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Serum levels of progastrin but not amidated gastrin or glycine-extended gastrin are elevated in patients with colorectal neoplasia
A1106 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
5116
511S
SERUM LEVELS OF PROGASTRIN BUT NOT AMIDATED GASTRIN OR GLYCINE-EXTENDED GASTRIN ARE ELEVATED IN PATIENTS WITH COLORECTAL NEOPLASIA. Karim B. Muhammad, Ravindra K. Siddheshwar, Janine Gray, Seamus B. Kelly, North Tyneside Gen Hosp, Newcastle, United Kingdom; Wansbeck Gen Hosp, Ashington, United Kingdom; Ctr for Health and Med Research, Middlesbrough, United Kingdom.
INVOLVEMENT OF THE SMALL GTP BINDING PROTEIN, RHO A AND THE CELL -CELL ADHERENCE PROTEINS, E-CAD· HERIN AND B-CATENIN IN SIGNALING PATHWAY OF PANCREATIC ACINI. Fumihiko Nozu, Shigeki Tanaka, Keiji Mitamura, Showa Univ Sch of Medicine, Tokyo, Japan.
Aim: To determine whether fasting serum levels of progastrin, amidated gastrin and glycine-extended gastrin are elevated in patients with colorectal cancer or colorectal polyps compared to controls. Background: It has been shown that fasting serum amidated gastrin levels are elevated in patients with colorectal cancer and decrease after apparently curative resection of the tumor. However, these studies failed to control for H.Pylori infection (a known cause of hypergastrinaemia). When confounding factors which increase serum gastrin levels are taken into account, it has been shown that serum gastrin levels are not elevated in patients with colorectal cancer. These studies have only measured amidated gastrin and not the more immature forms of gastrin. Total gastrin, which includes all unprocessed, partially processed and mature forms of gastrin has been shown to be elevated in patients with colorectal cancer. Methods: This study included 57 patients with colorectal cancer, 29 patients with colorectal polyps and 53 controls. Controls included symptomatic patients who underwent colonoscopy and were found to have normal mucosa or mild diverticular disease. Patients and controls with factors known to raise plasma gastrin levels were excluded from the study. Fasting serum levels of progastrin, amidated gastrin and glycine-extended gastrin were measured by radioimmunoassay. H.Pylori antibodies were measured by ELISA test. Statistical analysis was by Kruskal Wallis test. Results: This study shows that patients with colorectal cancers have elevated median levels of progastrin (27.5 pM) compared to polyps (:515 pM) or controls (:515 pM), (p=O.OOOI). There was no difference in amidated gastrin between cancer (median 13 pM), polyp (median 24 pM) or control (median 10 pM). H.Pylori positive patients had high amidated gastrin levels (median 19 pM) (p=0.OO22). No significant effect of H.Pylori on progastrin was seen. Median progastrin was significantly elevated in moderately dysplastic polyps (median 38 pM) compared to mildly dysplastic (median 15 pM) or severely dysplastic (median 15 pM) polyps, (p=0.05). There was no difference between median levels of progastrin and amidated gastrin in relation to sex, polyp type, cancer site, grade or Dukes' staging. Glycine-extended gastrin levels were below the limit of detection «20 pM) in all groups. Conclusion: Progastrin levels are elevated in patients with colorectal cancer compared to patients with polyps or controls. 5117 EFFECT OF NICOTINE ON EXPRESSION OF NACHR ON CD4+CELLS OF SPLENOCYTES AND THYMOCYTES IN OXAZOLONE-INDUCED COLITIS OF MURINE MODEL. Yuhji Murata, Jugoh Itoh, Ying Han, Akihiro Munakata, Yutaka Yoshida, Aomori Gen Hosp, Aomori, Japan; First Dept Medicine Hirosaki Univ Sch of Med, Hirosaki, Japan; Dept Gastroenterology, Beijing Army Gen Hosp, Beijing, P. R. China; Hirosaki Univ, Hirosaki, Japan. Aim:We have shown that nicotine enhance Thl-derived cytokine in DSSinduced colitis and suppress Th2-derived cytokine in oxazolone(OXZ)induced colitis. The presence of nicotinic acetylcholine receptor (nAchR) on lymphocytes has been demonstrated and nicotine may influence the function of lymphocytes through nAchR on their surface. However, no study has investigated the expression of nAchR of lymphocytes from spleen and thymus in murine models of IBD. The present study attempted to elucidate the possible role of nAchR in nicotine-related effect on IBD. Methods: 6 mg of OXZ(in 50% ethanol) was intrarectally administered in SJL/J mice to induce colitis. In nicotine group, 0.5mg/kg/day of nicotine was injected subcutaneously for three weeks. Isolated splenocytes and thymocytes as well as lamina propria lymphocytes(LPL) from small intestine were incubated with 0.1 U/ml of cholinesterase for over 8 hours and resuspended . Cells were stained with FITC-conjugated nornicotine ( I X lO,4M) and PE-conjugated anti-CD4, and analyzed by FACS. Results: The percentage of FITC-Iabeled nornicotine binding to splenocytes after the addition of nAchR antagonist mecamylamine in each concentrations of I X lO'4M, I X lO'3M and I X IO'2M was 94.8% ,60.9% and 8.3% respectively, indicating that nornicotine is able to bind with nAchR specifically. About 90-95% of splenocytes, thymocytes and LPL expressed nAchR on their surface.In OXZ-induced colitis, the percentage of CD4 +nAchR+cells in splenocytes (33.9::':1.0%) or thymocytes(50.7::':8.4%) tend to decrease compared with controls(55.3::':lO.l% and 80.9::':2.1%, respectively), but the nAchR on CD4+ cells expressed quantitatively as mean fluorescence intensity (MFI) had no significant change in either splenocytes (126.8::':17.7) or thymocytes (62.8::':6.9, respectively) compared with controls (118.9::':23.6 and 86.2::':8.9, respectively). In nicotine group, compared with OXZ group, the decreased proportion of CD4 + nAchR+cells was up-regulated in splenocytes(45.3::':3.7) and thymocytes (58.3::':2.7%), and amount of nAchR on CD4+ cells was down-regulated in splenocytes(75.9::':24.4) but was up-regulated in thymocytes (157.7::': 17.7). Conclusion: Nicotine could affect cytokine production by lymphocytes through a specific binding with nAchR on their surface; colonic inflammation might influence CD4+nAchR+cells; and nicotine might have an inhibitory effect on the expression of nAchR on CD4 +cells of splenocytes but a stimulatory effect on that of thymocytes.
Background: Several lines of evidence have suggested that the actin cytoskeleton is regulated by Rho family proteins. We previously reported that Rho A is involved during stimulus-secretion coupling of pancreatic acini (Am. 1. Physio!. 276:G915-G923, 1999). Although the actin cytoskeleton is known to play important roles in pancreatic acinar cell, its precise mechanisms are still uncertain. On the other hand, the roles of cell-cell adherence proteins, E-cadherin and ,B-catenin remain to be determined in pancreatic acini. Aim: In this study, we aimed to evaluate the interaction of Rho A, E-cadherin and ,B-catenin in signaling pathway of pancreatic acini. Methods: Pancreatic acini were incubated with or without cholecystokinin-octapeptide (CCK-8), carbachol (CCh) and 12-0-tetradecanoylphorbol 13-acetate (TPA). Western immunoblotting and immunoprecipitation of solubilized rat pancreatic acini were performed. Isolated acini were used for immunohistochemical study. Results: Using anti-Rho A antibody, anti-E-cadherin antibody and anti-,B-catenin antibody, clear bands were detected at the location of 21 KDa, 115 KDa and 90 KDa, respectively. CCK-8 (10 pM-lO nM) enhansed these bands from 3 min and sustained up to 30 min after stimulation. CCK-8 (10 pM-lO nM) and CCh (100 nM-lOO p.M) biphasically increased and TPA (10 nM-Ip.M ) dosedependently increased the intensities of Rho A bands. CCK-8 (10 pM-lO nM), CCh (100 nM-lOO p.M) and TPA (10 nM-Ip.M ) dose-dependently increased the intensities of E-cadherin and ,B-catenin bands. Removal of extracellular Ca 2+ during CCK-8, CCh and TPA stimulation inhibited the increase of E-cadherin bands. In immunoprecipitation study, E-cadherin formed imrnunocomplex with ,B-catenin after CCK-8 (10 nM) and CCh (lOOp.M) stimulation and Rho A also formed immunocomplex with ,B-catenin after CCK-8 (10 nM) and CCh (lOOp.M) stimulation. Immunohistochemical study indicated that Rho A localized in acinar luminal portion and E-cadherin and ,B-catenin localized in the basolateral portion after CCK-8 stimulation. Conclusion: Our study suggest that the activation and interaction of Rho A, E-cadherin and ,B-catenin are involved in signaling pathways of rat pancreatic acini evoked by CCK and CCh. 5119 FOLLOW-UP OF VAGAL FUNCTION IN DIABETIC PATIENTS AFTER PANCREAS TRANSPLANTATION. B. Otto, S. Kiehler, RI Riepl, C. Dieterle, R. Landgraf, Clin Innenstadt Ludwig Maximilians Univ, Munich, Germany. During insulin-induced hypoglycemia a strong increase of pancreatic polypeptide (PP) - a hormone under vagal control - is observed in healthy subjects. This increase can be abolished after truncal vagotomy or atropine. It has been shown that only in diabetic patients with an intact autonomic nervous system a postprandial increase of PP was observed. The intention of our study was to show whether autonomic neuropathy (vagal function) improves after pancreas transplantation in longstanding type I diabetic patients. Methods: In 12 patients (8 male, 4 female; 44.7::':8.2 years; mean::':SD) autonomic neuropathy was tested in a follow-up design: 2.9::':1.1 months (test A) and 20.6::':4.8 months (test B) after pancreas transplantation. After given written informed consent, vagal neuropathy was screened by measuring PP-increase during insulininduced hypoglycemia (0.15 IV insulin/kg body weight). Plasma PP concentrations were measured in regular intervals with an established radioimmunoassay. Results: There was no significant difference in serum creatinine (A: 1.2::':0.2 mg/dL, B: 1.3+-0.2 mg/dL), HbAI (A: 5.5::':0.6 %, B: 6.1 ::':0.6 %) or basal PP levels (A: 10.5::':4 pmollL, B: 18.2::':11.2 pmollL) at both time points. In two patients the insulinhypoglycemia-test had to be interrupted because of prolonged hypoglycemia. Early after pancreas transplantation (test A) no significant increase of plasma PP concentrations during hypoglycemia (delta after 30 min: -0.6::':2.9 pmol/L; delta after 45 min: 1.5::':5.2 pmollL) was observed. There was also no significant difference in hypoglycemiainduced PP release in test B (delta after 30 min: -2.0::':10.3 pmol/L; delta after 45 min: 4.1 ::':9.0 pmol/L). Conclusions: No improvement of vagal function could be observed in patients after successful pancreas transplantation (20.6:+ - 4.8 posttrmsplant.). It may be speculated that autonomic neuropathy of the vagus needs a longer time for recovery. 5120 PHAGOCYTIC KILLING OF THE PERITONEAL MACROPHAGES IS ENHANCED BY THE SIGNAL VIA MAC-t (CD11BI CDtS). Tetsuhiro Owaki, Sonshin Takao, Takashi Aikou, Gregory B. Bulkley, Andrew S. Klein, 1st Dept of Surg, Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Johns Hopkins Med Inst, Baltimore, MD. INTRODUCTION Mac-I (CD29/CDllb, CR3), a beta2 integrin expressed on macrophages (Mfs), is important in adhesion to endothelial cells. Mfs have a phagocytic and phagocytic killing function, and these functions require the adhesion with foreign substances. In the previous study, Mac-I affected the phagocytic function of Mfs, and increased superoxide anion (Oi) generation in neutrophils. Therefore we investigated phagocytic kill-
GASTROENTEROLOGY Vol. 118, No.4
5116
511S
SERUM LEVELS OF PROGASTRIN BUT NOT AMIDATED GASTRIN OR GLYCINE-EXTENDED GASTRIN ARE ELEVATED IN PATIENTS WITH COLORECTAL NEOPLASIA. Karim B. Muhammad, Ravindra K. Siddheshwar, Janine Gray, Seamus B. Kelly, North Tyneside Gen Hosp, Newcastle, United Kingdom; Wansbeck Gen Hosp, Ashington, United Kingdom; Ctr for Health and Med Research, Middlesbrough, United Kingdom.
INVOLVEMENT OF THE SMALL GTP BINDING PROTEIN, RHO A AND THE CELL -CELL ADHERENCE PROTEINS, E-CAD· HERIN AND B-CATENIN IN SIGNALING PATHWAY OF PANCREATIC ACINI. Fumihiko Nozu, Shigeki Tanaka, Keiji Mitamura, Showa Univ Sch of Medicine, Tokyo, Japan.
Aim: To determine whether fasting serum levels of progastrin, amidated gastrin and glycine-extended gastrin are elevated in patients with colorectal cancer or colorectal polyps compared to controls. Background: It has been shown that fasting serum amidated gastrin levels are elevated in patients with colorectal cancer and decrease after apparently curative resection of the tumor. However, these studies failed to control for H.Pylori infection (a known cause of hypergastrinaemia). When confounding factors which increase serum gastrin levels are taken into account, it has been shown that serum gastrin levels are not elevated in patients with colorectal cancer. These studies have only measured amidated gastrin and not the more immature forms of gastrin. Total gastrin, which includes all unprocessed, partially processed and mature forms of gastrin has been shown to be elevated in patients with colorectal cancer. Methods: This study included 57 patients with colorectal cancer, 29 patients with colorectal polyps and 53 controls. Controls included symptomatic patients who underwent colonoscopy and were found to have normal mucosa or mild diverticular disease. Patients and controls with factors known to raise plasma gastrin levels were excluded from the study. Fasting serum levels of progastrin, amidated gastrin and glycine-extended gastrin were measured by radioimmunoassay. H.Pylori antibodies were measured by ELISA test. Statistical analysis was by Kruskal Wallis test. Results: This study shows that patients with colorectal cancers have elevated median levels of progastrin (27.5 pM) compared to polyps (:515 pM) or controls (:515 pM), (p=O.OOOI). There was no difference in amidated gastrin between cancer (median 13 pM), polyp (median 24 pM) or control (median 10 pM). H.Pylori positive patients had high amidated gastrin levels (median 19 pM) (p=0.OO22). No significant effect of H.Pylori on progastrin was seen. Median progastrin was significantly elevated in moderately dysplastic polyps (median 38 pM) compared to mildly dysplastic (median 15 pM) or severely dysplastic (median 15 pM) polyps, (p=0.05). There was no difference between median levels of progastrin and amidated gastrin in relation to sex, polyp type, cancer site, grade or Dukes' staging. Glycine-extended gastrin levels were below the limit of detection «20 pM) in all groups. Conclusion: Progastrin levels are elevated in patients with colorectal cancer compared to patients with polyps or controls. 5117 EFFECT OF NICOTINE ON EXPRESSION OF NACHR ON CD4+CELLS OF SPLENOCYTES AND THYMOCYTES IN OXAZOLONE-INDUCED COLITIS OF MURINE MODEL. Yuhji Murata, Jugoh Itoh, Ying Han, Akihiro Munakata, Yutaka Yoshida, Aomori Gen Hosp, Aomori, Japan; First Dept Medicine Hirosaki Univ Sch of Med, Hirosaki, Japan; Dept Gastroenterology, Beijing Army Gen Hosp, Beijing, P. R. China; Hirosaki Univ, Hirosaki, Japan. Aim:We have shown that nicotine enhance Thl-derived cytokine in DSSinduced colitis and suppress Th2-derived cytokine in oxazolone(OXZ)induced colitis. The presence of nicotinic acetylcholine receptor (nAchR) on lymphocytes has been demonstrated and nicotine may influence the function of lymphocytes through nAchR on their surface. However, no study has investigated the expression of nAchR of lymphocytes from spleen and thymus in murine models of IBD. The present study attempted to elucidate the possible role of nAchR in nicotine-related effect on IBD. Methods: 6 mg of OXZ(in 50% ethanol) was intrarectally administered in SJL/J mice to induce colitis. In nicotine group, 0.5mg/kg/day of nicotine was injected subcutaneously for three weeks. Isolated splenocytes and thymocytes as well as lamina propria lymphocytes(LPL) from small intestine were incubated with 0.1 U/ml of cholinesterase for over 8 hours and resuspended . Cells were stained with FITC-conjugated nornicotine ( I X lO,4M) and PE-conjugated anti-CD4, and analyzed by FACS. Results: The percentage of FITC-Iabeled nornicotine binding to splenocytes after the addition of nAchR antagonist mecamylamine in each concentrations of I X lO'4M, I X lO'3M and I X IO'2M was 94.8% ,60.9% and 8.3% respectively, indicating that nornicotine is able to bind with nAchR specifically. About 90-95% of splenocytes, thymocytes and LPL expressed nAchR on their surface.In OXZ-induced colitis, the percentage of CD4 +nAchR+cells in splenocytes (33.9::':1.0%) or thymocytes(50.7::':8.4%) tend to decrease compared with controls(55.3::':lO.l% and 80.9::':2.1%, respectively), but the nAchR on CD4+ cells expressed quantitatively as mean fluorescence intensity (MFI) had no significant change in either splenocytes (126.8::':17.7) or thymocytes (62.8::':6.9, respectively) compared with controls (118.9::':23.6 and 86.2::':8.9, respectively). In nicotine group, compared with OXZ group, the decreased proportion of CD4 + nAchR+cells was up-regulated in splenocytes(45.3::':3.7) and thymocytes (58.3::':2.7%), and amount of nAchR on CD4+ cells was down-regulated in splenocytes(75.9::':24.4) but was up-regulated in thymocytes (157.7::': 17.7). Conclusion: Nicotine could affect cytokine production by lymphocytes through a specific binding with nAchR on their surface; colonic inflammation might influence CD4+nAchR+cells; and nicotine might have an inhibitory effect on the expression of nAchR on CD4 +cells of splenocytes but a stimulatory effect on that of thymocytes.
Background: Several lines of evidence have suggested that the actin cytoskeleton is regulated by Rho family proteins. We previously reported that Rho A is involved during stimulus-secretion coupling of pancreatic acini (Am. 1. Physio!. 276:G915-G923, 1999). Although the actin cytoskeleton is known to play important roles in pancreatic acinar cell, its precise mechanisms are still uncertain. On the other hand, the roles of cell-cell adherence proteins, E-cadherin and ,B-catenin remain to be determined in pancreatic acini. Aim: In this study, we aimed to evaluate the interaction of Rho A, E-cadherin and ,B-catenin in signaling pathway of pancreatic acini. Methods: Pancreatic acini were incubated with or without cholecystokinin-octapeptide (CCK-8), carbachol (CCh) and 12-0-tetradecanoylphorbol 13-acetate (TPA). Western immunoblotting and immunoprecipitation of solubilized rat pancreatic acini were performed. Isolated acini were used for immunohistochemical study. Results: Using anti-Rho A antibody, anti-E-cadherin antibody and anti-,B-catenin antibody, clear bands were detected at the location of 21 KDa, 115 KDa and 90 KDa, respectively. CCK-8 (10 pM-lO nM) enhansed these bands from 3 min and sustained up to 30 min after stimulation. CCK-8 (10 pM-lO nM) and CCh (100 nM-lOO p.M) biphasically increased and TPA (10 nM-Ip.M ) dosedependently increased the intensities of Rho A bands. CCK-8 (10 pM-lO nM), CCh (100 nM-lOO p.M) and TPA (10 nM-Ip.M ) dose-dependently increased the intensities of E-cadherin and ,B-catenin bands. Removal of extracellular Ca 2+ during CCK-8, CCh and TPA stimulation inhibited the increase of E-cadherin bands. In immunoprecipitation study, E-cadherin formed imrnunocomplex with ,B-catenin after CCK-8 (10 nM) and CCh (lOOp.M) stimulation and Rho A also formed immunocomplex with ,B-catenin after CCK-8 (10 nM) and CCh (lOOp.M) stimulation. Immunohistochemical study indicated that Rho A localized in acinar luminal portion and E-cadherin and ,B-catenin localized in the basolateral portion after CCK-8 stimulation. Conclusion: Our study suggest that the activation and interaction of Rho A, E-cadherin and ,B-catenin are involved in signaling pathways of rat pancreatic acini evoked by CCK and CCh. 5119 FOLLOW-UP OF VAGAL FUNCTION IN DIABETIC PATIENTS AFTER PANCREAS TRANSPLANTATION. B. Otto, S. Kiehler, RI Riepl, C. Dieterle, R. Landgraf, Clin Innenstadt Ludwig Maximilians Univ, Munich, Germany. During insulin-induced hypoglycemia a strong increase of pancreatic polypeptide (PP) - a hormone under vagal control - is observed in healthy subjects. This increase can be abolished after truncal vagotomy or atropine. It has been shown that only in diabetic patients with an intact autonomic nervous system a postprandial increase of PP was observed. The intention of our study was to show whether autonomic neuropathy (vagal function) improves after pancreas transplantation in longstanding type I diabetic patients. Methods: In 12 patients (8 male, 4 female; 44.7::':8.2 years; mean::':SD) autonomic neuropathy was tested in a follow-up design: 2.9::':1.1 months (test A) and 20.6::':4.8 months (test B) after pancreas transplantation. After given written informed consent, vagal neuropathy was screened by measuring PP-increase during insulininduced hypoglycemia (0.15 IV insulin/kg body weight). Plasma PP concentrations were measured in regular intervals with an established radioimmunoassay. Results: There was no significant difference in serum creatinine (A: 1.2::':0.2 mg/dL, B: 1.3+-0.2 mg/dL), HbAI (A: 5.5::':0.6 %, B: 6.1 ::':0.6 %) or basal PP levels (A: 10.5::':4 pmollL, B: 18.2::':11.2 pmollL) at both time points. In two patients the insulinhypoglycemia-test had to be interrupted because of prolonged hypoglycemia. Early after pancreas transplantation (test A) no significant increase of plasma PP concentrations during hypoglycemia (delta after 30 min: -0.6::':2.9 pmol/L; delta after 45 min: 1.5::':5.2 pmollL) was observed. There was also no significant difference in hypoglycemiainduced PP release in test B (delta after 30 min: -2.0::':10.3 pmol/L; delta after 45 min: 4.1 ::':9.0 pmol/L). Conclusions: No improvement of vagal function could be observed in patients after successful pancreas transplantation (20.6:+ - 4.8 posttrmsplant.). It may be speculated that autonomic neuropathy of the vagus needs a longer time for recovery. 5120 PHAGOCYTIC KILLING OF THE PERITONEAL MACROPHAGES IS ENHANCED BY THE SIGNAL VIA MAC-t (CD11BI CDtS). Tetsuhiro Owaki, Sonshin Takao, Takashi Aikou, Gregory B. Bulkley, Andrew S. Klein, 1st Dept of Surg, Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Kagoshima Univ Sch of Medicine, Kagoshima, Japan; Johns Hopkins Med Inst, Baltimore, MD. INTRODUCTION Mac-I (CD29/CDllb, CR3), a beta2 integrin expressed on macrophages (Mfs), is important in adhesion to endothelial cells. Mfs have a phagocytic and phagocytic killing function, and these functions require the adhesion with foreign substances. In the previous study, Mac-I affected the phagocytic function of Mfs, and increased superoxide anion (Oi) generation in neutrophils. Therefore we investigated phagocytic kill-