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598 Eosinophil Derived TGF-ß1 Activates Human Esophageal Mesenchymal Cells and Alters Esophageal Motility - Implications for Dysphagia in Eosinophilic Esophagitis (EoE)
AGA Abstracts
and were randomized to three treatment regimens: 14 days of lansoprazole, amoxicillin and clarithromycin (PAC-14); 5 days of lansoprazole and amoxicillin followed by 5 days of lansoprazole, clarithromycin and metronidazole (SEQ-10); or 5 days of lansoprazole, amoxicillin, clarithromycin, and metronidazole (PACM-5). Participants had UBTs 6-8 weeks after randomization, and retreatment with 14-day lansoprazole, bismuth, metronidazole and tetracycline was offered to those remaining positive. Program effectiveness was determined from the 1340 (91%) participants with a definitive UBT 48-52 weeks after randomization, and effectiveness absent retesting and retreatment was estimated assuming all UBT-positives at 6-8 weeks remained so. Estimated costs were $12 for PAC-14, $7 for SEQ-10, $6 for PACM-5, $10 for retreatment, and $10 for each UBT. RESULTS: Of the 281 UBT-positive participants at 6-8 weeks, 213 (76%) chose to be retreated, and 143 (51%) completed retreatment; 244 had a definitive 1-year UBT result, of whom 93 (38%) tested negative. The observed 1-year eradication probabilities and estimated costs per treatment success for the three treatment arms were 80.5% and $44 for PAC-14, 79.8% and $39 for SEQ-10, and 77.8% and $39 for PACM-5 (Table). Under the scenario of no retesting and retreatment, the estimated eradication probabilities were 5-8% lower, but the estimated costs per treatment success declined substantially more, to $32 for PAC-14 and $27 for SEQ-10 and PACM-5. The principal contributor to the cost differential was the cost of the follow-up UBTs rather than the low probability of retreatment success. CONCLUSIONS: Data from this Latin American study suggest that repeat UBT testing and retreatment after initial therapy add substantially to program costs but only modestly increase the probability of Hp eradication after one year. In settings where resources are scarce, retesting and retreating do not appear to be indicated for programs to prevent Hp-associated gastric cancer. Increasing the effectiveness of eradication therapies and lowering the risk of recurrence will be better approaches for improving program performance. SUPPORT: Bill & Melinda Gates Foundation grant 43930 and NIH grant CA037429. Estimated one-year outcome and costs by treatment arm with and without retesting and retreatment.
598 Eosinophil Derived TGF-β1 Activates Human Esophageal Mesenchymal Cells and Alters Esophageal Motility - Implications for Dysphagia in Eosinophilic Esophagitis (EoE) Florian Rieder, Ilche T. Nonevski, Jie Ma, Zhufeng Ouyang, Gail West, Cheryl A. Protheroe, Anja Schirbel, John R. Goldblum, Tracey L. Bonfield, Karen M. Harnett, James J. Lee, Ikuo Hirano, Gary W. Falk, Piero Biancani, Claudio Fiocchi Background: Dysphagia is a frequent symptom of EoE and may result from a combination of esophageal fibrosis and motility abnormalities. We investigated mechanisms of dysphagia in EoE by assessing the response of primary human esophageal fibroblasts (HEF), muscle cells (HEMC) and esophageal muscle strips to TGF-β1 and eosinophil-derived products. Spontaneous production of TGF-β1 in esophageal biopsies from EoE and control patients was also investigated. Methods: The presence of eosinophils in the esophageal wall of EoE was investigated by H&E and eosinophil peroxidase (EPX) stain of full thickness sections. Production of fibronectin (FN) and collagen I (Col I) by HEF and HEMC in response to TGF-β1 and eosinophil products was measured. αSMA, ICAM-1 and VCAM-1 expression was assessed by flow cytometry and immunocytochemistry. Eosinophil adhesion assays were performed using the human eosinophil cell line AML14.3D10. TGF-β1 and eosinophil sonicates were tested in esophageal muscle contraction assays assessing neurogenic and acetylcholine (ACh)-induced contraction. TGF-β1 was measured in undernatants of esophageal biopsy organ cultures by ELISA. Results: Activated eosinophils were present in all layers of the esophageal wall. TGF-β1 induced a dose-dependent increase in FN and Col I secretion as well as αSMA expression by HEF and HEMC, indicating transformation to a pro-fibrogenic phenotype. Sonicates of pre-activated eosinophils also increased FN and Col I production by HEF and HEMC, an effect that was inhibited by blocking TGF-β1 and p38MAKP signaling. Eosinophil binding increased by pre-treating HEF and HEMC with TGF-β1 or eosinophil sonicates. Combined TGF-β1 and p38MAKP blockade reduced sonicate-induced eosinophil adhesion and completely abrogated TGF-β1-induced adhesion. TGF-β1 did not modulate VCAM-1 or ICAM-1 expression. Eosinophil sonicates and TGF-β1 reduced electrical field-induced contraction of esophageal muscle strips. Neither eosinophil sonicates nor TGF-β1 influenced ACh-induced contraction, indicating a primary effect on neurotransmitter release without affecting the muscle itself. Significantly higher concentrations of TGF-β1 were present in EoE compared to control esophageal mucosal biopsy cultures. Conclusion: Eosinophils infiltrate all esophageal layers in EoE. Eosinophil-derived TGF-β1 increases matrix production by and eosinophil binding to esophageal mesenchymal cells and reduces neurogenic esophageal smooth muscle contraction. TGF-β1 is present in elevated amounts in EoE compared to controls. These results suggest a direct functional link of TGF-β1 expression with altered fibrogenesis and motility in EoE, both of which may contribute to development of dysphagia.
591 Obese Pediatric Patients Have Increased Cardiovascular Disease Risk Comparable to Pediatric Patients With Familial Dyslipidemia Angela Shannon, Suraj Thangada, Christine Carter-Kent, Ariel E. Feldstein, Michael Rocco, Leslie Cho, Stanley L. Hazen, Naim Alkhouri
599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter. Xi Zhang, Edaire Cheng, Xiaofang Huo, David H. Wang, Qiuyang Zhang, Chunhua Yu, Stuart J. Spechler, Rhonda F. Souza
Pediatric patients with familial dyslipidemia (FD) are known to be at increased risk of developing cardiovascular disease (CVD) and are often treated aggressively in order to prevent manifestations of CVD. Obese pediatric patients are also at increased cardiovascular risk but this risk is less well defined. Myeloperoxidase (MPO) and high-sensitive C-reactive protein (hsCRP), markers associated with cardiovascular disease(CVD) in adults, may also be predictive of future cardiovascular disease in predisposed children. Our aim was to investigate the association between MPO and hsCRP with pediatric obesity and then compare these markers between pediatric patients with FD and those with obesity in order to better describe the CVD risk in obese pediatric patients. Methods: Data was collected prospectively on consecutive patients attending the Pediatric Preventive Cardiology and Metabolic Clinic (PPCMC) and the Genetics Clinic at Cleveland Clinic. Data included demographics, anthropometric and laboratory data. The PPCMC cohort was further divided into groups based on BMI (overweight, obese, severely obese) to investigate the association between MPO and hsCRP and obesity. Then, the overall group of patients was divided based on having a diagnosis of FD or overweight/obesity to compare CVD markers between the two groups. Results: 323 patients were included for analysis- 151 in the overweight/obese group and 172 in the FD group. In the overweight/obese group, mean age was 12.8±3.5 yr, there were 56% males, and 63% of the patients were Caucasian. In the FD group, mean age was 12.2±4.4 yr, there were 52% males, and 80% of the patients were Caucasian. The mean LDL in the overweight/obese group was 107.7±4.3 mg/dL, and that of patients with familial hypercholesterolemia was 205.1±63.6 mg/dL (p<0.0001). Mean MPO in the overweight/ obese group was 417±162 pmol/L and in the FD group was 424±162 pmol/L [CL (55.1,69.7), p=0.41]. Mean hsCRP was 4.9±7.2 mg/L in the obese group and 5.6±26 mg/L in the FD group [CL (3.8, -5.2), p=0.38]. MPO and hsCRP levels both had a positive correlation with BMI%ile (rho 0.42 and 0.47, respectively, p<0.001). Conclusions: This is the first pediatric study evaluating the association between obesity and MPO and comparing CVD risk between overweight/obese pediatric patients and patients with FD. MPO and hsCRP both correlate with increasing BMI in pediatric patients. In addition, though overweight/ obese pediatric patients may have a lower LDL than pediatric patients with familial dyslipidemia, there is no significant difference when comparing nontraditional markers of CVD such as MPO and hsCRP. We conclude that MPO and hsCRP are useful markers to identify obese pediatric patients at increased CVD risk, and obesity may in fact confer a CVD risk burden comparable to patients with FD and earlier medical intervention may be warranted.
AGA Abstracts
Introduction: EoE and GERD can have similar clinical and histological features. It has been proposed that patients with these disorders can be distinguished by their response to proton pump inhibitors (PPIs). This is based on the assumption that reduced gastric acid secretion is the only important effect of PPIs and, therefore, only an acid-peptic disorder like GERD can respond to PPIs. In earlier studies, we showed that omeprazole in high concentration (100 μM) blocked cytokine-stimulated secretion of eotaxin-3 protein (a potent eosinophil chemoattractant) in esophageal squamous cell lines from patients with EoE. This suggests that PPIs might have anti-inflammatory actions independent of their effects on gastric acid secretion. Now, we have studied the effects of omeprazole in lower concentrations, including those that can be achieved in blood with conventional dosage, on IL-4-stimulated eotaxin3 production in primary cultures of esophageal squamous cells from EoE patients. To explore the anti-inflammatory mechanism further, we also studied the effects of omeprazole on IL4-stimulated transcriptional activation of eotaxin-3. Methods: Primary cultures of esophageal squamous cells (EoE1 and EoE2) and telomerase-immortalized esophageal squamous cell lines (EoE1-T and EoE2-T) derived from two patients with EoE were treated with IL-4 (10 ng/ml) in the presence or absence of 1-50 μM doses of acid-activated omeprazole for 48 hours. To address transcriptional activation, EoE1-T and EoE2-T cells were pretreated for 24 hours with 50 μM acid-activated omeprazole before the addition of IL-4. We measured eotaxin-3 protein secretion by ELISA, mRNA by real-time PCR, STAT6 phosphorylation and its nuclear translocation by Western blot, and eotaxin-3 promoter binding by ChIP assay. Results: In primary EoE cells, IL-4-stimulated eotaxin-3 secretion was blocked by omeprazole in a dose-dependent fashion (Table). Similar results were obtained in the EoE cell lines, although does-dependency was not as clear as in the primary cells. Omeprazole had no effect on STAT6 phosphorylation or its translocation to the nucleus. However, omeprazole significantly reduced IL-4 stimulated STAT6 binding to the eotaxin-3 promoter (to 61.4±6.6% in EoE1-T and to 56 ±9.8% in EoE2-T cells). In addition, omeprazole had no effect on eotaxin-3 mRNA stability. Conclusions: In esophageal squamous epithelial cells from EoE patients, omeprazole blocks IL-4-stimulated eotaxin-3 protein secretion by reducing binding of STAT6 to the eotaxin-3 promoter. In primary cells, we observed significant effects on IL4-stimulated eotaxin-3 secretion with omeprazole in concentrations that are achieved in blood with conventional dosage. These findings elucidate an acid-independent mechanism whereby PPIs might heal EoE, and cast doubt on the assumption that a response to PPI therapy distinguishes EoE from GERD. Eotaxin-3 Protein Secretion (pg/ml/250Kcells±SEM)
S-116
and were randomized to three treatment regimens: 14 days of lansoprazole, amoxicillin and clarithromycin (PAC-14); 5 days of lansoprazole and amoxicillin followed by 5 days of lansoprazole, clarithromycin and metronidazole (SEQ-10); or 5 days of lansoprazole, amoxicillin, clarithromycin, and metronidazole (PACM-5). Participants had UBTs 6-8 weeks after randomization, and retreatment with 14-day lansoprazole, bismuth, metronidazole and tetracycline was offered to those remaining positive. Program effectiveness was determined from the 1340 (91%) participants with a definitive UBT 48-52 weeks after randomization, and effectiveness absent retesting and retreatment was estimated assuming all UBT-positives at 6-8 weeks remained so. Estimated costs were $12 for PAC-14, $7 for SEQ-10, $6 for PACM-5, $10 for retreatment, and $10 for each UBT. RESULTS: Of the 281 UBT-positive participants at 6-8 weeks, 213 (76%) chose to be retreated, and 143 (51%) completed retreatment; 244 had a definitive 1-year UBT result, of whom 93 (38%) tested negative. The observed 1-year eradication probabilities and estimated costs per treatment success for the three treatment arms were 80.5% and $44 for PAC-14, 79.8% and $39 for SEQ-10, and 77.8% and $39 for PACM-5 (Table). Under the scenario of no retesting and retreatment, the estimated eradication probabilities were 5-8% lower, but the estimated costs per treatment success declined substantially more, to $32 for PAC-14 and $27 for SEQ-10 and PACM-5. The principal contributor to the cost differential was the cost of the follow-up UBTs rather than the low probability of retreatment success. CONCLUSIONS: Data from this Latin American study suggest that repeat UBT testing and retreatment after initial therapy add substantially to program costs but only modestly increase the probability of Hp eradication after one year. In settings where resources are scarce, retesting and retreating do not appear to be indicated for programs to prevent Hp-associated gastric cancer. Increasing the effectiveness of eradication therapies and lowering the risk of recurrence will be better approaches for improving program performance. SUPPORT: Bill & Melinda Gates Foundation grant 43930 and NIH grant CA037429. Estimated one-year outcome and costs by treatment arm with and without retesting and retreatment.
598 Eosinophil Derived TGF-β1 Activates Human Esophageal Mesenchymal Cells and Alters Esophageal Motility - Implications for Dysphagia in Eosinophilic Esophagitis (EoE) Florian Rieder, Ilche T. Nonevski, Jie Ma, Zhufeng Ouyang, Gail West, Cheryl A. Protheroe, Anja Schirbel, John R. Goldblum, Tracey L. Bonfield, Karen M. Harnett, James J. Lee, Ikuo Hirano, Gary W. Falk, Piero Biancani, Claudio Fiocchi Background: Dysphagia is a frequent symptom of EoE and may result from a combination of esophageal fibrosis and motility abnormalities. We investigated mechanisms of dysphagia in EoE by assessing the response of primary human esophageal fibroblasts (HEF), muscle cells (HEMC) and esophageal muscle strips to TGF-β1 and eosinophil-derived products. Spontaneous production of TGF-β1 in esophageal biopsies from EoE and control patients was also investigated. Methods: The presence of eosinophils in the esophageal wall of EoE was investigated by H&E and eosinophil peroxidase (EPX) stain of full thickness sections. Production of fibronectin (FN) and collagen I (Col I) by HEF and HEMC in response to TGF-β1 and eosinophil products was measured. αSMA, ICAM-1 and VCAM-1 expression was assessed by flow cytometry and immunocytochemistry. Eosinophil adhesion assays were performed using the human eosinophil cell line AML14.3D10. TGF-β1 and eosinophil sonicates were tested in esophageal muscle contraction assays assessing neurogenic and acetylcholine (ACh)-induced contraction. TGF-β1 was measured in undernatants of esophageal biopsy organ cultures by ELISA. Results: Activated eosinophils were present in all layers of the esophageal wall. TGF-β1 induced a dose-dependent increase in FN and Col I secretion as well as αSMA expression by HEF and HEMC, indicating transformation to a pro-fibrogenic phenotype. Sonicates of pre-activated eosinophils also increased FN and Col I production by HEF and HEMC, an effect that was inhibited by blocking TGF-β1 and p38MAKP signaling. Eosinophil binding increased by pre-treating HEF and HEMC with TGF-β1 or eosinophil sonicates. Combined TGF-β1 and p38MAKP blockade reduced sonicate-induced eosinophil adhesion and completely abrogated TGF-β1-induced adhesion. TGF-β1 did not modulate VCAM-1 or ICAM-1 expression. Eosinophil sonicates and TGF-β1 reduced electrical field-induced contraction of esophageal muscle strips. Neither eosinophil sonicates nor TGF-β1 influenced ACh-induced contraction, indicating a primary effect on neurotransmitter release without affecting the muscle itself. Significantly higher concentrations of TGF-β1 were present in EoE compared to control esophageal mucosal biopsy cultures. Conclusion: Eosinophils infiltrate all esophageal layers in EoE. Eosinophil-derived TGF-β1 increases matrix production by and eosinophil binding to esophageal mesenchymal cells and reduces neurogenic esophageal smooth muscle contraction. TGF-β1 is present in elevated amounts in EoE compared to controls. These results suggest a direct functional link of TGF-β1 expression with altered fibrogenesis and motility in EoE, both of which may contribute to development of dysphagia.
591 Obese Pediatric Patients Have Increased Cardiovascular Disease Risk Comparable to Pediatric Patients With Familial Dyslipidemia Angela Shannon, Suraj Thangada, Christine Carter-Kent, Ariel E. Feldstein, Michael Rocco, Leslie Cho, Stanley L. Hazen, Naim Alkhouri
599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter. Xi Zhang, Edaire Cheng, Xiaofang Huo, David H. Wang, Qiuyang Zhang, Chunhua Yu, Stuart J. Spechler, Rhonda F. Souza
Pediatric patients with familial dyslipidemia (FD) are known to be at increased risk of developing cardiovascular disease (CVD) and are often treated aggressively in order to prevent manifestations of CVD. Obese pediatric patients are also at increased cardiovascular risk but this risk is less well defined. Myeloperoxidase (MPO) and high-sensitive C-reactive protein (hsCRP), markers associated with cardiovascular disease(CVD) in adults, may also be predictive of future cardiovascular disease in predisposed children. Our aim was to investigate the association between MPO and hsCRP with pediatric obesity and then compare these markers between pediatric patients with FD and those with obesity in order to better describe the CVD risk in obese pediatric patients. Methods: Data was collected prospectively on consecutive patients attending the Pediatric Preventive Cardiology and Metabolic Clinic (PPCMC) and the Genetics Clinic at Cleveland Clinic. Data included demographics, anthropometric and laboratory data. The PPCMC cohort was further divided into groups based on BMI (overweight, obese, severely obese) to investigate the association between MPO and hsCRP and obesity. Then, the overall group of patients was divided based on having a diagnosis of FD or overweight/obesity to compare CVD markers between the two groups. Results: 323 patients were included for analysis- 151 in the overweight/obese group and 172 in the FD group. In the overweight/obese group, mean age was 12.8±3.5 yr, there were 56% males, and 63% of the patients were Caucasian. In the FD group, mean age was 12.2±4.4 yr, there were 52% males, and 80% of the patients were Caucasian. The mean LDL in the overweight/obese group was 107.7±4.3 mg/dL, and that of patients with familial hypercholesterolemia was 205.1±63.6 mg/dL (p<0.0001). Mean MPO in the overweight/ obese group was 417±162 pmol/L and in the FD group was 424±162 pmol/L [CL (55.1,69.7), p=0.41]. Mean hsCRP was 4.9±7.2 mg/L in the obese group and 5.6±26 mg/L in the FD group [CL (3.8, -5.2), p=0.38]. MPO and hsCRP levels both had a positive correlation with BMI%ile (rho 0.42 and 0.47, respectively, p<0.001). Conclusions: This is the first pediatric study evaluating the association between obesity and MPO and comparing CVD risk between overweight/obese pediatric patients and patients with FD. MPO and hsCRP both correlate with increasing BMI in pediatric patients. In addition, though overweight/ obese pediatric patients may have a lower LDL than pediatric patients with familial dyslipidemia, there is no significant difference when comparing nontraditional markers of CVD such as MPO and hsCRP. We conclude that MPO and hsCRP are useful markers to identify obese pediatric patients at increased CVD risk, and obesity may in fact confer a CVD risk burden comparable to patients with FD and earlier medical intervention may be warranted.
AGA Abstracts
Introduction: EoE and GERD can have similar clinical and histological features. It has been proposed that patients with these disorders can be distinguished by their response to proton pump inhibitors (PPIs). This is based on the assumption that reduced gastric acid secretion is the only important effect of PPIs and, therefore, only an acid-peptic disorder like GERD can respond to PPIs. In earlier studies, we showed that omeprazole in high concentration (100 μM) blocked cytokine-stimulated secretion of eotaxin-3 protein (a potent eosinophil chemoattractant) in esophageal squamous cell lines from patients with EoE. This suggests that PPIs might have anti-inflammatory actions independent of their effects on gastric acid secretion. Now, we have studied the effects of omeprazole in lower concentrations, including those that can be achieved in blood with conventional dosage, on IL-4-stimulated eotaxin3 production in primary cultures of esophageal squamous cells from EoE patients. To explore the anti-inflammatory mechanism further, we also studied the effects of omeprazole on IL4-stimulated transcriptional activation of eotaxin-3. Methods: Primary cultures of esophageal squamous cells (EoE1 and EoE2) and telomerase-immortalized esophageal squamous cell lines (EoE1-T and EoE2-T) derived from two patients with EoE were treated with IL-4 (10 ng/ml) in the presence or absence of 1-50 μM doses of acid-activated omeprazole for 48 hours. To address transcriptional activation, EoE1-T and EoE2-T cells were pretreated for 24 hours with 50 μM acid-activated omeprazole before the addition of IL-4. We measured eotaxin-3 protein secretion by ELISA, mRNA by real-time PCR, STAT6 phosphorylation and its nuclear translocation by Western blot, and eotaxin-3 promoter binding by ChIP assay. Results: In primary EoE cells, IL-4-stimulated eotaxin-3 secretion was blocked by omeprazole in a dose-dependent fashion (Table). Similar results were obtained in the EoE cell lines, although does-dependency was not as clear as in the primary cells. Omeprazole had no effect on STAT6 phosphorylation or its translocation to the nucleus. However, omeprazole significantly reduced IL-4 stimulated STAT6 binding to the eotaxin-3 promoter (to 61.4±6.6% in EoE1-T and to 56 ±9.8% in EoE2-T cells). In addition, omeprazole had no effect on eotaxin-3 mRNA stability. Conclusions: In esophageal squamous epithelial cells from EoE patients, omeprazole blocks IL-4-stimulated eotaxin-3 protein secretion by reducing binding of STAT6 to the eotaxin-3 promoter. In primary cells, we observed significant effects on IL4-stimulated eotaxin-3 secretion with omeprazole in concentrations that are achieved in blood with conventional dosage. These findings elucidate an acid-independent mechanism whereby PPIs might heal EoE, and cast doubt on the assumption that a response to PPI therapy distinguishes EoE from GERD. Eotaxin-3 Protein Secretion (pg/ml/250Kcells±SEM)
S-116