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Gabexate mesilate promotes neutrophil apoptosisand attenuates serum cytokines alterations inhuman acute pancreatitis
SSATAI043
April 2000
2300 ANTI·ANGIOGENESIS EFFECT OF P53·TARGET GENE, BAIl ON PANCREATIC CANCER. Makoto Sunamura, Dan G. Duda, Lucian Lozonschi, Tadamichi Yokoyama, Toshimasa Yatsuoka, Fuyuhiko Motoi, Hiromune Shimamura, Kazunori Takeda, Seiki Matsuno, Tohoku Univ Sch of Medicine, Sendai, Mi, Japan. Background: It is reported that the genetic alteration of p53 is associated with neovascularization during the progression of glioma to its more malignant form, glioblastoma. BAIl which was identified as a p53-target gene, is specifically expressed in the brain, however is not expressed in gioblastoma cell lines, suggesting that BAIl plays a significant role in angiogenesis inhibitor as a mediator of p53. We already demonstrated that BAIl transfected glioblastoma failed to form tumor neovascularization. Purpose: In an attempt to gain insight into this effect, we checked the expression of this gene in pancreatic cancer cell lines. Furthermore, we transfected BAIl gene to a pancreatic cancer cell line using an adenoviral vector and monitored the in vitro and in vivo growth in severe combined immune-deficient (SCID) mice. Materials and Methods: RT-PCR analysis revealed that BAIl gene was not expressed in any of the eight pancreatic cancer cell lines. After p53 transfection using an adenoviral vector, BAIl expression was induced in pancreatic cancer cell line (Pane1) as well as colon cancer cell lines (T84 and HT29). We transfected BAIl gene into a pancreatic cancer cell line (PancIBAIl) using an adenoviral vector and monitored both in vitro growth and in vivo growth using scm mice. LacZ gene-tranfected pancreatic cancer cell line was used as control (Pane/ LacZ). The in vitro growth curve of PanclBAIl was similar to that of PnaclLacZ or wild type tumor cells (Pane/wild). However, the in vivo growth of PanclBAIl was significantly delayed as compared to PanclLacZ and Pane/wild, We analysed the anti-angiogenesis effect using our skinfold chamber and vital microscopy system, which allowed the accurate assessment of tumor angiogenesis. Pane/wild or PanclLacZ in the chamber promoted the tumor neovascularization at day 14, whereas PanclBAIl did not. RT-PCR analysis confirmed the BAIl expression in the PanclBAIl and also showed that BAIl effect was not dependent on the endothelial cell mitogens, such as VEGF or bFGF activity. Conclusion: The expression of BAIl gene induced tumor dormancy in human pancreatic tumors through the inhibition of tumor angiogenesis. The anti-tumor effect of p53 gene therapy would be strengthened by the combination of BAIl pathway. The mechanism of this effect is currently under investigation. 2301 PARATHYROID HORMONE-RELATED PROTEIN EXPRESSION IN HUMAN PANCREATIC ADENOCARCINOMA. Michael Bouvet, Stephanie R. Nardin, Douglas W. Burton, Cynthia Behling, John M. Carethers, Robert M. Hoffman, A. R. Moossa, Leonard J. Deftos, UC San Diego, San Diego, CA. Parathyroid hormone-related protein (PTHrP) is expressed in many common malignancies such as breast and prostate cancer and can regulate their growth. Little is known, however, about the role of PTHrP in pancreatic adenocarcinoma. In order to study PTHrP expression in pancreatic cancer, eight human pancreatic carcinoma cell lines were evaluated including AsPC-l, BxPC-3, Capan-I, CFPAC-1, MIA PaCa-2, PANC-1, PANC-28, and PANC-48. Murine monoclonal antibodies to the amino-terminal amino acid-fragment (1-34), mid-region (38-64), and carboxy-terminal aminoacid fragment (l 09-141) of PTHrP were used to identify intracellular PTHrP. secreted PTHrP, and for western blotting and immunocytochemical staining for PTHrP from each cell line. Intracellular PTHrP 1-34 was detected in all cell lines by both western blotting and immunoassay at concentrations ranging from 2.6 to 30.5 pg/mg protein. CFPAC-I, derived from a pancreatic liver metastasis, had the highest concentration and MIA PaCa-2, derived from primary pancreatic adenocarcinoma, the lowest. Secretion of PTHrP 1-34 into cell media was also noted for each cell line and paralled intracellular PTHrP 1-34 levels. PTHrP was localized by immunocytochemical staining in the cytoplasm in all but one cell line and both nuclear and cytoplasmic immunostaining was observed in the MIA PaCa-2 and PANC-1 cells. Evidence for differential processing of PTHrP was provided by studies demonstrating that PTHrP 38-64 secretion was highest for BxPC-3 rather than CFPAC-I while the highest levels of PTHrP 109-141 secretion were seen in Capan-I. Immunostaining from archival tissue of patients with pancreatic adenocarcinoma revealed strong PTHrP staining in 14 of 14 specimens. These results demonstrate that PTHrP is commonly expressed in pancreatic cancer and may playa role in the pathogenesis of this disease.
2302 GABEXATE MESILATE PROMOTES NEUTROPHIL APOPTOSIS AND ATTENUATES SERUM CYTOKINES ALTERATIONS IN HUMAN ACUTE PANCREATITIS. Han-Ming Chen, Miin-Fu Chen, Tsann-Long Hwang, Yi-Yin Jan, Ji-Chan Chen, Dept of Gen Surg, Taipei, Taiwan, ROC; Dept of Emergency Medicine, Taipei, Taiwan, ROC. Introduction: Inappropriate acivation of neutrophils within tissues plays a crucial role in the development of organs dysfunction in acute pancreatitis. The delay of programmed cell death or apoptosis of neutrophils is associated with the failure in termination of neutrophil-mediated inflammatory response. We investigated the effects of gabexate mesilate on neutrophil apoptosis in the progress of severe acute pancreatitits. Methods: Patients with acute pancreatitis who met the following criteria: onset less than 48 hr and APACHE-II score>8 or/and Ranson score>3 were enrolled and randomly grouped in this study. All patients were admitted to the intensive care unit. Gabexate mesilate was delivered through a central vein catheterization at the dose of Img/kg/hr afteradmission (Group B, n= 15). Group A received vehecle infusion only (n= 11). Neutrophils and plasma were isolated from whole venous blood at 3 time points: at admision, 12 hr post-treatment and 48 hr post-treatment of acute pancreatitis. Neutrophil apoptosis was quantified by immunofluorescence flow cytometry of 3' terminal deoxynucleotidyl transferase nick end labeling and PI staining. Cytokine detection was through special commercialized kits, which were based on enzyme-linked immunosorbent assay. Results: Neutrophil apoptosis was significantly inhibited with an event evident 12 hr and 48 hr aftertreatment of acute pancreatitis. Gabexate mesilate infusion significantly promoted neutrophil apoptosis. Levels of inflarnrnatory mediators (TNF-aand IL-6) which levels increased 12 hr and 48 hr after treatment of acute pancreatitis was notably attenuated by gabexate mesilate. Levels of other inflarnrnatory mediators (Il.l-Band IL-8) which levels increased 12 hr after treatment of acute pancreatitis was unaltered. Whereas level of IL-IO, which was increased 48 hr after treatment of acute pancreatitis, was only slightly decreased. Conclusions: Gabexate mesilate given early in the course of acute pancreatitis lessens the magnitudes of changes of inflammatory mediators and the inhibition of neutrophil apoptosis. It has little effect on the anti-inflammatory cytokine IL-IO, however. 2303 SIMULTANEOUS KIDNEY-PANCREAS TRANSPLANTATION WITH ENTERIC EXOCRINE DRAINAGE: A PROSPECTIVE COMPARISON OF SYSTEMIC VERSUS PORTAL VENOUS DELIVERY OF INSULIN. Robert J. Stratta, Mohammed H. Shokouh-Amiri, Hani P. Grewal, Maria F. Egidi, Tarik A. Kizilisik, Lillian W. Gaber, Ahmed O. Gaber, Univ of Tennessee-Memphis, Memphis, TN; Univ of Tennessee, Memphis, TN. The results of pancreas transplantation (PTX) continue to improve due to advances in surgical techniques and immunosuppression. Although renewed interest has occurred in enteric exocrine drainage, most PTXs are performed with systemic venous delivery of insulin (systemic-enteric [SED. To improve the physiology of PTX, a new surgical technique was developed with portal venous delivery of insulin and enteric exocrine drainage (portal-enteric [PED. The purpose of this study was to compare PTX with SE vs PE drainage in a prospective fashion. Over a 16-month period, we alternately performed either SE (N = 18) or PE (N = 18) drainage in 36 consecutive simultaneous kidney-PTXs (SKPTs). The two groups were well matched for donor and recipient demographic, immunologic, dialysis, diabetes, and transplant characteristics. Maintenance immunosuppression in both groups consisted of tacrolimus, mycophenolate mofetil, and steroids. No antibody induction therapy occurred in about half of patients in each group, with the remainder equally divided between daclizumab and basiliximab adjuvant therapy. Results: Patient and kidney graft survival rates are 94% in both groups. One early death occurred in each group (both with functioning grafts). All but I kidney graft (PE group) had immediate function. PTX survival (complete insulin independence) is 89% in both groups with a mean follow-up of nearly 1 year (minimum 3 months). There was I non-immunologic pancreas graft loss in each group, but no grafts were lost to thrombosis. The mean length of initial hospital stay was 12 days in the SE and 14 days in the PE groups, respectively. The SE group was characterized by a slight increase in the number of readmissions (mean 2.5 SE vs 1.4 PE, P=NS). The incidence of major infection was similar (61% SE vs 50% PE), as was the relaparotomy rate (28% SE vs 33% PE). There was I CMV infection (6%) in the SE group. The incidence of intra-abdominal infection and enteric leaks was slightly higher in the SE group (28% SE vs 11% PE, P=NS). However, the need for prolonged vascular access was comparable (33% SE vs 28% PE). Mean hospital charges were comparable between groups ($100,102 SE vs $107,685 PE). The composite endpoint of no readmission, no re-operation, and no rejection was equally attained by four patients (22%) in each group. Conclusions: These preliminary results suggest that SKPT with SE and PE drainage can be performed with comparable short-term outcomes.
April 2000
2300 ANTI·ANGIOGENESIS EFFECT OF P53·TARGET GENE, BAIl ON PANCREATIC CANCER. Makoto Sunamura, Dan G. Duda, Lucian Lozonschi, Tadamichi Yokoyama, Toshimasa Yatsuoka, Fuyuhiko Motoi, Hiromune Shimamura, Kazunori Takeda, Seiki Matsuno, Tohoku Univ Sch of Medicine, Sendai, Mi, Japan. Background: It is reported that the genetic alteration of p53 is associated with neovascularization during the progression of glioma to its more malignant form, glioblastoma. BAIl which was identified as a p53-target gene, is specifically expressed in the brain, however is not expressed in gioblastoma cell lines, suggesting that BAIl plays a significant role in angiogenesis inhibitor as a mediator of p53. We already demonstrated that BAIl transfected glioblastoma failed to form tumor neovascularization. Purpose: In an attempt to gain insight into this effect, we checked the expression of this gene in pancreatic cancer cell lines. Furthermore, we transfected BAIl gene to a pancreatic cancer cell line using an adenoviral vector and monitored the in vitro and in vivo growth in severe combined immune-deficient (SCID) mice. Materials and Methods: RT-PCR analysis revealed that BAIl gene was not expressed in any of the eight pancreatic cancer cell lines. After p53 transfection using an adenoviral vector, BAIl expression was induced in pancreatic cancer cell line (Pane1) as well as colon cancer cell lines (T84 and HT29). We transfected BAIl gene into a pancreatic cancer cell line (PancIBAIl) using an adenoviral vector and monitored both in vitro growth and in vivo growth using scm mice. LacZ gene-tranfected pancreatic cancer cell line was used as control (Pane/ LacZ). The in vitro growth curve of PanclBAIl was similar to that of PnaclLacZ or wild type tumor cells (Pane/wild). However, the in vivo growth of PanclBAIl was significantly delayed as compared to PanclLacZ and Pane/wild, We analysed the anti-angiogenesis effect using our skinfold chamber and vital microscopy system, which allowed the accurate assessment of tumor angiogenesis. Pane/wild or PanclLacZ in the chamber promoted the tumor neovascularization at day 14, whereas PanclBAIl did not. RT-PCR analysis confirmed the BAIl expression in the PanclBAIl and also showed that BAIl effect was not dependent on the endothelial cell mitogens, such as VEGF or bFGF activity. Conclusion: The expression of BAIl gene induced tumor dormancy in human pancreatic tumors through the inhibition of tumor angiogenesis. The anti-tumor effect of p53 gene therapy would be strengthened by the combination of BAIl pathway. The mechanism of this effect is currently under investigation. 2301 PARATHYROID HORMONE-RELATED PROTEIN EXPRESSION IN HUMAN PANCREATIC ADENOCARCINOMA. Michael Bouvet, Stephanie R. Nardin, Douglas W. Burton, Cynthia Behling, John M. Carethers, Robert M. Hoffman, A. R. Moossa, Leonard J. Deftos, UC San Diego, San Diego, CA. Parathyroid hormone-related protein (PTHrP) is expressed in many common malignancies such as breast and prostate cancer and can regulate their growth. Little is known, however, about the role of PTHrP in pancreatic adenocarcinoma. In order to study PTHrP expression in pancreatic cancer, eight human pancreatic carcinoma cell lines were evaluated including AsPC-l, BxPC-3, Capan-I, CFPAC-1, MIA PaCa-2, PANC-1, PANC-28, and PANC-48. Murine monoclonal antibodies to the amino-terminal amino acid-fragment (1-34), mid-region (38-64), and carboxy-terminal aminoacid fragment (l 09-141) of PTHrP were used to identify intracellular PTHrP. secreted PTHrP, and for western blotting and immunocytochemical staining for PTHrP from each cell line. Intracellular PTHrP 1-34 was detected in all cell lines by both western blotting and immunoassay at concentrations ranging from 2.6 to 30.5 pg/mg protein. CFPAC-I, derived from a pancreatic liver metastasis, had the highest concentration and MIA PaCa-2, derived from primary pancreatic adenocarcinoma, the lowest. Secretion of PTHrP 1-34 into cell media was also noted for each cell line and paralled intracellular PTHrP 1-34 levels. PTHrP was localized by immunocytochemical staining in the cytoplasm in all but one cell line and both nuclear and cytoplasmic immunostaining was observed in the MIA PaCa-2 and PANC-1 cells. Evidence for differential processing of PTHrP was provided by studies demonstrating that PTHrP 38-64 secretion was highest for BxPC-3 rather than CFPAC-I while the highest levels of PTHrP 109-141 secretion were seen in Capan-I. Immunostaining from archival tissue of patients with pancreatic adenocarcinoma revealed strong PTHrP staining in 14 of 14 specimens. These results demonstrate that PTHrP is commonly expressed in pancreatic cancer and may playa role in the pathogenesis of this disease.
2302 GABEXATE MESILATE PROMOTES NEUTROPHIL APOPTOSIS AND ATTENUATES SERUM CYTOKINES ALTERATIONS IN HUMAN ACUTE PANCREATITIS. Han-Ming Chen, Miin-Fu Chen, Tsann-Long Hwang, Yi-Yin Jan, Ji-Chan Chen, Dept of Gen Surg, Taipei, Taiwan, ROC; Dept of Emergency Medicine, Taipei, Taiwan, ROC. Introduction: Inappropriate acivation of neutrophils within tissues plays a crucial role in the development of organs dysfunction in acute pancreatitis. The delay of programmed cell death or apoptosis of neutrophils is associated with the failure in termination of neutrophil-mediated inflammatory response. We investigated the effects of gabexate mesilate on neutrophil apoptosis in the progress of severe acute pancreatitits. Methods: Patients with acute pancreatitis who met the following criteria: onset less than 48 hr and APACHE-II score>8 or/and Ranson score>3 were enrolled and randomly grouped in this study. All patients were admitted to the intensive care unit. Gabexate mesilate was delivered through a central vein catheterization at the dose of Img/kg/hr afteradmission (Group B, n= 15). Group A received vehecle infusion only (n= 11). Neutrophils and plasma were isolated from whole venous blood at 3 time points: at admision, 12 hr post-treatment and 48 hr post-treatment of acute pancreatitis. Neutrophil apoptosis was quantified by immunofluorescence flow cytometry of 3' terminal deoxynucleotidyl transferase nick end labeling and PI staining. Cytokine detection was through special commercialized kits, which were based on enzyme-linked immunosorbent assay. Results: Neutrophil apoptosis was significantly inhibited with an event evident 12 hr and 48 hr aftertreatment of acute pancreatitis. Gabexate mesilate infusion significantly promoted neutrophil apoptosis. Levels of inflarnrnatory mediators (TNF-aand IL-6) which levels increased 12 hr and 48 hr after treatment of acute pancreatitis was notably attenuated by gabexate mesilate. Levels of other inflarnrnatory mediators (Il.l-Band IL-8) which levels increased 12 hr after treatment of acute pancreatitis was unaltered. Whereas level of IL-IO, which was increased 48 hr after treatment of acute pancreatitis, was only slightly decreased. Conclusions: Gabexate mesilate given early in the course of acute pancreatitis lessens the magnitudes of changes of inflammatory mediators and the inhibition of neutrophil apoptosis. It has little effect on the anti-inflammatory cytokine IL-IO, however. 2303 SIMULTANEOUS KIDNEY-PANCREAS TRANSPLANTATION WITH ENTERIC EXOCRINE DRAINAGE: A PROSPECTIVE COMPARISON OF SYSTEMIC VERSUS PORTAL VENOUS DELIVERY OF INSULIN. Robert J. Stratta, Mohammed H. Shokouh-Amiri, Hani P. Grewal, Maria F. Egidi, Tarik A. Kizilisik, Lillian W. Gaber, Ahmed O. Gaber, Univ of Tennessee-Memphis, Memphis, TN; Univ of Tennessee, Memphis, TN. The results of pancreas transplantation (PTX) continue to improve due to advances in surgical techniques and immunosuppression. Although renewed interest has occurred in enteric exocrine drainage, most PTXs are performed with systemic venous delivery of insulin (systemic-enteric [SED. To improve the physiology of PTX, a new surgical technique was developed with portal venous delivery of insulin and enteric exocrine drainage (portal-enteric [PED. The purpose of this study was to compare PTX with SE vs PE drainage in a prospective fashion. Over a 16-month period, we alternately performed either SE (N = 18) or PE (N = 18) drainage in 36 consecutive simultaneous kidney-PTXs (SKPTs). The two groups were well matched for donor and recipient demographic, immunologic, dialysis, diabetes, and transplant characteristics. Maintenance immunosuppression in both groups consisted of tacrolimus, mycophenolate mofetil, and steroids. No antibody induction therapy occurred in about half of patients in each group, with the remainder equally divided between daclizumab and basiliximab adjuvant therapy. Results: Patient and kidney graft survival rates are 94% in both groups. One early death occurred in each group (both with functioning grafts). All but I kidney graft (PE group) had immediate function. PTX survival (complete insulin independence) is 89% in both groups with a mean follow-up of nearly 1 year (minimum 3 months). There was I non-immunologic pancreas graft loss in each group, but no grafts were lost to thrombosis. The mean length of initial hospital stay was 12 days in the SE and 14 days in the PE groups, respectively. The SE group was characterized by a slight increase in the number of readmissions (mean 2.5 SE vs 1.4 PE, P=NS). The incidence of major infection was similar (61% SE vs 50% PE), as was the relaparotomy rate (28% SE vs 33% PE). There was I CMV infection (6%) in the SE group. The incidence of intra-abdominal infection and enteric leaks was slightly higher in the SE group (28% SE vs 11% PE, P=NS). However, the need for prolonged vascular access was comparable (33% SE vs 28% PE). Mean hospital charges were comparable between groups ($100,102 SE vs $107,685 PE). The composite endpoint of no readmission, no re-operation, and no rejection was equally attained by four patients (22%) in each group. Conclusions: These preliminary results suggest that SKPT with SE and PE drainage can be performed with comparable short-term outcomes.