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Sialic acid shields MUC1 peptide regions in pancreatic cancer cells
A360
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AGA ABSTRACTS
PEDIATRIC ERCP TREATMENT IN RELAPSING PANCREATITIS. R.E, Hintz¢, A. Adler, W. Veltzke, W. Luck*, M. Becker*. Central Interdisciplinary Endoscopy, Depts. of Medicine/Gastroenterology and *Pediatrics, University Hospital Rudolf Virehow, Free University of Berlin, Germany lntro~toqtion: Children with relapsing eholangitis or pancreatltis as well as unclear cholestasis need diagnosis by ERC(P) because in most of the eases the reason for the disease can not be clarified with other imaging techniques. Therapeutic experience is low in transpapillary endoscopy in childhood and youth until now. M~$hocls: As instruments we use the standard video.-sideviewing codescopes for adult-ERCP with the corresponding accessories. Because of the experiences of an initial series which was performed under sedativa, we now make all pediatric ERC(P)'s in insufflation anaesthesia. Re~Ils: From 3/92 to 10/94 we performed in 17 children (mean age 6.9 years, 9 male, 8 female) an ERC, in 11 combined with pancreaticography. The indication in 11 children was a relapsing pancreatitis, in 6 cholestasis and/or icterus The diagnostic imaging showed in 2 children a pancreas divisum, in 3 a choledochal cyst, thereof once the types 1, I1 and Ill, and in one a M. Byler (heredofamilial bile duet atresia). Three of them had a juvenile heredofamilial pancreatitis. Operative endoscopic treatment was necessary in 5 children: in 3 a precut-sphineterotomy, in 1 a stone-extraction out e r a big common ampullary duct system, in I a sphincterotomy in a M. Byler situs. One complication due to the endoscopic intervention oocured (chologenic pseudomorias sepsis) that could be treated successfully by conservative methods. Conclusions: ERC(P) in children is a very important diagnostic agent for the demonstration of the reason of diseases caused by hereditary' duct anomalies in the biliary or pancreatic tract. As direct canalieular presentation it is superior to other imaging techniques in such problems. Its result determines possible therapeutic steps that follow endoseopieally during the same procedure or surgically. Pediatric ERC(P) avoids in most cases explorative laparotomies or is precondition for the strategy of a planed operation. It can be seen as a safe proceduredown to an age of two years.
EFFECTS OF ETHANOL ON THE PANCREAS OF DISULFIRAMTREATED RATS. S.HONDA, T.FUJIOKA, K.SHIOTA, K.FUJIYAMA, r.KUBOTA, K.MURAKAMI and M.NASU. The Second Department of ~texnal Medicine, Oita Medical University, ella, Japan
A role for acetaldehyde, the lust product of ethanol metabolism, the pathogenesis of acute alcoholic pancreatitis has been investigated with ontrary results. In rats, blood acetaldehyde is slightly elevated after the oral dministration of ethanol. However disulfiram, an aldehyde dehydrogenase thibitor, markedly increases the concentration of blood acetaldehyde. W e udied effects of ethanol on the rat pancreas during the simultaneous lministration of disult'a-am. Method:Nine-week-old Wistar male rats were vided in five groups. Group 1 (n=5) served as control and all rats were crificed immediately prior to experiment. Group 2 (n=5) included untreated ts. Group 3 (n=4) was given a single dose of disulFwam (1,000 mg/kg) at y 1 and day 4. Group 4 (n=4) was given only ethanol (2,000 rag/kg) from y 2 to day 6 divided into two daily doses with 12 hours intervals. Group ~n---4) was treated with combination disulfli'am-ethanol as described above Group 3 and 4. All animals were sacrificed on the seventh day, 24 hours ;r the last dose of ethanol. The pancreas was removed, weighed, and ided into three pieces which were used for microscopic study, electron :roscopic study and determination of amylase activities and DNA content. ',ults: There were no significant differences in blood ethanol levels between tlfu'am-treated rats and disulf'wam-untreated rats after oral ethanol finistration. But blood acetaldehyde levels of disulf'wam-treated rats were ~ifieantly higher than those of disulfiram-untre,ated rats over twelve hours •,thanol admhfistratinn. Final body weights, pancreas weights and serum lase activities in Group 2, Group 3, Group 4 and Group 5 were ificantly low in relation to Group 1. Amylase activity of pancreatic tissue l m g of DNA was significantly low at Group 5 co.m,pared with other ps. Compared with Group 1, Group 2, 3 and 4 didn't reveal abnormal logical findings. However in Group 5 large vacuolizations appeared. In ion, a decrease in zymogen granules and the presence of diffuse small :ytoplasmie vacuolizations were demonstrated by electron microscopy. small vacuolizations were observed at the basal side of pancreatic r cells where zymogen granules were decreased or did absent. Similar vacuolizations were also observed in hepatic cells. Therefore these ,les are seemed to he lipid. Conclusion: High blood acetaldehyde content produce lipid inclusions in the acinat cells in a short time. And this of lipid inclusions may disturb the synthesis of zymogen granule. Our suggest that acetaldehyde may play an impbrtant role in the ,~enesls of alcoholic pancreatic injury.
GASTROENTEROLOGY, Vol. 108, No. 4
• SlALIC ACID SHIELDS MUCI PEPTIDE REGIONS IN PANCREATIC CANCER CELLS. J.J.L. Ho, S. Chen9, B. Siddiki, and Y.S. Kim. Univ. of Calif. and VA Medical Center, S.F., CA. Patients with pancreatic carcinomas produce cytotoxic T-cells and antibodies that recognize peptide regions of the MUCI mucin polypeptide core. We examined the accessibility of MUCI peptide regions on i n t r a c e l l u l a r , surface, and secreted mucins of the pancreatic cancer cell line, SWlg90. METHODS: Immunoassayswere performed with the following antibodies: DF3 (epitope: TRPAPGS), HMFG-] (PDTR), HMFG-2 (DTR), SM-3 (PDTRP), 139H2 (MUCI peptide), lISD8 (MUCI carbohydrate), 19-9 (sialylated Lewisa) and SNH-3 (sialylated LewisX). Mucins from SWI990 cytosol and spent medium were fractionated by CsCI buoyant density centrifugation on the basis of degree of glycosylation. Surface mucin was assayed on formalin fixed cells grown in 96-well plates. RESULTS: The low buoyant density (less glycosylated) fraction of i n t r a c e l l u l a r mucins contained most of the MUCI peptide immunoreactivity identified by HMFG-I, HMFG-2, and SM-3 as well as by polyclonal anti-sera against deglycosylated purified SWI990 mucins (anti-SWB). The high density (more glycosylated) fraction of both cytosol and medium contained most of the immunoreactivity with 139H2 and DF3, as well as most of the reactivity with antibodies specific for carbohydrate determinants such as sialylated Lewisa and Lewisx. This fraction also contained most of the reactivity with the MUCI carbohydrate specific antibody llSD8. In contrast to i n t r a c e l l u l a r mucins, medium mucins had very l i t t l e MUCI peptide immunoreactivity with low buoyant density. Treatment of the high buoyant density fraction of either intra L c e l l u l a r or medium mucins with neuraminida~e greatly increased the binding of HMFG-I, HMFG-2, SM-3 and anti-SWB (deglycosylated). Neuraminidase also greatly enhanced rea c t i v i t y of these antibodies with the cell surface, while decreasing DF3 reactivity slightly. CONCLUSIONS: Peptide regions of heavily glycosylated MUCI are accessible on swig90 pancreatic cancer mucins, but are normally shielded by s i a l i c acids.
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SORTING OF GP-2 IN P A N C R E A T I C ISLET [~-CELLS OF T R A N S G E N I C M I C E DIFFERS FROM A C I N A R CELLS T. C. Hoons. J. P. Kerr. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA. Cells that secrete proteins by the regulated pathway of secretion, package and store their products in dense-core granules for release upon cellular stimulation. The mechanisms responsible for the sorting of these proteins to the granules remain to be determined. GP-2, the major glycoprotein of the exocrine pancreas secretory granule membrane, is targeted specifically to this organelle. Expression studies in exocrinederived tissue culture models of regulated secretion with the eDNA encoding GP-2 have demonstrated that the protein is sorted appropriately to the endogenous secretory granules. By contrast, when expressed in two endocrine-derived tissue culture cell lines, GP-2 is excluded from the endogenous granules (Hoops, et al., J. Biol. Chem. (1993), 268:25694-5). Tt~is finding suggests that the targeting mechanisms employed by various tissues that secrete proteins by the regulated pathway of secretion may differ. In order to determine whether this difference could be demonstrated in normal nontransformed tissues, we expressed GP-2 in transgenic animals. To study the sorting of GP-2 in endocrine cells, we placed the eDNA for GP-2 under the control of the rat insulin promoter which directs the expression of the protein to I~-cells of pancreatic islets. Pancreata from offspring of founder mice were fixed and sections cut. These sections were incubated with anti-GP-2 antibodies and/or antiinsulin antibodies followed by appropriate fluorochrome-conjugated second antibodies and viewed with a fluorescence microscope. GP-2 staining was seen on the apical plasma membrane and in the apical cytoplasmic region of acinar ceils. In islets, insulin was detected in granules within the cells. By contrast, staining of GP-2 was present on the plasma membranes of insulin-containing cells with little intracellular labeling. These data corroborate our previous findings in U'ansformed cell lines. They demonstrate that in different tissues there is a diversity of sorting mechanisms to the regulated pathway of secretion for some secretory granule proteins. (Supported by the University of Pennsylvania Diabetes Research Center and NIH DK46940)
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AGA ABSTRACTS
PEDIATRIC ERCP TREATMENT IN RELAPSING PANCREATITIS. R.E, Hintz¢, A. Adler, W. Veltzke, W. Luck*, M. Becker*. Central Interdisciplinary Endoscopy, Depts. of Medicine/Gastroenterology and *Pediatrics, University Hospital Rudolf Virehow, Free University of Berlin, Germany lntro~toqtion: Children with relapsing eholangitis or pancreatltis as well as unclear cholestasis need diagnosis by ERC(P) because in most of the eases the reason for the disease can not be clarified with other imaging techniques. Therapeutic experience is low in transpapillary endoscopy in childhood and youth until now. M~$hocls: As instruments we use the standard video.-sideviewing codescopes for adult-ERCP with the corresponding accessories. Because of the experiences of an initial series which was performed under sedativa, we now make all pediatric ERC(P)'s in insufflation anaesthesia. Re~Ils: From 3/92 to 10/94 we performed in 17 children (mean age 6.9 years, 9 male, 8 female) an ERC, in 11 combined with pancreaticography. The indication in 11 children was a relapsing pancreatitis, in 6 cholestasis and/or icterus The diagnostic imaging showed in 2 children a pancreas divisum, in 3 a choledochal cyst, thereof once the types 1, I1 and Ill, and in one a M. Byler (heredofamilial bile duet atresia). Three of them had a juvenile heredofamilial pancreatitis. Operative endoscopic treatment was necessary in 5 children: in 3 a precut-sphineterotomy, in 1 a stone-extraction out e r a big common ampullary duct system, in I a sphincterotomy in a M. Byler situs. One complication due to the endoscopic intervention oocured (chologenic pseudomorias sepsis) that could be treated successfully by conservative methods. Conclusions: ERC(P) in children is a very important diagnostic agent for the demonstration of the reason of diseases caused by hereditary' duct anomalies in the biliary or pancreatic tract. As direct canalieular presentation it is superior to other imaging techniques in such problems. Its result determines possible therapeutic steps that follow endoseopieally during the same procedure or surgically. Pediatric ERC(P) avoids in most cases explorative laparotomies or is precondition for the strategy of a planed operation. It can be seen as a safe proceduredown to an age of two years.
EFFECTS OF ETHANOL ON THE PANCREAS OF DISULFIRAMTREATED RATS. S.HONDA, T.FUJIOKA, K.SHIOTA, K.FUJIYAMA, r.KUBOTA, K.MURAKAMI and M.NASU. The Second Department of ~texnal Medicine, Oita Medical University, ella, Japan
A role for acetaldehyde, the lust product of ethanol metabolism, the pathogenesis of acute alcoholic pancreatitis has been investigated with ontrary results. In rats, blood acetaldehyde is slightly elevated after the oral dministration of ethanol. However disulfiram, an aldehyde dehydrogenase thibitor, markedly increases the concentration of blood acetaldehyde. W e udied effects of ethanol on the rat pancreas during the simultaneous lministration of disult'a-am. Method:Nine-week-old Wistar male rats were vided in five groups. Group 1 (n=5) served as control and all rats were crificed immediately prior to experiment. Group 2 (n=5) included untreated ts. Group 3 (n=4) was given a single dose of disulFwam (1,000 mg/kg) at y 1 and day 4. Group 4 (n=4) was given only ethanol (2,000 rag/kg) from y 2 to day 6 divided into two daily doses with 12 hours intervals. Group ~n---4) was treated with combination disulfli'am-ethanol as described above Group 3 and 4. All animals were sacrificed on the seventh day, 24 hours ;r the last dose of ethanol. The pancreas was removed, weighed, and ided into three pieces which were used for microscopic study, electron :roscopic study and determination of amylase activities and DNA content. ',ults: There were no significant differences in blood ethanol levels between tlfu'am-treated rats and disulf'wam-untreated rats after oral ethanol finistration. But blood acetaldehyde levels of disulf'wam-treated rats were ~ifieantly higher than those of disulfiram-untre,ated rats over twelve hours •,thanol admhfistratinn. Final body weights, pancreas weights and serum lase activities in Group 2, Group 3, Group 4 and Group 5 were ificantly low in relation to Group 1. Amylase activity of pancreatic tissue l m g of DNA was significantly low at Group 5 co.m,pared with other ps. Compared with Group 1, Group 2, 3 and 4 didn't reveal abnormal logical findings. However in Group 5 large vacuolizations appeared. In ion, a decrease in zymogen granules and the presence of diffuse small :ytoplasmie vacuolizations were demonstrated by electron microscopy. small vacuolizations were observed at the basal side of pancreatic r cells where zymogen granules were decreased or did absent. Similar vacuolizations were also observed in hepatic cells. Therefore these ,les are seemed to he lipid. Conclusion: High blood acetaldehyde content produce lipid inclusions in the acinat cells in a short time. And this of lipid inclusions may disturb the synthesis of zymogen granule. Our suggest that acetaldehyde may play an impbrtant role in the ,~enesls of alcoholic pancreatic injury.
GASTROENTEROLOGY, Vol. 108, No. 4
• SlALIC ACID SHIELDS MUCI PEPTIDE REGIONS IN PANCREATIC CANCER CELLS. J.J.L. Ho, S. Chen9, B. Siddiki, and Y.S. Kim. Univ. of Calif. and VA Medical Center, S.F., CA. Patients with pancreatic carcinomas produce cytotoxic T-cells and antibodies that recognize peptide regions of the MUCI mucin polypeptide core. We examined the accessibility of MUCI peptide regions on i n t r a c e l l u l a r , surface, and secreted mucins of the pancreatic cancer cell line, SWlg90. METHODS: Immunoassayswere performed with the following antibodies: DF3 (epitope: TRPAPGS), HMFG-] (PDTR), HMFG-2 (DTR), SM-3 (PDTRP), 139H2 (MUCI peptide), lISD8 (MUCI carbohydrate), 19-9 (sialylated Lewisa) and SNH-3 (sialylated LewisX). Mucins from SWI990 cytosol and spent medium were fractionated by CsCI buoyant density centrifugation on the basis of degree of glycosylation. Surface mucin was assayed on formalin fixed cells grown in 96-well plates. RESULTS: The low buoyant density (less glycosylated) fraction of i n t r a c e l l u l a r mucins contained most of the MUCI peptide immunoreactivity identified by HMFG-I, HMFG-2, and SM-3 as well as by polyclonal anti-sera against deglycosylated purified SWI990 mucins (anti-SWB). The high density (more glycosylated) fraction of both cytosol and medium contained most of the immunoreactivity with 139H2 and DF3, as well as most of the reactivity with antibodies specific for carbohydrate determinants such as sialylated Lewisa and Lewisx. This fraction also contained most of the reactivity with the MUCI carbohydrate specific antibody llSD8. In contrast to i n t r a c e l l u l a r mucins, medium mucins had very l i t t l e MUCI peptide immunoreactivity with low buoyant density. Treatment of the high buoyant density fraction of either intra L c e l l u l a r or medium mucins with neuraminida~e greatly increased the binding of HMFG-I, HMFG-2, SM-3 and anti-SWB (deglycosylated). Neuraminidase also greatly enhanced rea c t i v i t y of these antibodies with the cell surface, while decreasing DF3 reactivity slightly. CONCLUSIONS: Peptide regions of heavily glycosylated MUCI are accessible on swig90 pancreatic cancer mucins, but are normally shielded by s i a l i c acids.
•
SORTING OF GP-2 IN P A N C R E A T I C ISLET [~-CELLS OF T R A N S G E N I C M I C E DIFFERS FROM A C I N A R CELLS T. C. Hoons. J. P. Kerr. Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA. Cells that secrete proteins by the regulated pathway of secretion, package and store their products in dense-core granules for release upon cellular stimulation. The mechanisms responsible for the sorting of these proteins to the granules remain to be determined. GP-2, the major glycoprotein of the exocrine pancreas secretory granule membrane, is targeted specifically to this organelle. Expression studies in exocrinederived tissue culture models of regulated secretion with the eDNA encoding GP-2 have demonstrated that the protein is sorted appropriately to the endogenous secretory granules. By contrast, when expressed in two endocrine-derived tissue culture cell lines, GP-2 is excluded from the endogenous granules (Hoops, et al., J. Biol. Chem. (1993), 268:25694-5). Tt~is finding suggests that the targeting mechanisms employed by various tissues that secrete proteins by the regulated pathway of secretion may differ. In order to determine whether this difference could be demonstrated in normal nontransformed tissues, we expressed GP-2 in transgenic animals. To study the sorting of GP-2 in endocrine cells, we placed the eDNA for GP-2 under the control of the rat insulin promoter which directs the expression of the protein to I~-cells of pancreatic islets. Pancreata from offspring of founder mice were fixed and sections cut. These sections were incubated with anti-GP-2 antibodies and/or antiinsulin antibodies followed by appropriate fluorochrome-conjugated second antibodies and viewed with a fluorescence microscope. GP-2 staining was seen on the apical plasma membrane and in the apical cytoplasmic region of acinar ceils. In islets, insulin was detected in granules within the cells. By contrast, staining of GP-2 was present on the plasma membranes of insulin-containing cells with little intracellular labeling. These data corroborate our previous findings in U'ansformed cell lines. They demonstrate that in different tissues there is a diversity of sorting mechanisms to the regulated pathway of secretion for some secretory granule proteins. (Supported by the University of Pennsylvania Diabetes Research Center and NIH DK46940)