, we elected an aggressive tumor volume doubling rate of 6 months (i.e., β=0.0013). Results: Among the 10,136 patients with either primary or metastatic colon cancer 2692 patients had undergone a total of 4743 colonoscopies at MCR. From this cohort, we identified 187 Cs in 145 patients who developed CCdespiteC. There was a subset in which the CC was truly missed: no lesion was seen or treated in the colonic segment during the preceding C: 120 Cs in 100 patients. Of these 120 truly missed tumors, we were able to retrieve the tumor size at the time of CC diagnosis of 96 tumors using the pathology and surgical reports. Out of 96 CCdespiteC tumors, 67 lesions have an estimated maximal diameter (obtained using the exponential growth model) of more than 10 mm at the time of preceding C. Nearly all lesions (94 out of 96) have an estimated maximal diameter of more than 5 mm at the time of the preceding C. Conclusions: Almost all CC diagnosed within 3 years after a negative or clearing colonoscopy have an estimated maximal tumor diameter greater than 5 mm using an exponential growth model and a very aggressive tumor volume doubling time. Our results strongly suggest that CCdespiteC is the result of a lesion truly being missed at the time of the preceding C and not the result of a de novo rapidly growing neoplasm.

Active transport of conjugated bile acids by the distal ileum is required for efficient enterohepatic cycling of bile acids. Experiments were performed in the rat to obtain accurate values for Tmax and Michaelis constant (Km) of the absorptive area of the rat ileum and to define the structural specificity of the transport system. The distal fifth (20 cm) of the small intestine from an anesthetized animal with a biliary fistula was perfused using solutions of 10 taurine-conjugated bile acids; a flow rate was used that was sufficiently high such that unstirred water layer effects were negligible and the intraluminal concentration remained unchanged throughout the perfused segment. The absorption rate was equated with the rate of hepatic bile acid secretion. Values of Tmax (mumol/min.kg) were markedly influenced by bile acid structure: cholyltaurine, 12.9; ursocholyltaurine, 9.6; ursodeoxycholyl taurine, 5.0; and lagodeoxycholyl-(3 alpha,12 beta-dihydroxy-cholanoic acid)-taurine, 1.2. Decreasing the length of the side chain of ursodeoxycholate conjugates from 8 to 6 carbon atoms was associated with a modest increase in Tmax values from 5.0 to 9.1 mumols/min.kg. Values of Km correlated with Tmax values and ranged from 0.5 to 5 mmol/L, being highest for those bile acids that were best transported. The Tmax for cholyltaurine transport was not reached when the intraluminal concentration was as high as its critical micellization concentration, precluding the definition of its Tmax; however, for ursocholyltaurine, with a critical micellization concentration of 40 mmol/L, saturation of transport was clearly shown. Kinetic parameters could not be obtained for two common dihydroxy conjugates (chenodeoxycholyltaurine and deoxycholyltaurine) because at a transport rate of 2 mumols/min.kg systemic toxicity and death occurred. These studies define the maximal transport capacity of the rat ileum for taurine-conjugated bile acids; they indicate that the ileal transport system in the rat is of low affinity and high capacity for taurine conjugates of hydrophilic bile acids, and they show that both nuclear substituents and side chain length influence the transport rate of taurine-conjugated bile acids.

To define whether side-chain length influences the physiologic properties of bile acids, nor-ursodeoxycholate (nor-UDC), the C23-nor derivative of ursodeoxycholate (UDC), was synthesized in both nonradioactive and radioactive forms (23-14C). Its hepatic translocation, hepatic biotransformation, and effect on bile flow, biliary bicarbonate, and biliary lipid secretion were compared with that of UDC and those of their respective glycine and taurine conjugates in anesthetized biliary fistula hamsters, rats, and guinea pigs, as well as the isolated perfused hamster liver. Hepatic uptake and biliary output of nor-UDC was slower than that of UDC or cholyltaurine in the isolated perfused hamster liver. In biliary fistula animals, nor-UDC was secreted only in bile. Biliary recovery of nor-UDC as compared to that of UDC was prolonged in the rat and hamster, although not in the guinea pig. Hepatic biotransformation, assessed by chromatography of bile, showed that conjugation of nor-UDC was inefficient, as unconjugated nor-UDC was present in bile; there was little amidation with glycine or taurine in any species, but sulfates and glucuronides, as well as other metabolites, were formed, with the pattern of biotransformation varying among species. When infused over a dosage range of 0.2-30 mumol/kg X min, nor-UDC induced a striking choleresis of canalicular origin. The bile acid-dependent flow was increased threefold in hamsters, ninefold in rats, and nearly twofold in guinea pigs when compared to that induced by UDC. The choleresis was associated with a linear increase in bicarbonate output and concentration in bile, and little phospholipid or cholesterol secretion was induced. A competition experiment in the bile fistula hamster indicated that nor-UDC or its metabolites, or both, appeared to compete for canalicular transport of ursocholyltaurine (a cholyltaurine epimer) when the latter was secreted under its Vmax conditions. Conjugates of nor-UDC and UDC were promptly and almost completely recovered in bile without appreciable hepatic biotransformation; the conjugates did not induce a hypercholeresis or increase biliary bicarbonate concentration. It is proposed that a fraction of nor-UDC is secreted into canalicular bile in the unconjugated form and is protonated by a hydrogen ion derived from carbonic acid that was generated by the hydration of luminal CO2 by carbonic anhydrase present in biliary ductular cells. The protonated bile acid is absorbed, thus generating a bicarbonate anion. The bile acid passes through the cholangiocyte, returns to the sinusoids via the periductular capillary plexus, and is resecreted into bile.(ABSTRACT TRUNCATED AT 400 WORDS)

The release of gastric somatostatinlike immunoreactivity and gastrin was studied in rats with chronic achlorhydria induced by the substituted benzimidazole BY 308. In vitro, stimulation of gastrin release by acetylcholine was slightly enhanced after 1 day of treatment but no further effects were observed compared to placebo controls. Four weeks of treatment evoked marked gastrin hypersecretion, which was atropine-resistant. Stimulation of gastrin release was inversely correlated to enhancement of basal gastrin levels. Chronic achlorhydria distinctly reduced somatostatin responses to isoproterenol, whereas potent stimulation was observed in controls. Treatment with BY 308 for 1 wk was associated with fully developed gastrin hypersecretion but isoproterenol-stimulated somatostatin release was still unaffected. Hypergastrinemia accompanied by increased antral gastrin and reduced antral and fundic somatostatin concentrations was also found in vivo after 4 wk of treatment with BY 308. It is concluded that chronic achlorhydria not only enhances storage and secretion of gastrin but also diminishes the secretion and tissue stores of somatostatin; adaptive changes of the somatostatin cell occur, however, with a much longer delay.