- Email: [email protected]
Identification of CFTR chloride (Cl) channels on laterobasal membranes (LBM) of pancreatic duct epithelial cells (PDEC)
Materials and methods: anti peptides antibodies technique has been chosen. A specific HPLRP1 decapeptide was used for rabbit immunization. The anti HPLRP1 antibodies were purified by immunoaffinity, using a column of immobilized recombinant HPLRP1. The specificity of antibodies was assessed by western blot. A double sandwich ELISA was performed with the purified anti peptide antibody and a biotinylated HPLRPI polyclonal antibody. 20 human sera with elevated hpasemia were preliminary tested, lmmunochemical study was also performed with human pancreas. Results: the reactivity and specificity of the antibody against HPLRP1 was good, and no cross reactivity with HPLRP2 and pancreatic lipase was observed. The ELISA was assessed by precision and variation tests, The sensitivity of the test was 60 ng/L with 90 % precision in serum. Each semm tested exhibited HPLRP1 with a mean value of 18 microg/L. Immunochemical studies demonstrated that HPLRP1 was present in pancreatic acinar cells. Conclusion : we show for the first time that HPLRP1 is present in human serum and in pancreatic acinar cells. The ELISA will allow us to study the secretion of this protein in several physiological and pathological conditions,
performed in 7-13 rats. Pancreatic juice volume (PJ) (~l/h) and output of bicarbonate (Bic) (mE@) and amylase (Amy) (1u/h) ware determined. Serum insulin and glucose levels were measured also. Results: intraduodenal administration of oleic acid significantly increased PJ (115,9 +/- 9 5), Bic (6.1 +/- 0.7) and Amy (2075.6 +/- 134.1 ), comparing to the control (p<0.001). Intravenous infusion of glucose in a high dose was resulted in sigmficant augmentation of oleic acid-stimulated pancreatic secretion in terms of PJ (161.3 +/- 10.6), gic (85 +/- 06) and Amy (3150,0 +/- 287.1) (p
M2218 Substance P Inhibits Bicarbonate Secretion in Guinea Pig Panceatic Ducts by Modulating an Aniort Exchanger Peter Hegyi, Mike A. Gray, Bar~' E, Argent
M2221 Effects of Cyclooxygenase-2 on Angiogenesis in Pancreatic Carcinoma Xing-Peng Wang, Chuan-Gao Xie
Introduction. The stimulatury pathways controlling the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, wbich may play, an important role in the physiological control of the pancreas. Aim. The aim of this study was to investigate the eftects of Substance P on HCO~ secretion from guinea pig pancreatic ducts. Methods. Small mtraBnterlobular ducts were isolated from the pancreas of guinea pigs weighing 150-250g~ by enzymatic digestion, microdisseetion and then cukured over night. In order to estimate the rate of HCO3 secretion in the isolated ducts, we measured the minal rate of intracelluiar acidification (using the pH sensitive dye BCECF and tmcrofluurometry) niter the basolateral pH regulatory mechanisms of the duct cells were suddenly' inhibited, The Na+/H + exchanger was blocked by amiloride and the Na*/HCO~ cotransporter was blocked by DIDS. The initial rate of intracenular acidification was measured twice in each duct, the initial measurement being the control and the second the test. The ratio of these two changes of pH was calculated in each duct 20 nM secretin and/or Substance P were given for 10 minutes between the two measurements. In addition, ducts cells were loaded by exposure to 3-minute pulses 20 mM NH4CI. The initial rate of recovery phases (from alkalosis and also from acidosis) were calculated in each experiments, Results and Conclusions. Our data clearly demonstrate, that secrefin can stimulate HCO3 secretion in guinea pig pancreatic duct about 5-fold, Moreover, Substance P can totally block this secretin-stimulated HCO3 secretion. Substance P has no effect on either the Na+/ H C Q cotransporter or the Na+/H + exchanger. However, Substance P can inhibit a C1 dependent H C Q efflux (secretory) mechanism probably the CITHCO3 exchanger on the apical membrane of the duct cell. This work was supported by the Wellcome Trust.
Objectives To investigate tire effects of cyclooxygenase-2 (COX-2) on angiogenesis in pancreatic carcinoma, and to clarify"the mechanisms of selective COX-2 inhibitor on the chemoprevention of pancreatic carcinoma. Methods The inhibitor), eftects of Celebrex, a selective cydooxygenase-2 inhibitor, on the expression of vascular endothelial growth tactor (VEGF) and PGE2 in pancreatic carcinoma cell lines PC-3 were studied by using reverse transcription polynaerase chain reaction (RT-PCR), enzyme-finked inlmuno-adsordent assay (ELISA) and radioimmunoassay (RIM, respectively. Effects of Celebrex on the expression of VEGF and PGE2 in pancreatic tumor of xeuografted nude mice induced by PC-3 cell hnes were investigated by immunohistochemistry, RT-PCR and Western blot. Microvessel density (MVD) was also determined under microscope. The COX-2 ant/sense oligdeoxynucletids (ODNs) was designed, synthesized, and transfected into PC-3 cell lines. The transfection effects were confirmed by fluorescence microscope, RT-PCR and Western blot. Chorioallantoic membrane (CAM) grafted model was used to evaluate the effects of COX-2 anti-ODNs and PGE2 on the angiogenests in pancreatic carcinoma, Results The expression of VEGF and PGE2 was inhibited by Celebrex in a certain degree with time-dependent and dosedependent manner. Celebrex could inhibit the expression of VEGF and PGE2 in the xenografted tumor of PC-3 cell lines in nude mice, and also could decrease the average MX/~ significantly in tumor tissue (P
M2219 Identification of CFTR Chloride (Cl) Channels on Laterobasal Membranes (LBM) of Pancreatic Duct Epithelial Cells (PDEC) Toan D, Ngnyen, Toby Wu
M2222 Characterization of Serum Biomarkers for Pancreatic Cancer Using SELDI-TOF Mass Spectrometry: Preliminary Findings Randy S. Haun, Sndeepa Bhattacharyya, LarD' J Suva, Suresh T. Chari, Gloria Petersen
Introduction: The exact mechanism for bicarbonate (HCO3) secretion by PDEC remains to be clarified, as it has to account for i) HCO3 reaching 145 mM in the pancreatic juice of many species, ii) dependence of HCO~ secretion on cAMP-stimulated CFTR CI channel function, and in) reciprocal changes in HCO~ and C1 concentrations in stimulated pancreatic juice Additional characterization of PDEC ion transport pathways may further clarify this mechainsm We previously identified CFTR and calcium-activated CI channels on the apical membrane (AM) of dog PDEC. We now report that CFTR is also expressed on the IBM of these cens, where it may contribute to transepithefial CI absorption in exchange for HCO3 secretion. Methods: Cultured dog PDEC were mounted in Ussing chambers and studied either intact (transepithelkal transport) or follomng AM permeabifization to monovalent ions with 0.2 mg/ml luminal nystatin (transport across IBM). A CI gradient was generated by substituting CI with ghiconate. Results: When dog PDEC were studied in the presence of a 135 mM serosal-to-lummal C1 gradient and following AM permeabilization, 100 ~M forskolin stimulated a large and sustained Ise increase of up to 85 IxA/cm2, This increase was dependent on the CI gradient, inverted with a reversed gradient, and unaffected by substitution of Na with cesium, reflecting C1 transport across the IBM through a pathway that does not involve Na co-transport. It was inhibited by the C1 channel inhibiturs, NPPB (500 ~tM) and glibenclamide (500 IxM), hut resistant to DIDS (500 p.M), a C1 channel inhibitor which does not inhibit CFTR, and SITS (500 ixM), an inhibitor of the HCO3/C[ exchanger. These properties are characteristic of CFTR. V~41enintact dog PDEC were studied in Ussing chamber and subject to a serosal-to-luminal CI gradient, forskolin also stimulated an Isc increase that was similarly dependent on the C1 gradient and inhibited by NPPB and glibendamide, bot not DIDS and SITS. Conclusion: We have identified on the IBM of dog PDEC CI transport pathways with known CFTR properties. Along with its apical counterpart, LBM CFTR may regulate transepitfielial diffusion of Cl, In cAMP-stimulated PDEC, the combined activation of CFTR on both AM and IBM will allow reabsorption of Cl, providing an efficient mechanism tbr transepithelial exchange of C1 for HCO~. Because these dog PDEC are derived fi-om the main pancreatic duct, this CI reabsorptive capacity may also be a characteristic of large duct PDEC. Funded by NIH RO1 DK55885 and VA Merit Review,
Background: There is a desperate need for a specific and sensitive diagnostic test for early stages of pancreatic cancer. Recent techniques of mass spectrometry, such as surface-enhanced laser desorption/ioinzation time-of-flight (SELDI-TOF) mass spectrometry (MS), have been used to generate "fingerprints" of cancer cells and identify proteins elicited by tumors to yield new biomarkers for earl), detection of cancers. Aims: The goal of this study was to screen for potential serum biomarkers and identify a unique signature of serum proteins for early detection of pancreatic cancer using the SELDI-TOF mass spectrometric technique. Methods: In the initial phase of this study reported here a "training set" of sera of 62 pancreatic cancer patients and 10 controls w~asstudied using SELDI-TOF mass spectrometry to generate serum protein profiles. Briefly, serum samples were applied in triplicate to IMAC3 ProteinChips (Ciphergen) and bomM proteins were detected with a ProteinChip Reader (Ciphergen PBS ll) after laser desorption/ionization. The resulting spectra were compiled and mass peaks with mass-tu-charge ratios (roJz) between 2000 and 20000 were identified using ProteinChip Reader Software (Ciphergen). Results: Peak masses with a signal-to-noise ratio > 2 were identified, clnstered, and analyzed. This process revealed 136 peaks that had significant differences (p < 0.05) in the average peak intensities between sera of patients with pancreatic cancer compared with those without pancreatic disease. S/xty-three of these specific protein peaks were elevated in pancreatic cancer serum samples (1.1- to 3.2-|bid, pancreatic cancer vs. normal) and the remaining 73 peaks were higher in unaffected individuals (1.1- to 19-fold, controls vs. pancreatic cancer). Conclusions: These preliminary' data suggest that there are significant differences in protein profiles of pancreatic cancer patients compared to controls. Upon validation of these preliminary findings we will a) quantify and identify the individual proteins, or a subset thereof, within the signature profile generated by SELDI-TOP MS and prepare antibodies to these biomarkers for validation of the dlagnnstic potential in more conventional immunological-based assays (ELISA, RIA) and b) use the signature profiles to study a "masked set" of sera from pancreatic cancer and controls.
M2220
M2223
Immunological Detection and Measurement of Pancreatic Lipase Related Protein l (HPLRP1) in Human Pancreas and Serum Grandval Philippe, De Caro Alain, Garcia Stephane, kaugier Rene
Predictive Factors for Malignancy in Intraductal Papillary-mucinous Tumors of the Pancreas Masanori Sugiyama, Yutaka Suzuki, Yumi Izumisato, Nobntsugu Abe, Tadahiko Masaki, Toshiyuki Mori, Yutaka Atomi
introduction : Pancreatic lipase related protein 1 and 2 (HPkRP1 and 2) belong to the pancreatic lipase family and share 65 to 68% of identity. HPLRP1 have already been detected in human pancreatic jmce, but its physiological role remains unknown. The aim the study was to produce a specific antibody to HPLRP1 tbr ELISA and immunocbemical detection,
Background: lntraductal papdlary-mucinous tumor (IPMT) of the pancreas exhibits a broad histohigic spectrum, ranging from adenoma to invasive carcinoma. Accurate assessment of grade of malignancy is required for appropriate management of IPMT. However, preoperative
A-439
AGA
Abstracts
performed in 7-13 rats. Pancreatic juice volume (PJ) (~l/h) and output of bicarbonate (Bic) (mE@) and amylase (Amy) (1u/h) ware determined. Serum insulin and glucose levels were measured also. Results: intraduodenal administration of oleic acid significantly increased PJ (115,9 +/- 9 5), Bic (6.1 +/- 0.7) and Amy (2075.6 +/- 134.1 ), comparing to the control (p<0.001). Intravenous infusion of glucose in a high dose was resulted in sigmficant augmentation of oleic acid-stimulated pancreatic secretion in terms of PJ (161.3 +/- 10.6), gic (85 +/- 06) and Amy (3150,0 +/- 287.1) (p
M2218 Substance P Inhibits Bicarbonate Secretion in Guinea Pig Panceatic Ducts by Modulating an Aniort Exchanger Peter Hegyi, Mike A. Gray, Bar~' E, Argent
M2221 Effects of Cyclooxygenase-2 on Angiogenesis in Pancreatic Carcinoma Xing-Peng Wang, Chuan-Gao Xie
Introduction. The stimulatury pathways controlling the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, wbich may play, an important role in the physiological control of the pancreas. Aim. The aim of this study was to investigate the eftects of Substance P on HCO~ secretion from guinea pig pancreatic ducts. Methods. Small mtraBnterlobular ducts were isolated from the pancreas of guinea pigs weighing 150-250g~ by enzymatic digestion, microdisseetion and then cukured over night. In order to estimate the rate of HCO3 secretion in the isolated ducts, we measured the minal rate of intracelluiar acidification (using the pH sensitive dye BCECF and tmcrofluurometry) niter the basolateral pH regulatory mechanisms of the duct cells were suddenly' inhibited, The Na+/H + exchanger was blocked by amiloride and the Na*/HCO~ cotransporter was blocked by DIDS. The initial rate of intracenular acidification was measured twice in each duct, the initial measurement being the control and the second the test. The ratio of these two changes of pH was calculated in each duct 20 nM secretin and/or Substance P were given for 10 minutes between the two measurements. In addition, ducts cells were loaded by exposure to 3-minute pulses 20 mM NH4CI. The initial rate of recovery phases (from alkalosis and also from acidosis) were calculated in each experiments, Results and Conclusions. Our data clearly demonstrate, that secrefin can stimulate HCO3 secretion in guinea pig pancreatic duct about 5-fold, Moreover, Substance P can totally block this secretin-stimulated HCO3 secretion. Substance P has no effect on either the Na+/ H C Q cotransporter or the Na+/H + exchanger. However, Substance P can inhibit a C1 dependent H C Q efflux (secretory) mechanism probably the CITHCO3 exchanger on the apical membrane of the duct cell. This work was supported by the Wellcome Trust.
Objectives To investigate tire effects of cyclooxygenase-2 (COX-2) on angiogenesis in pancreatic carcinoma, and to clarify"the mechanisms of selective COX-2 inhibitor on the chemoprevention of pancreatic carcinoma. Methods The inhibitor), eftects of Celebrex, a selective cydooxygenase-2 inhibitor, on the expression of vascular endothelial growth tactor (VEGF) and PGE2 in pancreatic carcinoma cell lines PC-3 were studied by using reverse transcription polynaerase chain reaction (RT-PCR), enzyme-finked inlmuno-adsordent assay (ELISA) and radioimmunoassay (RIM, respectively. Effects of Celebrex on the expression of VEGF and PGE2 in pancreatic tumor of xeuografted nude mice induced by PC-3 cell hnes were investigated by immunohistochemistry, RT-PCR and Western blot. Microvessel density (MVD) was also determined under microscope. The COX-2 ant/sense oligdeoxynucletids (ODNs) was designed, synthesized, and transfected into PC-3 cell lines. The transfection effects were confirmed by fluorescence microscope, RT-PCR and Western blot. Chorioallantoic membrane (CAM) grafted model was used to evaluate the effects of COX-2 anti-ODNs and PGE2 on the angiogenests in pancreatic carcinoma, Results The expression of VEGF and PGE2 was inhibited by Celebrex in a certain degree with time-dependent and dosedependent manner. Celebrex could inhibit the expression of VEGF and PGE2 in the xenografted tumor of PC-3 cell lines in nude mice, and also could decrease the average MX/~ significantly in tumor tissue (P
M2219 Identification of CFTR Chloride (Cl) Channels on Laterobasal Membranes (LBM) of Pancreatic Duct Epithelial Cells (PDEC) Toan D, Ngnyen, Toby Wu
M2222 Characterization of Serum Biomarkers for Pancreatic Cancer Using SELDI-TOF Mass Spectrometry: Preliminary Findings Randy S. Haun, Sndeepa Bhattacharyya, LarD' J Suva, Suresh T. Chari, Gloria Petersen
Introduction: The exact mechanism for bicarbonate (HCO3) secretion by PDEC remains to be clarified, as it has to account for i) HCO3 reaching 145 mM in the pancreatic juice of many species, ii) dependence of HCO~ secretion on cAMP-stimulated CFTR CI channel function, and in) reciprocal changes in HCO~ and C1 concentrations in stimulated pancreatic juice Additional characterization of PDEC ion transport pathways may further clarify this mechainsm We previously identified CFTR and calcium-activated CI channels on the apical membrane (AM) of dog PDEC. We now report that CFTR is also expressed on the IBM of these cens, where it may contribute to transepithefial CI absorption in exchange for HCO3 secretion. Methods: Cultured dog PDEC were mounted in Ussing chambers and studied either intact (transepithelkal transport) or follomng AM permeabifization to monovalent ions with 0.2 mg/ml luminal nystatin (transport across IBM). A CI gradient was generated by substituting CI with ghiconate. Results: When dog PDEC were studied in the presence of a 135 mM serosal-to-lummal C1 gradient and following AM permeabilization, 100 ~M forskolin stimulated a large and sustained Ise increase of up to 85 IxA/cm2, This increase was dependent on the CI gradient, inverted with a reversed gradient, and unaffected by substitution of Na with cesium, reflecting C1 transport across the IBM through a pathway that does not involve Na co-transport. It was inhibited by the C1 channel inhibiturs, NPPB (500 ~tM) and glibenclamide (500 IxM), hut resistant to DIDS (500 p.M), a C1 channel inhibitor which does not inhibit CFTR, and SITS (500 ixM), an inhibitor of the HCO3/C[ exchanger. These properties are characteristic of CFTR. V~41enintact dog PDEC were studied in Ussing chamber and subject to a serosal-to-luminal CI gradient, forskolin also stimulated an Isc increase that was similarly dependent on the C1 gradient and inhibited by NPPB and glibendamide, bot not DIDS and SITS. Conclusion: We have identified on the IBM of dog PDEC CI transport pathways with known CFTR properties. Along with its apical counterpart, LBM CFTR may regulate transepitfielial diffusion of Cl, In cAMP-stimulated PDEC, the combined activation of CFTR on both AM and IBM will allow reabsorption of Cl, providing an efficient mechanism tbr transepithelial exchange of C1 for HCO~. Because these dog PDEC are derived fi-om the main pancreatic duct, this CI reabsorptive capacity may also be a characteristic of large duct PDEC. Funded by NIH RO1 DK55885 and VA Merit Review,
Background: There is a desperate need for a specific and sensitive diagnostic test for early stages of pancreatic cancer. Recent techniques of mass spectrometry, such as surface-enhanced laser desorption/ioinzation time-of-flight (SELDI-TOF) mass spectrometry (MS), have been used to generate "fingerprints" of cancer cells and identify proteins elicited by tumors to yield new biomarkers for earl), detection of cancers. Aims: The goal of this study was to screen for potential serum biomarkers and identify a unique signature of serum proteins for early detection of pancreatic cancer using the SELDI-TOF mass spectrometric technique. Methods: In the initial phase of this study reported here a "training set" of sera of 62 pancreatic cancer patients and 10 controls w~asstudied using SELDI-TOF mass spectrometry to generate serum protein profiles. Briefly, serum samples were applied in triplicate to IMAC3 ProteinChips (Ciphergen) and bomM proteins were detected with a ProteinChip Reader (Ciphergen PBS ll) after laser desorption/ionization. The resulting spectra were compiled and mass peaks with mass-tu-charge ratios (roJz) between 2000 and 20000 were identified using ProteinChip Reader Software (Ciphergen). Results: Peak masses with a signal-to-noise ratio > 2 were identified, clnstered, and analyzed. This process revealed 136 peaks that had significant differences (p < 0.05) in the average peak intensities between sera of patients with pancreatic cancer compared with those without pancreatic disease. S/xty-three of these specific protein peaks were elevated in pancreatic cancer serum samples (1.1- to 3.2-|bid, pancreatic cancer vs. normal) and the remaining 73 peaks were higher in unaffected individuals (1.1- to 19-fold, controls vs. pancreatic cancer). Conclusions: These preliminary' data suggest that there are significant differences in protein profiles of pancreatic cancer patients compared to controls. Upon validation of these preliminary findings we will a) quantify and identify the individual proteins, or a subset thereof, within the signature profile generated by SELDI-TOP MS and prepare antibodies to these biomarkers for validation of the dlagnnstic potential in more conventional immunological-based assays (ELISA, RIA) and b) use the signature profiles to study a "masked set" of sera from pancreatic cancer and controls.
M2220
M2223
Immunological Detection and Measurement of Pancreatic Lipase Related Protein l (HPLRP1) in Human Pancreas and Serum Grandval Philippe, De Caro Alain, Garcia Stephane, kaugier Rene
Predictive Factors for Malignancy in Intraductal Papillary-mucinous Tumors of the Pancreas Masanori Sugiyama, Yutaka Suzuki, Yumi Izumisato, Nobntsugu Abe, Tadahiko Masaki, Toshiyuki Mori, Yutaka Atomi
introduction : Pancreatic lipase related protein 1 and 2 (HPkRP1 and 2) belong to the pancreatic lipase family and share 65 to 68% of identity. HPLRP1 have already been detected in human pancreatic jmce, but its physiological role remains unknown. The aim the study was to produce a specific antibody to HPLRP1 tbr ELISA and immunocbemical detection,
Background: lntraductal papdlary-mucinous tumor (IPMT) of the pancreas exhibits a broad histohigic spectrum, ranging from adenoma to invasive carcinoma. Accurate assessment of grade of malignancy is required for appropriate management of IPMT. However, preoperative
A-439
AGA
Abstracts