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Phospholipase A2 - inhibition: A new concept to treat acute pancreatitis
A390
AGA
ABSTRACTS
GASTROENTEROLOGY,
• P H O S P H O L I P A S E A 2 - I N H I B I T I O N : A N E W CONCEPT TO TREAT ACUTE PANCREATIT1S.
~
W.H. Uhl, H. Friess, J. Aufenanger 1, T.J. Nevalainen2, U. Tibes3, W. -G Friebe3, M.W. B0clder. Clinic of Visceral and Transplantation Surgery, University of Berne, Switzerland; llnstitute Of Clinical Chemistry, University of Mannheim, Germany 2Deparunent of Pathology, University' of Turku, Finland 3Department of Preclinical, Biochemical and Chemical Research, Boehringer Mannheim GmbH, Germany. In acute pancreatitis(AP)two differenttypesof phospholipaseA2 havebeen found,namelythe pancreatictype I (PLA2-1I)and an extmpancreatictype II (PLA2-H)which is responsiblefor a complicatedcourseof this disease.Therefore,we investigateda new potentlow molecularweight PLA2-inhibitor(CBM 16.2056)in experimentalnecrotizingAP. Methods: 50 female Wistarrats were dividedinto two groups.Group1: intraductalinjectionof 0.2 ml of 3%-sodium-taurocholate, control (0.9% NaCI)- mad inhibitor-group(each n=t0). The inhibitor(341.8Dalton) was givenin a therapeuticmanner, i.v. 10 min after induction of AP (16 mg/kgbodywt.), 6 and 12 hrs later (9 mg/kg body wt. respectively). Group 2: from controls (n=15) and the inhibitor-group(n=15) the whole pancreas was removed for histology 0.25, 3 and 12 hrs after inductionof taurochoiateAP. The inhibitorin this experimentwas givenin a prophylacticmanner, i.v. 10 min before APinduction(32 mg/kg body wt.) and the findings were assessed by a modified scoring system -24 (Path. Res.Praet.184, 507-13,1989). The followingparameterswere measuredin rats sera: nln~,~,mo pan-PLA2 by fluoroimmtmoassay,total PLA2activity by radiometric E. coil-assay and CAr ~ - ' ~ PLA2-Iand ][Idifferentiationby heat inactivation of CA-PLA2-]I(60°). Results: The significant ~~0 ~ ................. : ........ " • ....... ............. inhibition of CA-PLA2Was mainly due to the ~ is ......... ............ inhibition of CA-PLA2-II, with significant ~ lo ......... ............. , ........ decreases of CA-PLA2-IIat the time points 3 ' s (*p<0.002), 6(*p<0.002), 12(**p<0.0l), o t~a C~I~L/a 18(***p<0.02)hrs. The pan-PLA2concentration o 3 6 ~ui did not changed significantly (control vs. inhibitorng/ml:0h-22,45:17,75;3h-683,7:622,5; 6h-187,35:166,75; 12h-136,55:148,45; 18h57,95:70,25;24h-35,08:26,51).Tissue damageand inflammationincreasedrapidlyin the controls in contrast to the treated animals (hrs:control vs. inhibitor score: 0.25:41/55; 3:67/45; 12:120/78).Canclusian:The results show, i) that PLA2 type lI is markedly elevated in the necrotizingmodelof AP comparableto humanacutepancreatitis,ii) withregardto the inhibitionof CA-PLA2-IIthe new low molecularweight PLA2-inhibitoris very potent and effective,iii) this inhibitorprotectsagainstpancreatictissue-damage.
ERCP FINDINGS PATIENTS WITH
CORRELATE RESECTABLE
WITH TUMOR PANCREATIC
SIZE IN CANCER.
S.A. Shah, J. Movson, I. Waxman. Depts. of Medicine & R a d i o l o g y , Beth Israel Hospital, Harvard Medical School, Boston,/viA. P u r p o s e : T u m o r size has been shown to be an important prognostic factor in resectable pancreatic cancer. Although ERCP has proven utility in diagnosis, its potential as a pro~,nostic tool in resectable pancreatic cancer has not been evaluated. The aim of this study was to investigate whether findings on E R C P correlated with tumor size on rasected specimens in pancreatic cancer. M e t h o d s : Between January, 1991 and August, 1994, 14 patients admitted to our hospital who had resectahle pancreatic cancer (i.e. no evidence of metastases or vessel invasion on CT scan) a n d had u n d e r g o n e E R C P p r i o r to an a t t e m p t at c u r a t i v e resection w e r e retrospectively identified. C o m m o n bile duct (CBD) stricture length, pancreatic duct (PD) stricture length and greatest distance between strictures (Dist) were measured on E R C P and compared with actual size of mass on surgical pathology. Magnification was controlled for by comparison to actual endoscope diameter. Stricture length (if stricture present) was plotted against actual tumor size and a correlation coefficient was calculated. R e s u l t s : Table 1 shows stricture length compared with tumor size on pathology and CT scan, in millimeters. The largest measurement on ERCP correlated best with actual tumor size on surgical pathology. Table 2 shows the cormlal~on coefficient (r) for each measurement. )arient ! 1 Z ,3 i4
10 11 12 13 14
CBD 5.9 18,7 7.8 17.1 0.0 23.3 24.7 15.8 17.S 0,0 t2.4 8.2 3.0 4.7
PD 9,6 0.0 2.3 0.0 14,7 11.7 26.4 0.0 8,8 43.2 8.2 5.8 0.0 0.(~
table1 Dist Lar~lest Tumorsize CT~mass 10.3 10,3 20.0 18 0.0 18.7 20.0 none 23.3 23.3 55.0 none 10.8 17.1 22,0 none 0,0 14.7 30,0 none Z.3 a3.3 37.0 20 15.6 26.4 40.0 10 0.0 15,8 30.0 none 13.1 17.5 32.0 none 0.0 43.2 60.0 none 1.6 12,4 15.0 28 2.1 8.2 27.0 25 0.(] 3,0 35,0 10 5.4 5,4 12,0 not done
table ;~ Measurement r CBD O.t8 PD 0.54 Dist 0.70 Largest 0.74 LOO=.pcl.fl~tcon.cla$io n
E R C P findings correlated with tumor size on surgical pathology. In no case was tumor size overestimated. In addition, E R C P was more accurate in predicting actual tumor size than CT. Thus, E R C P may provide important prognostic as well as diagnostic information in pancreatic cancer.
Conclusions:
Vol.
108,
NO. 4
• A B N O R M A L PANCREATIC POLYPEPTIDE AND S O M A T O S T A T I N IMMUNOSTAINING IN EXPERIMENTAL CHRONIC PANCREATITIS. N.E. S e y m o u r , A.R. Volpert, L.R. Inman, E.L. Lee, F.C. Brunicardi. Depts. of Surgery & Pathology, Veterans Affairs Medical Center and the University of Texas Southwestern Medical Center, Dallas, Texas. Chronic pancreatitiS (CP) in rats after operative pancreatic duct infusion.with oleic acid is associated with exocrine and endocrine abnormalities. These include diminished immunohistochemieal staining for insulin and glucagon, T o f u r t h e r Characterize h o r m o n a l d e f i c i e n c i e s in this m o d e l , immunohistochemical staining of endocrine tissue was studied 6-8 weeks after induction of pancreatitis or sham operation in fully-recovered 3 0 0 - 3 5 0 gm Sprague-Dawley rats. Formalin-fixed pancreata were serially sectioned and immunostained with peptide-specific antibody to pancreatic polypeptide (PP) or somatostafin (SST) w h i c h were d e t e c t e d by a secondary antibody/ABC-peroxidase procedure~ Sections were counterstained with hematoxylin and examined microscopically. Pepti.de staining in Islets of Langerhans was quantified by the Weibel point counting method and expressed as stained volume fraction of islet. Islet cross sectional area was also determined. Results represent pooled data from 5 sham-operated control and 5 CP animals and are expressed as m e a n + standard error. Stained Volume Fraction PP SST Mean Islet Area (/~2) Control 0.045+_0.006(147islets) 0~056+01006(129islets) 14595+_1177 CP 0.013+0.003"['(98 islets) 0.009+0.002"~(129islets) 16637+_1541 J'p < 0.001 versus sham,operation, Student's unpaired t-test. Histologic changes in CP sections included acinar replacement with fibrosis and adipose tissue. Islets from animals with CP showed significantly less staining for both pancreatic polypeptide and somatostatin than those from animals after sham operation. The proportion of evaluated i s l e t s demonstrating detectable immunostaining was markedly diminished in CP for both PP (60% in controls versus 34% in CP) and somatostatin (82% in controls versus 4 7 % in CP). There was no significant difference in islet cross-sectional area in experimental and control groups,, but 62% of islets in CP specimens were remote from normal exocrine tissue (0% in controls) and demonstrated diminished immunostaining for both hormones as compared to intraparenchymal islets. We conclude that this model of chronic pancreatitis results in diminished immunohistochemically detectable islet staining for PP and somatostatin which is most prominent when islet and acinar tissue are separated after acinar destruction.
I M M U N O I S O L A T I O N AND CULTURE OF R A T P A N C R E A T I C DUCTAL EPITHELIAL CELLS. L.B. Shalon, T.A, Konkin, R.A. Faris . . . . . Depts. off Pediatrics & Medical Oncology, Brown University School of Medicine/RI Hospital, Providence, RI. The established methodology for expanding the rat pancreatic ductal epithelial cell If'DEC) population requires culturing the fresh pancreatic isolate in order to purify ductal fragments. This technique is limited by the possibility that pancreatic acinar cells may dedifferentiate into duct-like cells in culture. We have modified this technique in order to enhance PDEC purity. In the original technique, after digesting the pancreas with collagenase, newly isolated interlobularductal fragments are embedded in a collagen gel with DMEM/F12 media supplemented with dexamethasone, cholera toxin, insulin, epidermal growth factor and Nu-Serum (Heimann TG, Githens S. Pancreas 6:514r 21;1991). The duct fragments become distinguishable from other tissue t y p e s after the epithelial ends of the ducts seal and continued fluid secretion results in the formation of cystic ductal structures within 3 days. The collagen gel is solubilized and the cysts are manually collected using a pasteur pipet with the aid of a dissecting microscope. After dissociation by divalent cation chelation and collagenase digestior/, epithelial fragments are plated onto a collagen gel surface and subsequently form an epithelial monolayer. In the modified technique, after digesting the pancreas with collagenase the newly isolated ductal fragments were briefly incubated with pronase, which preferentially destroys acinar ceils. Following pronase digestion, the ductal fragments were immunoisolated using magnetic beads coated with the monoclonal antibody (MAb) 236.3. This Mab recognizes the setine exoprotease dipeptidyl peptidase (DPP) IV. In the pancreas, expression of the DPPIV epitope recognized by MAb 236.3 is restricted to ductal cells. The bead-bound fragments were embedded in a collagen gel where they formed cystic ductal structures within 3 days. The bead-bound cysts were subsequently removed with a magnet a n d then dissociated by divalent cation chelation and collagenase digestion; The epithelial fragments were plated onto a collagen gel surface where they formed an epithelial monolayer. There was minimal contamination of the monolayer with other tissue types. Immunohistochemical analysis of ductal epithelial monolayers demonstrated continued expression of DPPIV as well as other antigenic ductal markers. Our studies suggest that isolation of PDEC using magnetic beads conjugated to Mab 236.3 will enhance the purity of PDEC cultures. Future studies will attempt to identify culture conditions which support the long-term propagation of DPPIV+ pancreatic duct cells.
AGA
ABSTRACTS
GASTROENTEROLOGY,
• P H O S P H O L I P A S E A 2 - I N H I B I T I O N : A N E W CONCEPT TO TREAT ACUTE PANCREATIT1S.
~
W.H. Uhl, H. Friess, J. Aufenanger 1, T.J. Nevalainen2, U. Tibes3, W. -G Friebe3, M.W. B0clder. Clinic of Visceral and Transplantation Surgery, University of Berne, Switzerland; llnstitute Of Clinical Chemistry, University of Mannheim, Germany 2Deparunent of Pathology, University' of Turku, Finland 3Department of Preclinical, Biochemical and Chemical Research, Boehringer Mannheim GmbH, Germany. In acute pancreatitis(AP)two differenttypesof phospholipaseA2 havebeen found,namelythe pancreatictype I (PLA2-1I)and an extmpancreatictype II (PLA2-H)which is responsiblefor a complicatedcourseof this disease.Therefore,we investigateda new potentlow molecularweight PLA2-inhibitor(CBM 16.2056)in experimentalnecrotizingAP. Methods: 50 female Wistarrats were dividedinto two groups.Group1: intraductalinjectionof 0.2 ml of 3%-sodium-taurocholate, control (0.9% NaCI)- mad inhibitor-group(each n=t0). The inhibitor(341.8Dalton) was givenin a therapeuticmanner, i.v. 10 min after induction of AP (16 mg/kgbodywt.), 6 and 12 hrs later (9 mg/kg body wt. respectively). Group 2: from controls (n=15) and the inhibitor-group(n=15) the whole pancreas was removed for histology 0.25, 3 and 12 hrs after inductionof taurochoiateAP. The inhibitorin this experimentwas givenin a prophylacticmanner, i.v. 10 min before APinduction(32 mg/kg body wt.) and the findings were assessed by a modified scoring system -24 (Path. Res.Praet.184, 507-13,1989). The followingparameterswere measuredin rats sera: nln~,~,mo pan-PLA2 by fluoroimmtmoassay,total PLA2activity by radiometric E. coil-assay and CAr ~ - ' ~ PLA2-Iand ][Idifferentiationby heat inactivation of CA-PLA2-]I(60°). Results: The significant ~~0 ~ ................. : ........ " • ....... ............. inhibition of CA-PLA2Was mainly due to the ~ is ......... ............ inhibition of CA-PLA2-II, with significant ~ lo ......... ............. , ........ decreases of CA-PLA2-IIat the time points 3 ' s (*p<0.002), 6(*p<0.002), 12(**p<0.0l), o t~a C~I~L/a 18(***p<0.02)hrs. The pan-PLA2concentration o 3 6 ~ui did not changed significantly (control vs. inhibitorng/ml:0h-22,45:17,75;3h-683,7:622,5; 6h-187,35:166,75; 12h-136,55:148,45; 18h57,95:70,25;24h-35,08:26,51).Tissue damageand inflammationincreasedrapidlyin the controls in contrast to the treated animals (hrs:control vs. inhibitor score: 0.25:41/55; 3:67/45; 12:120/78).Canclusian:The results show, i) that PLA2 type lI is markedly elevated in the necrotizingmodelof AP comparableto humanacutepancreatitis,ii) withregardto the inhibitionof CA-PLA2-IIthe new low molecularweight PLA2-inhibitoris very potent and effective,iii) this inhibitorprotectsagainstpancreatictissue-damage.
ERCP FINDINGS PATIENTS WITH
CORRELATE RESECTABLE
WITH TUMOR PANCREATIC
SIZE IN CANCER.
S.A. Shah, J. Movson, I. Waxman. Depts. of Medicine & R a d i o l o g y , Beth Israel Hospital, Harvard Medical School, Boston,/viA. P u r p o s e : T u m o r size has been shown to be an important prognostic factor in resectable pancreatic cancer. Although ERCP has proven utility in diagnosis, its potential as a pro~,nostic tool in resectable pancreatic cancer has not been evaluated. The aim of this study was to investigate whether findings on E R C P correlated with tumor size on rasected specimens in pancreatic cancer. M e t h o d s : Between January, 1991 and August, 1994, 14 patients admitted to our hospital who had resectahle pancreatic cancer (i.e. no evidence of metastases or vessel invasion on CT scan) a n d had u n d e r g o n e E R C P p r i o r to an a t t e m p t at c u r a t i v e resection w e r e retrospectively identified. C o m m o n bile duct (CBD) stricture length, pancreatic duct (PD) stricture length and greatest distance between strictures (Dist) were measured on E R C P and compared with actual size of mass on surgical pathology. Magnification was controlled for by comparison to actual endoscope diameter. Stricture length (if stricture present) was plotted against actual tumor size and a correlation coefficient was calculated. R e s u l t s : Table 1 shows stricture length compared with tumor size on pathology and CT scan, in millimeters. The largest measurement on ERCP correlated best with actual tumor size on surgical pathology. Table 2 shows the cormlal~on coefficient (r) for each measurement. )arient ! 1 Z ,3 i4
10 11 12 13 14
CBD 5.9 18,7 7.8 17.1 0.0 23.3 24.7 15.8 17.S 0,0 t2.4 8.2 3.0 4.7
PD 9,6 0.0 2.3 0.0 14,7 11.7 26.4 0.0 8,8 43.2 8.2 5.8 0.0 0.(~
table1 Dist Lar~lest Tumorsize CT~mass 10.3 10,3 20.0 18 0.0 18.7 20.0 none 23.3 23.3 55.0 none 10.8 17.1 22,0 none 0,0 14.7 30,0 none Z.3 a3.3 37.0 20 15.6 26.4 40.0 10 0.0 15,8 30.0 none 13.1 17.5 32.0 none 0.0 43.2 60.0 none 1.6 12,4 15.0 28 2.1 8.2 27.0 25 0.(] 3,0 35,0 10 5.4 5,4 12,0 not done
table ;~ Measurement r CBD O.t8 PD 0.54 Dist 0.70 Largest 0.74 LOO=.pcl.fl~tcon.cla$io n
E R C P findings correlated with tumor size on surgical pathology. In no case was tumor size overestimated. In addition, E R C P was more accurate in predicting actual tumor size than CT. Thus, E R C P may provide important prognostic as well as diagnostic information in pancreatic cancer.
Conclusions:
Vol.
108,
NO. 4
• A B N O R M A L PANCREATIC POLYPEPTIDE AND S O M A T O S T A T I N IMMUNOSTAINING IN EXPERIMENTAL CHRONIC PANCREATITIS. N.E. S e y m o u r , A.R. Volpert, L.R. Inman, E.L. Lee, F.C. Brunicardi. Depts. of Surgery & Pathology, Veterans Affairs Medical Center and the University of Texas Southwestern Medical Center, Dallas, Texas. Chronic pancreatitiS (CP) in rats after operative pancreatic duct infusion.with oleic acid is associated with exocrine and endocrine abnormalities. These include diminished immunohistochemieal staining for insulin and glucagon, T o f u r t h e r Characterize h o r m o n a l d e f i c i e n c i e s in this m o d e l , immunohistochemical staining of endocrine tissue was studied 6-8 weeks after induction of pancreatitis or sham operation in fully-recovered 3 0 0 - 3 5 0 gm Sprague-Dawley rats. Formalin-fixed pancreata were serially sectioned and immunostained with peptide-specific antibody to pancreatic polypeptide (PP) or somatostafin (SST) w h i c h were d e t e c t e d by a secondary antibody/ABC-peroxidase procedure~ Sections were counterstained with hematoxylin and examined microscopically. Pepti.de staining in Islets of Langerhans was quantified by the Weibel point counting method and expressed as stained volume fraction of islet. Islet cross sectional area was also determined. Results represent pooled data from 5 sham-operated control and 5 CP animals and are expressed as m e a n + standard error. Stained Volume Fraction PP SST Mean Islet Area (/~2) Control 0.045+_0.006(147islets) 0~056+01006(129islets) 14595+_1177 CP 0.013+0.003"['(98 islets) 0.009+0.002"~(129islets) 16637+_1541 J'p < 0.001 versus sham,operation, Student's unpaired t-test. Histologic changes in CP sections included acinar replacement with fibrosis and adipose tissue. Islets from animals with CP showed significantly less staining for both pancreatic polypeptide and somatostatin than those from animals after sham operation. The proportion of evaluated i s l e t s demonstrating detectable immunostaining was markedly diminished in CP for both PP (60% in controls versus 34% in CP) and somatostatin (82% in controls versus 4 7 % in CP). There was no significant difference in islet cross-sectional area in experimental and control groups,, but 62% of islets in CP specimens were remote from normal exocrine tissue (0% in controls) and demonstrated diminished immunostaining for both hormones as compared to intraparenchymal islets. We conclude that this model of chronic pancreatitis results in diminished immunohistochemically detectable islet staining for PP and somatostatin which is most prominent when islet and acinar tissue are separated after acinar destruction.
I M M U N O I S O L A T I O N AND CULTURE OF R A T P A N C R E A T I C DUCTAL EPITHELIAL CELLS. L.B. Shalon, T.A, Konkin, R.A. Faris . . . . . Depts. off Pediatrics & Medical Oncology, Brown University School of Medicine/RI Hospital, Providence, RI. The established methodology for expanding the rat pancreatic ductal epithelial cell If'DEC) population requires culturing the fresh pancreatic isolate in order to purify ductal fragments. This technique is limited by the possibility that pancreatic acinar cells may dedifferentiate into duct-like cells in culture. We have modified this technique in order to enhance PDEC purity. In the original technique, after digesting the pancreas with collagenase, newly isolated interlobularductal fragments are embedded in a collagen gel with DMEM/F12 media supplemented with dexamethasone, cholera toxin, insulin, epidermal growth factor and Nu-Serum (Heimann TG, Githens S. Pancreas 6:514r 21;1991). The duct fragments become distinguishable from other tissue t y p e s after the epithelial ends of the ducts seal and continued fluid secretion results in the formation of cystic ductal structures within 3 days. The collagen gel is solubilized and the cysts are manually collected using a pasteur pipet with the aid of a dissecting microscope. After dissociation by divalent cation chelation and collagenase digestior/, epithelial fragments are plated onto a collagen gel surface and subsequently form an epithelial monolayer. In the modified technique, after digesting the pancreas with collagenase the newly isolated ductal fragments were briefly incubated with pronase, which preferentially destroys acinar ceils. Following pronase digestion, the ductal fragments were immunoisolated using magnetic beads coated with the monoclonal antibody (MAb) 236.3. This Mab recognizes the setine exoprotease dipeptidyl peptidase (DPP) IV. In the pancreas, expression of the DPPIV epitope recognized by MAb 236.3 is restricted to ductal cells. The bead-bound fragments were embedded in a collagen gel where they formed cystic ductal structures within 3 days. The bead-bound cysts were subsequently removed with a magnet a n d then dissociated by divalent cation chelation and collagenase digestion; The epithelial fragments were plated onto a collagen gel surface where they formed an epithelial monolayer. There was minimal contamination of the monolayer with other tissue types. Immunohistochemical analysis of ductal epithelial monolayers demonstrated continued expression of DPPIV as well as other antigenic ductal markers. Our studies suggest that isolation of PDEC using magnetic beads conjugated to Mab 236.3 will enhance the purity of PDEC cultures. Future studies will attempt to identify culture conditions which support the long-term propagation of DPPIV+ pancreatic duct cells.