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304 Effect of Gastric Banding on Esophageal Function, Gastroesophageal Reflux and Gastric Emptying in Morbidly Obese Adolescents
baseline to pre-surgery (31.96 ± 4.23 vs 16.63 ± 2.18, p=0.011) and then increased following surgery (16.63 ± 2.18 vs 30.77 ± 4.45, p=0.015). There were no differences in gastric emptying rate, total acid reflux index and number of non-acidic GOR episodes. Acidic GOR episodes at post surgery follow up were reduced (32.3 ± 6.5 vs 14.6 ± 3.8, p=0.007). For patients, only physical QOL scores improved both after the 4wk VLCD and at post surgery follow up. Conclusions:Gastric banding appears to be effective short term weight loss therapy. Reduced esophageal bolus flow resistance indicating improved esophageal function after the 4wk VLCD probably relates to reduced gastric pressure secondary to weight loss. Gastric banding placement had no effect on GOR and gastric emptying rate while improving QOL scores. 305 Predictive Model of Mortality for Bariatric Patients Carlos A. Macias, Bryan J. Sandler, Joslin Cheverie, Tazo Inui, Alisa M. Coker, Juan S. Barajas-Gamboa, Mark A. Talamini, David Chang, Santiago Horgan, Garth R. Jacobsen INTRODUCTION: Over the past 20 years, bariatric surgery has grown exponentially. By year 2000 with the increased number techniques and performing surgeons, mortality of bariatric patients showed huge disparities. Our aim was to evaluate several factors in order to predict mortality among bariatric patients. METHODS: We utilized the Office of Statewide Health Planning and Development of California database from year 1995 till 2010 and identified all obese patients that underwent a bariatric procedure. We analyzed the following parameters using multivariate logistic regression models in an attempt to predict mortality: age, gender, race, tobacco use, coexisting conditions (including cardiac, pulmonary, coagulation and mental disorders, diabetes, renal failure, cancer, collagen diseases, drug and alcohol abuse, obesity, weight loss, and fluid and electrolyte disorders), type of bariatric procedure: laparoscopic and open gastric band, laparoscopic and open gastric bypass and open sleeve gastrectomy. RESULTS: We identified 151,567 patients that underwent a bariatric operation in California from 1995 till 2010. The Area Under the Receiving Operator Curve (ROC) for our model was 0.89, suggesting very good discrimination. We identified the following predictors of mortality in bariatric patients: age (OR 1.06, 95%CI 1.05-1.08), male gender (OR 2.26 95%CI 1.7-3.0), coexisting cardiac heart failure (OR 3.98, 95%CI 2.65-5.97), coexisting cardiac arrhythmias (OR1.80, 95%CI 1.20-2.71), coexisting chronic renal failure (OR4.19, 95%CI 2.66-6.62). Type of surgery was a significant predictor of mortality. Patients who underwent open procedures had higher odds of dying compared to those who had a laparoscopic approach (OR 14.93, 95%CI 7.55-29.52). After accounting for differences between open/laparoscopic approach, there was no difference between procedures.
281 Mutant K-RAS in the Epithelial Stem Cell Compartment Promotes Symmetric Stem Cell Division and Crypt Fission but Not Tumorigenesis in the Mouse Intestine and Colon Dana J. Lukin, Samuel Asfaha, Christoph B. Westphalen, Anil K. Rustgi, Timothy C. Wang K-ras is a proto-oncogene frequently mutated in colorectal neoplasms. Tissue-specific expression of mutant K-ras in the colon promotes hyperplasia and activated K-ras is associated with accelerated tumor progression. However, activated K-ras does not lead to expansion of the Lgr5+ crypt base columnar (CBC) stem cell compartment. In the intestine, generation of new crypts is believed to occur through symmetric division of stem cells, leading to crypt fission. Using a new K19-BAC-CreER transgenic line crossed to a ROSA26r(LacZ or GFP) reporter line, we recently demonstrated that cytokeratin-19 (Krt19), an intermediate filament protein, is a marker of stem cells in the intestine and colon. To further study the role of mutant K-ras in intestinal homeostasis and cancer, conditional expression of mutant K-Ras in intestinal stem cells was achieved by crossing Krt19-BAC-CreER or Lgr5-EGFP-IRES-CreER mice to Lox-STOP-Lox-K-rasG12D mice. Additional introduction of the R26-TomatoGFP and R26-LacZ alleles allowed formal lineage tracing from K-ras mutated stem cells. Consistent with the labeling of long-lived stem cells both Krt19 and Lgr5 marked cells gave rise to positively labeled crypt glands for greater than 52 weeks following tamoxifen induction. Upon expression of K-rasG12D within the epithelial stem cell compartment, mice remained viable and did not spontaneously develop tumors of the intestine or colon. Lineage tracing in control animals not expressing mutant K-ras yielded primarily single and double labeled crypt units, whereas, K-rasG12D expressing mice demonstrated increased contiguously labeled glands in the small intestine and colon, consistent with increased crypt fission. Accordingly, Ki67 proliferative index was increased within colonic (Lgr5 lineage) and small intestinal (Lgr5 and Krt-19 lineage) crypts expressing K-rasG12D. Interestingly, we also observed branching of small intestinal villi exclusively within the crypt-villus units of animals expressing mutant K-ras. Colons from mice expressing K-rasG12D had higher numbers of branching crypts and greater crypt height than control animals. These data indicate that, in addition to increased epithelial proliferation, expression of mutant K-ras in intestinal stem cells induces symmetric stem cell division leading to crypt fission, that on its own is not sufficient to induce tumorigenesis.
306 Randomized Double-Blind Sham-Controlled Trial of Transoral Outlet Reduction for Weight Regain After Roux-en-Y Gastric Bypass: Single-Center Results Nitin Kumar, Michele B. Ryan, Christopher C. Thompson Background: Weight regain after Roux-en-Y gastric bypass (RYGB) is correlated with dilated gastrojejunal anastomosis (GJA). Endoscopic sutured transoral outlet reduction (TORe) is potentially a safe and effective modality for management of dilated GJA. Aim: To analyze the effectiveness of TORe using an endoscopic suction-based mucosal suturing device for therapy of weight regain in patients post-RYGB. Methods: 25 post-RYGB patients with weight regain and GJA diameter .2cm were randomly assigned to TORe using a suctionbased suturing device (n=16) or to a sham procedure (n=9). Patients and investigators were blinded for six months. Means were compared using Student's t-test and proportions were compared using Fisher's Exact test. All statistics are reported as mean ± SEM. Results: Baseline characteristics are reported in Table 1. There were no significant differences in baseline demographics, weight regain, or postoperative anatomy. TORe procedure time was 96.3 ±9.3 minutes. GJA was reduced to 3.6 ±0.3 mm and pouch diameter was reduced to 38.8 ±2.5 mm after placement of 4.3 ±0.3 plications. There were no device-related complications or adverse outcomes. Results are reported in Table 2. Patients in the TORe group experienced significantly greater excess weight loss (%EWL) over three months and six months than patients in the sham group. Waist size reduction was significantly greater in the TORe group at 3 months, and this persisted at six months. On a 10-point visual analog satiety scale, there was a trend towards greater satiety in the TORe group at six months. Based on weight loss results, the number needed to treat to achieve 15% EWL at six months is 3. Conclusions: The device demonstrated safety for transoral outlet reduction. Patients undergoing TORe for weight regain demonstrated significantly higher %EWL and significantly more waist size reduction than patients undergoing the sham procedure. Longer-term studies are needed to assess the durability of these results. Use of newer full-thickness suturing devices may result in improved outcomes. Table 1: Baseline characteristics
304 Effect of Gastric Banding on Esophageal Function, Gastroesophageal Reflux and Gastric Emptying in Morbidly Obese Adolescents Stamatiki Kritas, Sanjeev Khurana, Jacob Chisholm, Richard T. Couper, Lilian Kow, Taher Omari Background: Obesity in adolescents has reached epidemic proportions. Gastric banding is being considered as a treatment as it offers the advantages of adjustability and reversibility and is very effective in reducing food intake leading to successful weight loss in adults. Currently there is a poor understanding of the physiological effects of this procedure and the factors that lead to efficacy as well as predispose patients to complications. The aim of this study was to determine the effect of gastric banding on esophageal function, gastroesophageal reflux (GOR) and gastric emptying in morbidly obese adolescents. Methods: 15 morbidly obese adolescents (6 male, 16.8±1.2yrs), undergoing laparoscopic gastric banding were recruited. All patients were investigated by high resolution impedance manometry (HRIM), 24h pH/impedance monitoring and gastric emptying 13C-Na octanoate breath test. Investigations were performed at 3 study timepoints (baseline, pre surgery and 6-12 months post-surgery). At pre-surgery all patients had completed 4 weeks of a very low calorie diet (VLCD, Optifast®). In addition at all 3 study timepoints patients completed quality of life (QOL) questionnaires on perception of physical and psychosocial status. Results: After the 4wk VLCD patients achieved a significant weight loss compared to baseline (134.2 ± 6.4 vs 126.6 ± 6.9kg, p=0.004) and at post surgery follow up (105.0 ±7.2kg, p ,0.001). Esophageal pressure flow index (PFI), a marker of bolus flow resistance, reduced from
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AGA Abstracts
AGA Abstracts
from endoscopic biopsies without the need for additional stem cell enrichment based on cell sorting and 3) significantly expand organoids as either enriched for stem cells or as differentiated cells that retain a site-specific program of differentiation and are free from mesenchymal contamination. Here we present an improved isolation and culture method that addresses these needs. METHOD: Human gastrointestinal epithelial cells were cultured using an adaptation of our published mouse epithelium culturing system. This system utilizes conditioned media collected from an L cell line (L-WRN) engineered to secrete the factors required for epithelial stem cell growth (Wnt3a, R-spondin3 and Noggin). Importantly, cultures were established from biopsy tissue (2-3 biopsies with area of 6-24 mm2) collected during routine endoscopy. Cell sorting enrichment was not required. Epithelial cells were maintained as 3-dimensional organoids in basement membrane matrix (Matrigel) with 50% conditioned media (1:1 dilution with fresh crypt culture media). Analysis of cellular markers of proliferative and differentiated cells was performed using qRT-PCR, immunofluorescence and flow assisted cell sorting. RESULTS: Human epithelial cultures were established from multiple regions of the gastrointestinal tract, including small intestine (n=9), colon (n=23), stomach (n=2) and Barrett's esophagus (n=2). Biopsy donors included healthy and IBD patients. Mesenchymal cells carried over from the isolation process were eliminated during initial passages. Subsequent studies focused on intestinal organoids, which were maintained for .40 passages (.120 days) and were able to be stored as frozen stocks. In 50% conditioned media, the majority of cells were proliferative and expressed high levels of the stem cell markers, LGR5 and OLFM4. Accordingly, organoids expanded at a rapid rate and were split 1:3 every 3 days. An intestine-specific program of differentiation was achieved using lower percentage conditioned media plus additional factors, including the Notch inhibitor DAPT. CONCLUSIONS: We have generated a culture system that will allow for large-scale, functional experiments of human intestinal epithelial cells. This system will enable us to create a biobank of human epithelial cell lines. Moreover, because cultures can be established from endoscopic biopsies, tissue donors could potentially be pre-selected based on genotype to facilitate studies of the interactions between genotype and environmental factors.
281 Mutant K-RAS in the Epithelial Stem Cell Compartment Promotes Symmetric Stem Cell Division and Crypt Fission but Not Tumorigenesis in the Mouse Intestine and Colon Dana J. Lukin, Samuel Asfaha, Christoph B. Westphalen, Anil K. Rustgi, Timothy C. Wang K-ras is a proto-oncogene frequently mutated in colorectal neoplasms. Tissue-specific expression of mutant K-ras in the colon promotes hyperplasia and activated K-ras is associated with accelerated tumor progression. However, activated K-ras does not lead to expansion of the Lgr5+ crypt base columnar (CBC) stem cell compartment. In the intestine, generation of new crypts is believed to occur through symmetric division of stem cells, leading to crypt fission. Using a new K19-BAC-CreER transgenic line crossed to a ROSA26r(LacZ or GFP) reporter line, we recently demonstrated that cytokeratin-19 (Krt19), an intermediate filament protein, is a marker of stem cells in the intestine and colon. To further study the role of mutant K-ras in intestinal homeostasis and cancer, conditional expression of mutant K-Ras in intestinal stem cells was achieved by crossing Krt19-BAC-CreER or Lgr5-EGFP-IRES-CreER mice to Lox-STOP-Lox-K-rasG12D mice. Additional introduction of the R26-TomatoGFP and R26-LacZ alleles allowed formal lineage tracing from K-ras mutated stem cells. Consistent with the labeling of long-lived stem cells both Krt19 and Lgr5 marked cells gave rise to positively labeled crypt glands for greater than 52 weeks following tamoxifen induction. Upon expression of K-rasG12D within the epithelial stem cell compartment, mice remained viable and did not spontaneously develop tumors of the intestine or colon. Lineage tracing in control animals not expressing mutant K-ras yielded primarily single and double labeled crypt units, whereas, K-rasG12D expressing mice demonstrated increased contiguously labeled glands in the small intestine and colon, consistent with increased crypt fission. Accordingly, Ki67 proliferative index was increased within colonic (Lgr5 lineage) and small intestinal (Lgr5 and Krt-19 lineage) crypts expressing K-rasG12D. Interestingly, we also observed branching of small intestinal villi exclusively within the crypt-villus units of animals expressing mutant K-ras. Colons from mice expressing K-rasG12D had higher numbers of branching crypts and greater crypt height than control animals. These data indicate that, in addition to increased epithelial proliferation, expression of mutant K-ras in intestinal stem cells induces symmetric stem cell division leading to crypt fission, that on its own is not sufficient to induce tumorigenesis.
306 Randomized Double-Blind Sham-Controlled Trial of Transoral Outlet Reduction for Weight Regain After Roux-en-Y Gastric Bypass: Single-Center Results Nitin Kumar, Michele B. Ryan, Christopher C. Thompson Background: Weight regain after Roux-en-Y gastric bypass (RYGB) is correlated with dilated gastrojejunal anastomosis (GJA). Endoscopic sutured transoral outlet reduction (TORe) is potentially a safe and effective modality for management of dilated GJA. Aim: To analyze the effectiveness of TORe using an endoscopic suction-based mucosal suturing device for therapy of weight regain in patients post-RYGB. Methods: 25 post-RYGB patients with weight regain and GJA diameter .2cm were randomly assigned to TORe using a suctionbased suturing device (n=16) or to a sham procedure (n=9). Patients and investigators were blinded for six months. Means were compared using Student's t-test and proportions were compared using Fisher's Exact test. All statistics are reported as mean ± SEM. Results: Baseline characteristics are reported in Table 1. There were no significant differences in baseline demographics, weight regain, or postoperative anatomy. TORe procedure time was 96.3 ±9.3 minutes. GJA was reduced to 3.6 ±0.3 mm and pouch diameter was reduced to 38.8 ±2.5 mm after placement of 4.3 ±0.3 plications. There were no device-related complications or adverse outcomes. Results are reported in Table 2. Patients in the TORe group experienced significantly greater excess weight loss (%EWL) over three months and six months than patients in the sham group. Waist size reduction was significantly greater in the TORe group at 3 months, and this persisted at six months. On a 10-point visual analog satiety scale, there was a trend towards greater satiety in the TORe group at six months. Based on weight loss results, the number needed to treat to achieve 15% EWL at six months is 3. Conclusions: The device demonstrated safety for transoral outlet reduction. Patients undergoing TORe for weight regain demonstrated significantly higher %EWL and significantly more waist size reduction than patients undergoing the sham procedure. Longer-term studies are needed to assess the durability of these results. Use of newer full-thickness suturing devices may result in improved outcomes. Table 1: Baseline characteristics
304 Effect of Gastric Banding on Esophageal Function, Gastroesophageal Reflux and Gastric Emptying in Morbidly Obese Adolescents Stamatiki Kritas, Sanjeev Khurana, Jacob Chisholm, Richard T. Couper, Lilian Kow, Taher Omari Background: Obesity in adolescents has reached epidemic proportions. Gastric banding is being considered as a treatment as it offers the advantages of adjustability and reversibility and is very effective in reducing food intake leading to successful weight loss in adults. Currently there is a poor understanding of the physiological effects of this procedure and the factors that lead to efficacy as well as predispose patients to complications. The aim of this study was to determine the effect of gastric banding on esophageal function, gastroesophageal reflux (GOR) and gastric emptying in morbidly obese adolescents. Methods: 15 morbidly obese adolescents (6 male, 16.8±1.2yrs), undergoing laparoscopic gastric banding were recruited. All patients were investigated by high resolution impedance manometry (HRIM), 24h pH/impedance monitoring and gastric emptying 13C-Na octanoate breath test. Investigations were performed at 3 study timepoints (baseline, pre surgery and 6-12 months post-surgery). At pre-surgery all patients had completed 4 weeks of a very low calorie diet (VLCD, Optifast®). In addition at all 3 study timepoints patients completed quality of life (QOL) questionnaires on perception of physical and psychosocial status. Results: After the 4wk VLCD patients achieved a significant weight loss compared to baseline (134.2 ± 6.4 vs 126.6 ± 6.9kg, p=0.004) and at post surgery follow up (105.0 ±7.2kg, p ,0.001). Esophageal pressure flow index (PFI), a marker of bolus flow resistance, reduced from
S-63
AGA Abstracts
AGA Abstracts
from endoscopic biopsies without the need for additional stem cell enrichment based on cell sorting and 3) significantly expand organoids as either enriched for stem cells or as differentiated cells that retain a site-specific program of differentiation and are free from mesenchymal contamination. Here we present an improved isolation and culture method that addresses these needs. METHOD: Human gastrointestinal epithelial cells were cultured using an adaptation of our published mouse epithelium culturing system. This system utilizes conditioned media collected from an L cell line (L-WRN) engineered to secrete the factors required for epithelial stem cell growth (Wnt3a, R-spondin3 and Noggin). Importantly, cultures were established from biopsy tissue (2-3 biopsies with area of 6-24 mm2) collected during routine endoscopy. Cell sorting enrichment was not required. Epithelial cells were maintained as 3-dimensional organoids in basement membrane matrix (Matrigel) with 50% conditioned media (1:1 dilution with fresh crypt culture media). Analysis of cellular markers of proliferative and differentiated cells was performed using qRT-PCR, immunofluorescence and flow assisted cell sorting. RESULTS: Human epithelial cultures were established from multiple regions of the gastrointestinal tract, including small intestine (n=9), colon (n=23), stomach (n=2) and Barrett's esophagus (n=2). Biopsy donors included healthy and IBD patients. Mesenchymal cells carried over from the isolation process were eliminated during initial passages. Subsequent studies focused on intestinal organoids, which were maintained for .40 passages (.120 days) and were able to be stored as frozen stocks. In 50% conditioned media, the majority of cells were proliferative and expressed high levels of the stem cell markers, LGR5 and OLFM4. Accordingly, organoids expanded at a rapid rate and were split 1:3 every 3 days. An intestine-specific program of differentiation was achieved using lower percentage conditioned media plus additional factors, including the Notch inhibitor DAPT. CONCLUSIONS: We have generated a culture system that will allow for large-scale, functional experiments of human intestinal epithelial cells. This system will enable us to create a biobank of human epithelial cell lines. Moreover, because cultures can be established from endoscopic biopsies, tissue donors could potentially be pre-selected based on genotype to facilitate studies of the interactions between genotype and environmental factors.