Thai H. Pham

Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells

Background Patients who have esophageal eosinophilia without gastroesophageal reflux disease (GERD) nevertheless can respond to proton pump inhibitors (PPIs), which can have anti-inflammatory actions independent of effects on gastric acid secretion. In esophageal cell cultures, omeprazole has been reported to inhibit Th2 cytokine-stimulated expression of eotaxin-3, an eosinophil chemoattractant contributing to esophageal eosinophilia in eosinophilic esophagitis (EoE). The objective of this study was to elucidate molecular mechanisms underlying PPI inhibition of IL-4-stimulated eotaxin-3 production by esophageal cells. Methods/Findings Telomerase-immortalized and primary cultures of esophageal squamous cells from EoE patients were treated with IL-4 in the presence or absence of acid-activated omeprazole or lansoprazole. We measured eotaxin-3 protein secretion by ELISA, mRNA expression by PCR, STAT6 phosphorylation and nuclear translocation by Western blotting, eotaxin-3 promoter activation by an exogenous reporter construct, and STAT6, RNA polymerase II, and trimethylated H3K4 binding to the endogenous eotaxin-3 promoter by ChIP assay. Omeprazole in concentrations ≥5 µM significantly decreased IL-4-stimulated eotaxin-3 protein secretion and mRNA expression. Lansoprazole also blocked eotaxin-3 protein secretion. Omeprazole had no effect on eotaxin-3 mRNA stability or on STAT6 phosphorylation and STAT6 nuclear translocation. Rather, omeprazole blocked binding of IL-4-stimulated STAT6, RNA polymerase II, and trimethylated H3K4 to the eotaxin-3 promoter. Conclusions/Significance PPIs, in concentrations achieved in blood with conventional dosing, significantly inhibit IL-4-stimulated eotaxin-3 expression in EoE esophageal cells and block STAT6 binding to the promoter. These findings elucidate molecular mechanisms whereby patients with Th2 cytokine-driven esophageal eosinophilia can respond to PPIs, independent of effects on gastric acid secretion.

The impact of surgical site infection on the development of incisional hernia and small bowel obstruction in colorectal surgery

INTRODUCTION The purpose of this study was to evaluate the long-term complications of surgical site infection (SSI) in the colorectal population, specifically its association with incisional hernia and small bowel obstruction. METHODS Using standardized definitions of SSI, a retrospective review of patients undergoing transabdominal colorectal surgery from January 2002 to December 2005 was performed. Primary outcomes included incisional hernia and small bowel obstruction in patients with SSIs. RESULTS A total of 443 patients were analyzed. The median surgical follow-up was 12 months (2-3,091 days). Infections were identified in 101 (23%) cases. There were 99 cases (22%) of incisional hernia and 32 cases (7%) of small bowel obstruction. Logistic regression revealed SSI to be independently associated with incisional hernia after adjusting for clinical covariates (adjusted odds ratio = 2.23, P = .003; 95% confidence interval, 1.3-3.8). Patients with incisional hernia were 1.9 times more likely to have had an SSI (36.3% vs 18.8%, P ≤ .01). They required a longer operative time (224 minutes vs 198 minutes, P = .03), had an increased body mass index (29.0 vs 26.8, P ≤ .01), and had increased estimated blood loss (363 vs 289, mL, P = .03). Small bowel obstruction was significantly associated with operations involving the rectum (11.5% in operations involving the rectum vs 5.9% in nonrectal operations, P = .05), increased estimated blood loss (409 ml vs 297 ml, P = .04), and red blood cell transfusion (15.5% with transfusion vs 5.7% without, P = .01). SSI was not an independent predictor of small bowel obstruction (adjusted odds ratio = 1.05, P = .91; 95% confidence interval, .45-2.5). CONCLUSIONS Patients with an SSI were 1.9 times more likely to have an incisional hernia than those without an SSI. An SSI after colorectal surgery was a risk factor for the development of incisional hernia but was not a risk factor for small bowel obstruction in our population.

Comparison of perioperative outcomes after combined thoracoscopic-laparoscopic esophagectomy and open Ivor–Lewis esophagectomy

BACKGROUND Thoracoscopic-laparoscopic esophagectomy (TLE) has gained popularity in specialized centers. This study compares the perioperative outcomes of TLE and Ivor-Lewis esophagectomy (ILE). METHODS Forty-four consecutive TLEs were compared with 46 historical ILEs. Outcomes included surgical time and blood loss, hospital length of stay, 30-day mortality rate, and complications. RESULTS TLE took longer to perform (543 vs 437 min; P < .01) than ILE, but produced less blood loss (407 vs 780 mL; P < .01). The median length of stay and 30-day mortality did not differ between groups. Cardiovascular (41% for TLE vs 30% for ILE; P = .19) and pulmonary complications (31% TLE vs 30% ILE; P = 1.0) occurred frequently in both groups, but TLE patients had fewer wound complications (4% TLE vs 17% ILE; P = .05). CONCLUSIONS Despite longer surgical times, TLE produced decreased intraoperative blood loss and wound complications. These findings suggest that with further technical refinement TLE may ameliorate the morbidity seen with ILE.

Association of Acute Gastroesophageal Reflux Disease With Esophageal Histologic Changes

IMPORTANCE The histologic changes associated with acute gastroesophageal reflux disease (GERD) have not been studied prospectively in humans. Recent studies in animals have challenged the traditional notion that reflux esophagitis develops when esophageal surface epithelial cells are exposed to lethal chemical injury from refluxed acid. OBJECTIVE To evaluate histologic features of esophageal inflammation in acute GERD to study its pathogenesis. DESIGN, SETTING, AND PARTICIPANTS Patients from the Dallas Veterans Affairs Medical Center who had reflux esophagitis successfully treated with proton pump inhibitors (PPIs) began 24-hour esophageal pH and impedance monitoring and esophagoscopy (including confocal laser endomicroscopy [CLE]) with biopsies from noneroded areas of distal esophagus at baseline (taking PPIs) and at 1 week and 2 weeks after stopping the PPI medication. Enrollment began May 2013 and follow-up ended July 2015. INTERVENTIONS PPIs stopped for 2 weeks. MAIN OUTCOMES AND MEASURES Twelve patients (men, 11; mean age, 57.6 year [SD, 13.1]) completed the study. Primary outcome was change in esophageal inflammation 2 weeks after stopping the PPI medication, determined by comparing lymphocyte, eosinophil, and neutrophil infiltrates (each scored on a 0-3 scale) in esophageal biopsies. Also evaluated were changes in epithelial basal cell and papillary hyperplasia, surface erosions, intercellular space width, endoscopic grade of esophagitis, esophageal acid exposure, and mucosal impedance (an index of mucosal integrity). RESULTS At 1 week and 2 weeks after discontinuation of PPIs, biopsies showed significant increases in intraepithelial lymphocytes, which were predominantly T cells (median [range]: 0 (0-2) at baseline vs 1 (1-2) at both 1 week [P = .005] and 2 weeks [P = .002]); neutrophils and eosinophils were few or absent. Biopsies also showed widening of intercellular spaces (confirmed by CLE), and basal cell and papillary hyperplasia developed without surface erosions. Two weeks after stopping the PPI medication, esophageal acid exposure increased (median: 1.2% at baseline to 17.8% at 2 weeks; Δ, 16.2% [95% CI, 4.4%-26.5%], P = .005), mucosal impedance decreased (mean: 2671.3 Ω at baseline to 1508.4 Ω at 2 weeks; Δ, 1162.9 Ω [95% CI, 629.9-1695.9], P = .001), and all patients had evidence of esophagitis. CONCLUSIONS AND RELEVANCE In this preliminary study of 12 patients with severe reflux esophagitis successfully treated with PPI therapy, stopping PPI medication was associated with T lymphocyte-predominant esophageal inflammation and basal cell and papillary hyperplasia without loss of surface cells. If replicated, these findings suggest that the pathogenesis of reflux esophagitis may be cytokine-mediated rather than the result of chemical injury. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01733810.

Comparison of Laparoscopic Inversion Esophagectomy and Open Transhiatal Esophagectomy for High-Grade Dysplasia and Stage I Esophageal Adenocarcinoma

HYPOTHESIS The perioperative outcomes of laparoscopic inversion esophagectomy (LIE) are comparable to those of open transhiatal esophagectomy (THE), with potential benefits related to the use of minimally invasive techniques. DESIGN Case-control study. SETTING Tertiary care university hospital. PATIENTS AND INTERVENTIONS From July 1, 2003, through March 31, 2008, 21 consecutive patients underwent LIE for high-grade dysplasia or clinical stage I esophageal cancer. We compared these patients with 21 stage-matched control patients treated with THE from August 1, 1995, through August 31, 2003. MAIN OUTCOME MEASURES Operative time, blood loss, length of hospital stay, perioperative complications, and disease-free survival. RESULTS Mean (SD) operative times for LIE (399 [86] minutes) and THE (407 [127] minutes) were not significantly different (P = .80). Patients undergoing LIE had significantly lower intraoperative blood loss (168 mL; P < .001) and overall length of hospital stay (10 days; P = .03) compared with those in the THE group (526 mL and 14 days, respectively). Complication rates were not significantly different between the groups. With a median follow-up of 29 months, there has been 1 systemic recurrence in the LIE group. CONCLUSIONS Laparoscopic inversion esophagectomy is a safe and effective approach to the treatment of high-grade dysplasia and early esophageal adenocarcinoma. Compared with THE, LIE decreases operative blood loss and length of hospital stay without increasing the operative time, morbidity, or mortality related to esophagectomy.

Minimally Invasive Esophagectomy Provides Equivalent Oncologic Outcomes to Open Esophagectomy for Locally Advanced (Stage II or III) Esophageal Carcinoma

BACKGROUND Minimally invasive esophagectomy (MIE) has been performed at specialized centers for 15 years, but few studies have looked at outcomes in patients with locally advanced cancers, and few studies have provided long-term survival comparison with Ivor Lewis esophagectomy (ILE) to determine oncologic benefit or equivalence of MIE. HYPOTHESIS Minimally invasive esophagectomy for locally advanced esophageal carcinoma has similar oncologic outcomes to traditional open ILE with less associated short-term morbidity and mortality. DESIGN Retrospective comparison of patients with stage II or III esophageal carcinoma undergoing 3-field MIE compared with open ILE. SETTING University medical center. PATIENTS From 1995 to 2009, 64 patients who underwent MIE (33 patients) or ILE (31 patients) with clinical stage II or III esophageal cancer were compared. MAIN OUTCOME MEASURES Primary end points included operative performance, morbidity, mortality, hospital stay, and survival. RESULTS No differences were noted between the groups in demographics, neoadjuvant therapy use (P = .22), resection completeness (R0:R1) (P = .57), length of stay (P = .59), intensive care unit stay (P = .36), anastomotic leak (P = 1.0), pulmonary morbidity (P = .26), and mortality (P = 1.0). Median follow-up was 19 months for MIE and 17 months for ILE. Survival at 2 years was 55% for MIE (18 of 33 patients) and 32% for ILE (10 of 31 patients) while disease-free survival was 55% for MIE (18) and 26% for ILE (8). CONCLUSIONS Our survival analysis shows divergent curves that favor MIE but have not yet reached statistical significance. The oncologic outcomes of MIE are comparable to that of ILE 2 years after resection.

In oesophageal squamous cells exposed to acidic bile salt medium, omeprazole inhibits IL-8 expression through effects on nuclear factor-κB and activator protein-1

Objective Oesophagitis might result from the effects of chemokines produced by oesophageal cells in response to gastro-oesophageal reflux, and not solely from the direct, caustic effects of refluxed gastric juice. Proton pump inhibitors (PPI) can block chemokine production through mechanisms independent of their antisecretory effects. We studied omeprazole effects on chemokine production by oesophageal epithelial cells exposed to acidic bile salts. Design Human primary and telomerase-immortalised oesophageal squamous cells were exposed to acidic bile salt medium with or without omeprazole pretreatment. Interleukin (IL)-8 expression was determined by RT-PCR and ELISA. IL-8 promoter activity was measured by luciferase reporter assay. Binding of NF-κB and AP-1 subunits to the IL-8 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Immune cell migration induced by conditioned medium was determined by a double-chamber migration assay system. Results Acidic bile salt medium caused oesophageal epithelial cells to express IL-8 mRNA and protein by activating the IL-8 promoter through NF-κB and AP-1 binding. Omeprazole inhibited that acidic bile salt-stimulated IL-8 expression by blocking the nuclear translocation of p65 (an NF-κB subunit), and by blocking the binding of p65, c-jun and c-fos (AP-1 subunits) to the IL-8 promoter. Omeprazole also blocked the ability of conditioned medium from cells exposed to acidic bile salts to induce immune cell migration. Conclusions In oesophageal squamous epithelial cells, omeprazole inhibits IL-8 expression through effects on NF-κB and AP-1 that are entirely independent of effects on gastric acid secretion. These previously unrecognised PPI effects might contribute to the healing of reflux oesophagitis.

Autocrine VEGF Signaling Promotes Proliferation of Neoplastic Barrett's Epithelial Cells Through a PLC-Dependent Pathway

BACKGROUND & AIMS Tumor cells express vascular endothelial growth factor (VEGF), which induces angiogenesis. VEGF also activates VEGF receptors (VEGFRs) on or within tumor cells to promote their proliferation in an autocrine fashion. We studied the mechanisms of autocrine VEGF signaling in Barretts esophagus cells. METHODS Using Barretts epithelial cell lines, we measured VEGF and VEGFR messenger RNA and protein, and studied the effects of VEGF signaling on cell proliferation and VEGF secretion. We studied the effects of inhibiting factors in this pathway on levels of phosphorylated phospholipase Cγ1 (PLCG1), protein kinase C, and extracellular signal-regulated kinases (ERK)1/2. We performed immunohistochemical analysis of phosphorylated VEGFR2 on esophageal adenocarcinoma tissues. We studied effects of sunitinib, a VEGFR2 inhibitor, on proliferation of neoplastic cells and growth of xenograft tumors in mice. RESULTS Neoplastic and non-neoplastic Barretts cells expressed VEGF and VEGFR2 messenger RNA and protein, with higher levels in neoplastic cells. Incubation with recombinant human VEGF significantly increased secretion of VEGF protein and cell number; knockdown of PLCG1 markedly reduced the recombinant human VEGF-stimulated increase in levels of phosphorylated PLCG1 and phosphorylated ERK1/2 in neoplastic cells. Esophageal adenocarcinoma tissues showed immunostaining for phosphorylated VEGFR2. Sunitinib inhibited VEGF signaling in neoplastic cells and reduced weight and volume of xenograft tumors in mice. CONCLUSIONS Neoplastic and non-neoplastic Barretts epithelial cells have autocrine VEGF signaling. In neoplastic Barretts cells, VEGF activation of VEGFR2 initiates a PLCG1-protein kinase C-ERK pathway that promotes proliferation and is self-sustaining (by causing more VEGF production). Strategies to reduce autocrine VEGF signaling (eg, with sunitinib) might be used to prevent or treat cancer in patients with Barretts esophagus.

Decreased Conduit Perfusion Measured by Spectroscopy Is Associated With Anastomotic Complications

BACKGROUND Gastric conduit ischemia during esophagectomy likely contributes to high anastomotic complication rates, yet we lack a reliable method to assess gastric conduit perfusion. We hypothesize that optical fiber spectroscopy (OFS) can reliably assess conduit perfusion and that the degree of intraoperative gastric ischemia is associated with subsequent anastomotic complications. METHODS During esophagectomy, OFS was used to measure oxygen saturation (SaO(2)) and blood volume fraction (BVF) in the distal gastric conduit at baseline and after gastric devascularization, conduit formation, and transposition. The SaO(2) and BVF readings were correlated to clinical outcomes. RESULTS The OFS measurements were obtained in 23 patients during esophagectomy, four of whom previously underwent gastric ischemic conditioning. Eight patients developed anastomotic complications. Compared with baseline, conduit creation produced a 29.4% reduction in SaO(2) (p < 0.01), while BVF increased by 28% (p = 0.06). Patients with subsequent anastomotic complications demonstrated a 52.5% decrease in SaO(2) upon conduit creation compared with 15.1% in patients without complications (p = 0.01). Patients who underwent ischemic conditioning did not develop significant changes in SaO(2) (p = 0.72) or BVF (p = 0.5) upon gastric conduit creation. CONCLUSIONS Intraoperative OFS demonstrates significant alterations in gastric conduit oxygenation during esophageal replacement, which may be tempered by gastric ischemic conditioning. The degree of intraoperative gastric ischemia resulting from gastric conduit creation is associated with the development of anastomotic complications, suggesting that OFS is useful for assessing changes in conduit oxygenation during esophagectomy. Further studies are needed to refine this technology and investigate the clinical utility of intraoperative conduit oxygenation data.

In Barrett's esophagus patients and Barrett's cell lines, ursodeoxycholic acid increases antioxidant expression and prevents DNA damage by bile acids

Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-κB in Barretts metaplasia, might contribute to carcinogenesis in Barretts esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using nonneoplastic, telomerase-immortalized Barretts cell lines. We took biopsies of Barretts esophagus from 21 patients before and after esophageal perfusion with DCA (250 μM) at baseline and after 8 wk of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral CometAssay, and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barretts metaplasia. Oral UDCA increased GPX1 and catalase levels in Barretts metaplasia and prevented DCA perfusion from inducing DNA damage and NF-κB activation. In cells, DCA-induced DNA damage and NF-κB activation was prevented by 24-h pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-κB activation in Barretts metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barretts esophagus.