Impaired reciprocal regulation between SIRT6 and TGF-ß signaling in fatty liver.

Dysregulated transforming growth factor-beta (TGF-ß) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF-ß signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild-type mice, high-fat diet-induced fatty liver is worse in TGF-ß signaling-deficient mice (SPTBN1+/- ) and the mutant mice had reduced SIRT6 abundance in the liver. Therefore, we hypothesized that altered reciprocal regulation between TGF-ß signaling and SIRT6 contributes to these liver pathologies. We found that deficiency in SMAD3 or SPTBN1 reduced SIRT6 mRNA and protein abundance and impaired TGF-ß induction of SIRT6 transcripts, and that SMAD3 bound to the SIRT6 promoter, suggesting that an SMAD3-SPTBN1 pathway mediated the induction of SIRT6 in response to TGF-ß. Overexpression of SIRT6 in HCC cells reduced the expression of TGF-ß-induced genes, consistent with the suppressive role of SIRT6 on TGF-ß signaling. Manipulation of SIRT6 abundance in HCC cells altered sterol regulatory element-binding protein (SREBP) activity and overexpression of SIRT6 reduced the amount of acetylated SPTBN1 and the abundance of both SMAD3 and SPTBN1. Furthermore, induction of SREBP target genes in response to SIRT6 overexpression was impaired in SPTBN1 heterozygous cells. Thus, we identified a regulatory loop between SIRT6 and SPTBN1 that represents a potential mechanism for susceptibility to fatty liver in the presence of dysfunctional TGF-ß signaling.

Digital cholangioscopy can detect residual biliary stones missed by occlusion cholangiogram in ERCP: a prospective tandem study.

Background and study aims After stone removal in endoscopic retrograde cholangiopancreatography (ERCP), an occlusion cholangiogram (OC) is performed to confirm bile duct clearance. OC can miss residual stones that can lead to recurrent biliary symptoms. The aim of this study was to assess if digital peroral cholangioscopy (POC) increased the diagnostic yield of residual biliary stones that are missed with OC. Patients and methods Patients having ERCP performed for choledocholithiasis were enrolled into the study only if they had one of the following criteria: dilated bile duct ≥ 12 mm and/or if lithotripsy was being performed. An OC was performed to confirm duct clearance after removal of stones followed by POC, based on inclusion criteria. The incremental yield of biliary stones missed by OC but confirmed by POC was then measured. A total of 96 POC procedures were performed on 93 patients in two tertiary care centers. Results Residual biliary stones were found in 34 % of cases. The average bile duct size in cases with residual stones was 15.1 mm ±â€Š0.7 mm. One- to three-mm stones were found in 41 % of cases, 4- to 7-mm stones in 45 % of cases, and ≥ 8-mm stones in 14 % of cases. Lithotripsy was performed in 13 % of cases and was significantly associated with residual stones (30 % vs. 3 %, P  < 0.001). Conclusions Occlusion cholangiogram can miss residual stones in patients with dilated bile ducts and those receiving lithotripsy. Digital POC can increase the yield of residual stone detection in these patients and should be considered to confirm clearance of stones. (ClinicalTrials.gov-NCT03482375).

Incremental yield of dysplasia detection in Barrett's esophagus using volumetric laser endomicroscopy with and without laser marking compared with a standardized random biopsy protocol.

BACKGROUND AND AIMS: Volumetric laser endomicroscopy (VLE) is a new wide-field advanced imaging technology for Barrett's esophagus (BE). No data exist on incremental yield of dysplasia detection. Our aim is to report the incremental yield of dysplasia detection in BE using VLE. METHODS: This is a retrospective study from a prospectively maintained database from 2011 to 2017 comparing the dysplasia yield of 4 different surveillance strategies in an academic BE tertiary care referral center. The groups were (1) random biopsies (RB), (2) Seattle protocol random biopsies (SP), (3) VLE without laser marking (VLE), and (4) VLE with laser marking (VLEL). RESULTS: A total of 448 consecutive patients (79 RB, 95 SP, 168 VLE, and 106 VLEL) met the inclusion criteria. After adjusting for visible lesions, the total dysplasia yield was 5.7%, 19.6%, 24.8%, and 33.7%, respectively. When compared with just the SP group, the VLEL group had statistically higher rates of overall dysplasia yield (19.6% vs 33.7%, P = .03; odds ratio, 2.1, P = .03). Both the VLEL and VLE groups had statistically significant differences in neoplasia (high-grade dysplasia and intramucosal cancer) detection compared with the SP group (14% vs 1%, P = .001 and 11% vs 1%, P = .003). CONCLUSION: A surveillance strategy involving VLEL led to a statistically significant higher yield of dysplasia and neoplasia detection compared with a standard random biopsy protocol. These results support the use of VLEL for surveillance in BE in academic centers.

Defective mucosal movement at the gastroesophageal junction in patients with gastroesophageal reflux disease.

BACKGROUND: Little is known about the role of muscularis mucosa at the gastroesophageal junction (GEJ). AIM: To evaluate the movement of the mucosa/muscularis-mucosa/submucosa (MMS) at the GEJ in normal subjects and in patients with gastroesophageal reflux disease (GERD). METHODS: Gastroesophageal junctions of 20 non-GERD subjects and 10 patients with GERD were evaluated during 5 mL swallows using two methods: in high-resolution endoluminal ultrasound and manometry, the change in the GEJ luminal pressures and cross-sectional area of esophageal wall layers were measured; in abdominal ultrasound, the MMS movement at the GEJ was analyzed. RESULTS: Endoluminal ultrasound: In the non-GERD subjects, the gastric MMS moved rostrally into the distal esophagus at 2.17 s after the bolus first reached the GEJ. In GERD patients, the gastric MMS did not move rostrally into the distal esophagus. The maximum change in cross-sectional area of gastroesophageal MMS in non-GERD subjects and in GERD patients was 289 % and 183%, respectively. Abdominal ultrasound: In non-GERD subjects, the gastric MMS starts to move rostrally significantly earlier and to a greater distance than muscularis propria (MP) after the initiation of the swallow (1.75 vs. 3.00 s) and (13.97 vs. 8.91 mm). In GERD patients, there is no significant difference in the movement of gastric MMS compared to MP (6.74 vs. 6.09 mm). The independent movement of the gastric MMS in GERD subjects was significantly less than in non-GERD subjects. CONCLUSION: In non-GERD subjects, the gastric MMS moves rostrally into the distal esophagus during deglutitive inhibition and forms a barrier. This movement of the MMS is defective in patients with GERD.

Ileocecal valve dysfunction in small intestinal bacterial overgrowth: a pilot study.

AIM: To explore whether patients with a defective ileocecal valve (ICV)/cecal distension reflex have small intestinal bacterial overgrowth. METHODS: Using a colonoscope, under conscious sedation, the ICV was intubated and the colonoscope was placed within the terminal ileum (TI). A manometry catheter with 4 pressure channels, spaced 1 cm apart, was passed through the biopsy channel of the colonoscope into the TI. The colonoscope was slowly withdrawn from the TI while the manometry catheter was advanced. The catheter was placed across the ICV so that at least one pressure port was within the TI, ICV and the cecum respectively. Pressures were continuously measured during air insufflation into the cecum, under direct endoscopic visualization, in 19 volunteers. Air was insufflated to a maximum of 40 mmHg to prevent barotrauma. All subjects underwent lactulose breath testing one month after the colonoscopy. The results of the breath tests were compared with the results of the pressures within the ICV during air insufflation. RESULTS: Nineteen subjects underwent colonoscopy with measurements of the ICV pressures after intubation of the ICV with a colonoscope. Initial baseline readings showed no statistical difference in the pressures of the TI and ICV, between subjects with positive lactulose breath tests and normal lactulose breath tests. The average peak ICV pressure during air insufflation into the cecum in subjects with normal lactulose breath tests was significantly higher than cecal pressures during air insufflation (49.33 ± 7.99 mmHg vs 16.40 ± 2.14 mmHg, P = 0.0011). The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflations in subjects with normal lactulose breath tests was significantly higher (280.72% ± 43.29% vs 100% ± 0%, P = 0.0006). The average peak ICV pressure during air insufflation into the cecum in subjects with positive lactulose breath tests was not significantly different than cecal pressures during air insufflation 21.23 ± 3.52 mmHg vs 16.10 ± 3.39 mmHg. The average percentage difference of the area under the pressure curve of the ICV from the cecum during air insufflation was not significantly different 101.08% ± 7.96% vs 100% ± 0%. The total symptom score for subjects with normal lactulose breath tests and subjects with positive lactulose breath tests was not statistically different (13.30 ± 4.09 vs 24.14 ± 6.58). The ICV peak pressures during air insufflations were significantly higher in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests (P = 0.005). The average percent difference of the area under the pressure curve in the ICV from cecum was significantly higher in subjects with normal lactulose breath tests than in subjects with positive lactulose breath tests (P = 0.0012). Individuals with positive lactulose breath tests demonstrated symptom scores which were significantly higher for the following symptoms: not able to finish normal sized meal, feeling excessively full after meals, loss of appetite and bloating. CONCLUSION: Compared to normal, subjects with a positive lactulose breath test have a defective ICV cecal distension reflex. These subjects also more commonly have higher symptom scores.

Circular smooth muscle contributes to esophageal shortening during peristalsis.

AIM: To study the angle between the circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) fibers in the distal esophagus. METHODS: In order to identify possible mechanisms for greater shortening in the distal compared to proximal esophagus during peristalsis, the angles between the LSM and CSM layers were measured in 9 cadavers. The outer longitudinal layer of the muscularis propria was exposed after stripping the outer serosa. The inner circular layer of the muscularis propria was then revealed after dissection of the esophageal mucosa and the underlying muscularis mucosa. Photographs of each specimen were taken with half of the open esophagus folded back showing both the outer longitudinal and inner circular muscle layers. Angles were measured every one cm for 10 cm proximal to the squamocolumnar junction (SCJ) by two independent investigators. Two human esophagi were obtained from organ transplant donors and the angles between the circular and longitudinal smooth muscle layers were measured using micro-computed tomography (micro CT) and Image J software. RESULTS: All data are presented as mean ± SE. The CSM to LSM angle at the SCJ and 1 cm proximal to SCJ on the autopsy specimens was 69.3 ± 4.62 degrees vs 74.9 ± 3.09 degrees, P = 0.32. The CSM to LSM angle at SCJ were statistically significantly lower than at 2, 3, 4 and 5 cm proximal to the SCJ, 69.3 ± 4.62 degrees vs 82.58 ± 1.34 degrees, 84.04 ± 1.64 degrees, 84.87 ± 1.04 degrees and 83.72 ± 1.42 degrees, P = 0.013, P = 0.008, P = 0.004, P = 0.009 respectively. The CSM to LSM angle at SCJ was also statistically significantly lower than the angles at 6, 7 and 8 cm proximal to the SCJ, 69.3 ± 4.62 degrees vs 80.18 ± 2.09 degrees, 81.81 ± 1.75 degrees and 80.96 ± 2.04 degrees, P = 0.05, P = 0.02, P = 0.03 respectively. The CSM to LSM angle at 1 cm proximal to SCJ was statistically significantly lower than at 3, 4 and 5 cm proximal to the SCJ, 74.94 ± 3.09 degrees vs 84.04 ± 1.64 degrees, 84.87 ± 1.04 degrees and 83.72 ± 1.42 degrees, P = 0.019, P = 0.008, P = 0.02 respectively. At 10 cm above SCJ the angle was 80.06 ± 2.13 degrees which is close to being perpendicular but less than 90 degrees. The CSM to LSM angles measured on virtual dissection of the esophagus and the stomach on micro CT at the SCJ and 1 cm proximal to the SCJ were 48.39 ± 0.72 degrees and 50.81 ± 1.59 degrees. Rather than the angle of the CSM and LSM being perpendicular in the esophagus we found an acute angulation between these two muscle groups throughout the lower 10 cm of the esophagus. CONCLUSION: The oblique angulation of the CSM may contribute to the significantly greater shortening of distal esophagus when compared to the mid and proximal esophagus during peristalsis.

The esophagogastric junction.

The following discussion of the esophagogastric junctions includes commentaries on the three component structures of the sphincteric segment between the stomach and the esophagus; the pressure contributions from the three sphincteric components in normal subjects and in gastroesophageal reflux (GERD) patients; the mechanism of action of endoscopic plication to determine the underlying pathophysiology of GERD; and in vitro muscle strip studies of defects within the gastroesophageal sphincteric segment potentially leading to GERD.

Pharmacologic specificity of nicotinic receptor-mediated relaxation of muscarinic receptor precontracted human gastric clasp and sling muscle fibers within the gastroesophageal junction.

Relaxation of gastric clasp and sling muscle fibers is involved the transient lower esophageal sphincter relaxations underlying the pathophysiology of gastroesophageal reflux disease (GERD). These fibers do not contribute tone to the high-pressure zone in GERD patients, indicating their role in pathophysiology. This study identifies some mediators of the nicotine-induced relaxation of muscarinic receptor precontracted gastric clasp and sling fibers. Muscle strips from organ donors precontracted with bethanechol were relaxed with nicotine and then rechallenged after washing and adding inhibitors tetrodotoxin (TTX), the nitric-oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME), the ß-adrenoceptor antagonist propranolol, the glycine receptor antagonist strychnine or ginkgolide B, and the GABA(A) receptor antagonist bicuculline or 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide [(gabazine) SR95531]. TTX only inhibited clasp fiber relaxations. L-NAME and propranolol inhibited, and ginkgolide B was ineffective in both. SR95531 was ineffective in clasp fibers and partially effective in sling fibers. Strychnine and bicuculline prevented relaxations with low potency, indicating actions not on glycine or GABA(A) receptors but more consistent with nicotinic receptor blockade. Bethanechol-precontracted fibers were relaxed by the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine and by the ß-adrenergic agonist isoproterenol (clasp fibers only) but not by the glycine receptor agonist taurine or glycine or the GABA(A) agonist muscimol. These data indicate that nicotinic receptor activation mediates relaxation via release of nitric oxide in clasp and sling fibers, norepinephrine acting on ß-adrenoceptors in clasp fibers, and GABA acting on GABA(A) receptors in sling fibers. Agents that selectively prevent these relaxations may be useful in the treatment of GERD.

Comparison of human and porcine gastric clasp and sling fiber contraction by M2 and M3 muscarinic receptors.

To compare the gastroesophageal junction of the human with the pig, M(2) and M(3) receptor densities and the potencies of M(2) and M(3) muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and M(2) receptors are higher in pig than human clasp and sling fibers. M(3) receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer M(3) receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that M(2) receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that M(3) receptors mediate contraction in pig sling fibers and that M(2) and M(3) receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both M(2) and M(3) muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers.

Minimally invasive measurement of esophageal variceal pressure and wall tension (with video).

BACKGROUND: There is no simple method to measure intravariceal pressure in patients with esophageal varices. OBJECTIVE: Our purpose was to develop a new noninvasive technique to measure resting intravariceal pressure and wall tension. DESIGN: A model was developed. A long balloon (varix) was fitted inside an airtight cylinder (esophagus). Fluid ran through the model varices to maintain 5 different constant pressures. An endoscope was placed in the model esophagus, and pressure was increased by air insufflation. The endoscopy and pressure readings from the esophagus and varix were recorded continuously until variceal collapse. SETTING: Patient studies were done in an endoscopy suite with the patient under fentanyl and midazolam sedation. PATIENTS: Esophageal pressure was measured during air insufflation in patients with varices until the varices collapsed. EUS was used to measure radius and wall thickness to calculate wall tension. RESULTS: In the varix model, the mean (SD) intraluminal esophageal pressures at variceal flattening for the model varices at 5, 10, 15, 20, and 25 mm Hg were 5.69 (0.34), 11 (0.32), 15.72 (0.51), 21.55 (0.63), and 25.8 (0.14) mm Hg. The correlation between actual and measured variceal pressure in the model at variceal flattening was r = 0.98. In the patients, a total of 10 varices in 3 patients were evaluated. The mean (SD) for the varices in each subject was 12.16 (2.4), 23.2 (1.3), and 6.5 (2.2) mm Hg for subjects 1, 2, and 3, respectively. CONCLUSION: Standard endoscopy with air insufflation and manometry can be used as an accurate, simple, and reproducible method to measure intravariceal pressure.

Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.