- Email: [email protected]
Acute pancreatitis induces FasL gene expression and upregulates apoptotic pathways in the liver
256
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
Following transplantation, VE-cadherin became undetectable until P.O day 7 in isografts and P.O.day 5 in allografts at which time it became detectable at low levels on endothelial cells. Beta-1 integrin showed a pattern similar to VE-cadherin, except that it became barely detectable in either iso or allografts after transplantation. ET-1 did not show any detectable changes in either allo or isografts after transplantation. Conclusion: VE-cadherin and Beta-1 integrin expression on endothelial cells is diminished after cardiac transplantation, while ET-1 expression continues after transplantation. These data provide molecular insights into the loss of vascular endothelial integrity in cardiac allografts.
GASTROINTESTINAL/NUTRITION PARALLEL SESSION I 53. Adverse Effects of Total Parenteral Nutrition (TPN) on Small Intestine and Immune System: TPN vs. Lack of Enteral Nutrition. B. E. Wildhaber, M.D., H. Yang, M.D., D. H. Teitelbaum, M.D. University of Michigan. Introduction: TPN leads to phenotypic and functional changes in the mucosal intraepithelial lymphocytes (IEL). Such IEL changes may cause a loss of epithelial barrier function and lead to an bacterial translocation (BT). This study addresses if these changes were due to a lack of enteral feeding, or to the TPN, itself. Methods: Mice received either oral feeding (Control), TPN alone (TPN), or TPN plus oral feeding (TPN ⫹ Food). Mice were sacrificed at 7 days, and bacteriological cultures from spleen, liver and mesenteric lymph nodes were obtained. Positive cultures were considered consistent with BT. The intestine was harvested, IEL isolated, stained for lymphocyte markers, and phenotypic distribution was analyzed by flow cytometry. IEL mRNA cytokine expression was assessed by RT-PCR. Apoptosis was histochemically detected by TUNEL staining. Results are expressed as mean ⫾ SD, or mean (range). Statistics used ANOVA, P ⬍ 0.05 being significant. Results: BT significantly (P ⬍ 0.05) increased in the TPN group (53%) compared to Control (9%) and TPN ⫹ Food (14%) groups. TPN also resulted in a significant (P ⬍ 0.01) increase of epithelial cell apoptosis: TPN 7.6 ⫾ 1.1% versus Control 2.9 ⫾ 1.1%, and TPN ⫹ Food 2.1 ⫾ 0.3%. Height of the villus/crypt complex was significantly (P ⬍ 0.05) decreased in TPN mice (315 ⫾ 16 m) compared to Control (431 ⫾ 27 m), and TPN ⫹ Food (421 ⫾ 26 m) groups. Tables show other results (*P ⬍ 0.05 TPN vs. other 2 groups):
TABLE—ABSTRACT 53 Group
CD4⫹CD8⫺
CD8␣ ⫹ thymus-dependent
CD8⫹CD44⫹ mature
Control TPN TPN ⫹ Food
4.7% (3.9–6.1) 0.6% (0.2–0.7)* 2.5% (1.5–2.2)
4.8% (3.0–6.5) 0.4% (0.1–0.8)* 7.4% (5.6–8.6)
29% (28–37) 10% (1.0–18)* 40% (38–46)
Cytokines
IL-2
IL-4
INF-␥
TNF-␣
Control TPN TPN ⫹ Food
0.32 ⫾ 0.2 0.13 ⫾ 0.1* 0.31 ⫾ 0.2
0.10 ⫾ 0.1 0.73 ⫾ 0.4* 0.11 ⫾ 0.1
0.15 ⫾ 0.1 0.29 ⫾ 0.1* 0.14 ⫾ 0.0
0.34 ⫾ 0.1 0.55 ⫾ 0.1* 0.33 ⫾ 0.1
Conclusions: This study demonstrates that the major factor responsible for TPN-induced BT and IEL changes is the lack of enteral feeding, and not the administration of the PN solution.
54. Carbon Monoxide (co) Downregulates Hepatic Inducible Nitric Oxide Synthase (inos) by a Mitogen-Activated Protein Kinase (mapk) Dependent Mechanism. E. L. Marderstein, M.D., Z. Guo, M.D., L. Sonis, B.S., L. Shao, B.S., K. Reid, M.D., B. Bucher, B.S., D. A. Geller, M.D. University of Pittsburgh Medical Center, Pittsburgh, PA. Background: Low dose CO is non-toxic and has been shown to possess potent anti-inflammatory properties. We hypothesize that CO would decrease cytokine-stimulated iNOS induction in rat hepatocytes. Methods: Primary hepatocytes from Sprague-Dawley rats were exposed to combinations of cytokines (TNF␣, IL-1, IF␥) in the presence or absence of CO pre- and co- treatment at 250 ppm. Protein was harvested for Western blotting at 0, 1 and 6 hours after treatment. Greiss reaction was used to quantify NO production and crystal violet staining was used to measure cell viability. Experiments were performed 2-3 times each and t-test was used to assess significance between groups. Results: CO exposure at 250 ppm did not decrease hepatocyte viability measured by crystal violet staining at 24 hours. Pretreatment with CO resulted in a statistically significant decrease in cytokine mixture (TNF␣ 500 U/ml, IL-1 200 U/ml, IF␥ 100 U/ml) stimulated hepatocyte NO production measured by Greiss reaction (37.8 ⫾ 7.4 to 20.8 ⫾ 5.7 M, p ⬍ 0.05). Western blot 6 hours after stimulation resulted in a similar decrease in iNOS protein in the CO exposed hepatocytes. Pretreatment with CO upregulated mitogen activated protein kinase phosphatase-1 (MKP-1) protein and decreased TNF␣ induced c-Jun N-terminal kinase (JNK) activation measured by Western blot. Conclusions: CO pretreatment decreased cytokine stimulated hepatocyte iNOS protein synthesis and NO production. A possible mechanism for this effect is the up-regulation of MKP-1 protein and subsequent decrease in cytokine stimulated JNK activation. The study provides a new mechanism for the anti-inflammatory effects of CO and validates the importance of JNK activation in pro-inflammatory signaling. 55. Acute Pancreatitis Induces FasL Gene Expression and Upregulates Apoptotic Pathways in the Liver. S. F. Gallagher, M.D., J. Yang, M.D., P. K. Epling-Burnette, Ph.D., W. R. Gower, Jr., Ph.D., F. Bai, M.D., K. Baksh, K. Haines, B.A., J. Norman, M. M. Murr, M.D. University of South Florida. Background: Liver injury is of prognostic importance in acute pancreatitis. We previously demonstrated that Kupffer cell-derived cytokines mediate liver injury. Aim: To determine the role of Fas Ligand (FasL) in liver injury during acute pancreatitis. Methods: CCK was used to induce pancreatitis in mice; serum FasL (ELISA), AST, and liver FasL, p38-MAPK and Caspase-3 (western) were measured. Rat Kupffer cells were treated with elastase (1U/ml) to mimic acute pancreatitis; FasL and its receptor (Fas) (western) and FasL mRNA (RT-PCR) were determined. Apoptosis was measured by flow cytometry. Immunoblots were done in triplicates and quantified with densitometry. Results: CCK-induced pancreatitis increased serum AST and FasL, upregulated liver FasL (1315 ⫾ 111, vs. 310 ⫾ 164, p ⫽ 0.002 vs. sham), and induced phosphorylation of p38-MAPK (p ⬍ 0.01 vs. sham) and cleavage of Caspase-3 (p ⬍ 0.04 vs. sham); all of which were attenuated by pre-treatment with the Kupffer cell inhibitor, Gadolinium (all p ⬍ 0.003). In-vitro, elastase induced a timedependent increase in Kupffer cell FasL protein (FasL⫽ 512 ⫾ 53 vs. 170 ⫾ 40, p ⫽ 0.01, 2hrs vs. control), a 100-fold increase in FasL mRNA and upregulated FasL receptor (Fas) (Figure; E: elastase, G: Gadolinium, ⬘: min). Gadolinium significantly attenuated the elastase-induced increase in FasL and FasL mRNA (FasL⫽ 230 ⫾ 20 vs. 512 ⫾ 53, p ⫽ 0.01, Gadolinium vs. elastase) but had little effect on Fas (receptor). The medium of elastase-primed Kupffer cells induced apoptosis in fresh rat hepatocytes (29 ⫾ 1 vs. 16 ⫾ 1%; vs. control, p ⬍ 0.001). Conclusions: Acute pancreatitis induced liver injury and hepatocyte death, upregulated FasL production and activated hepatic p38-MAPK and Caspase-3. Utilizing Gadolinium,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Kupffer cell-derived FasL production was significantly attenuated both in-vivo and in-vitro. Similar to other immune cells, FasL receptor (Fas) was upregulated within Kupffer cells suggesting that FasL may auto-regulate its production by inducing its originator-cell death. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications.
56. Interference of Hepatitis B Virus Gene Expression and Replication by siRNA. D. Yang, M.D., Ph.D., B. Sun, Ph.D., Z. Zhang, M.D., Y. Tian, M.D., X. Lin, Ph.D., X. Feng, Ph.D. Tongji Medical College, Wuhan, China (D. Yang, Z. Zhang, Y. Tian), Baylor College of Medicine (B. Sun, X. Lin, X. Feng). Introduction: Hepatitis B virus (HBV) is a public health problem in the world. HBV-infected carriers, over 300 million people worldwide, are at high risk of developing end-stage liver diseases such as cirrhosis and have also dramatically increased the risk of liver cancer. Current treatments for HBV infections are not efficient. The enormous disease burden caused by the HBV infections makes the development of novel therapies critical. Molecular therapies for HBV can be directed at reducing viral load by interfering with the life cycle of the viruses. Objective: To explore the potential use of RNA interference (siRNA) as a therapeutic approach to inhibit HBV viral gene expression and replication. Methods: We first chose the target sequences for siRNA along the coding region of S antigen of HBV adw strain (HBsAg). A selected target sequence contained AA leader followed 19-nt. Double-stranded RNA oligos containing the 19-nt with added TT at 3⬘ ends were synthesized. DNA constructs containing the siRNA were also made in a vector called pSUPERretro. Effects of these siRNA or DNA construct on the expression of HBsAg were examined by anti-HBsAg Western blot on cell lysates of a variety of cell lines. Results: We have designed a series of small interfering RNA (siRNA) targeted to various regions of HBV genome and first tested the efficacy of these siRNAs in suppression of HBV gene expression. Three of four siRNAs directed to the coding region of HBsAg (siHBsAg) were very effective in knocking down the expression of co-transfected HBsAg in a variety of cell lines, including hepatocellular carcinoma HepG2 and Hep3B cells as well as nonliver cell lines such as HeLa and 293T cells. siHBsAg-mediated gene suppression is in a dose-dependent manner with 1 nM of siHBsAg1 being sufficient to inhibit HBsAg accumulation in cells. We are currently testing the ability of these siRNAs in inhibiting HBV viral replication in cell lines carrying whole HBV genome and animal models. Conclusion: siRNA-directed gene silencing is effective in inhibiting HBV gene expression, suggesting that it can be potentially used as a successful therapeutic approach to the prevention and treatment of HBV in the foreseeable future. 57. Glutamine Stimulates Amino Acid Transport During Ischemia-Reperfusion in Human Intestinal Epithelial Cells. M. Wasa, M.D., H. S. Wang, M.D., Y. Shimizu, M.D., M. Fukuzawa, M.D. Osaka University Department of Pediatric Surgery, Osaka, Japan. Introduction: The potential mechanism of intestinal ischemiareperfusion (I/R) injury includes oxygen-derived toxic free radicals. We tested the hypothesis that glutamine (Gln) increases intracellular glutathione, a protective substrate against oxidative stress, by stimulating membrane amino acid transport during I/R using hu-
257
man intestinal cell line Caco-2. Methods: Ischemic conditions were obtained by combining both hypoxic (1%O 2-5%CO 2-94% N 2) and nutrient-deprived (PBS) conditions. After two hours of ischemia, reoxygenation (5%CO 2-95% air) was initiated and the culture medium was changed to PBS or amino acid solution (A.A.) with or without Gln (2 mM) (reperfusion). After ischemia and four hours of reperfusion, transport of 3H-Gln and 3H-glutamate was assayed and intracellular glutathione was measured. The expression of glutamine transporter (system ATB o) was analyzed by using RT-PCR. Data (mean ⫾ SD) were analyzed by ANOVA. Results: Ischemia decreased Gln and glutamate transport compared with control by a mechanism that down-regulates the expression of system ATB o mRNA. After reperfusion, Gln and glutamate transport in the PBS and A.A. without Gln groups decreased significantly compared with control (p ⬍ 0.01), whereas glutamine supplementation increased Gln transport to the levels found in control (p ⬍ 0.01) and partially increased glutamate transport (p ⬍ 0.01) (Fig. 1). Gln significantly increased intracellular glutathione compared with the PBS and A.A. without Gln groups (p ⬍ 0.05) (Fig. 2). Conclusions: These results suggest that Gln would be protective against intestinal I/R injury through preservation of intracellular amino acid essential for glutathione synthesis and energy production.
58. Regulation of Intestinal Glutamine Absorption by Transforming Growth Factor-Beta. Q. Meng, M.D., W. W. Souba, M.D., S.C.D., M. J. Epler, M.D., A. M. Karinch, Ph.D., C. Lin, Ph.D., T. C. Vary, Ph.D., M. Pan, M.D., Ph.D. Penn State College of Medicine. Introduction: Transforming growth factor-beta (TGF-) plays a central role in regulating intestinal epithelial cell proliferation, migration and wound repair but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulation mechanisms and intracellular signaling pathways involved in the regulation of TGF- on glutamine transport in cultured intestinal cells. Methods: [ 3H]L-glutamine (50 M) transport activity and mRNA levels for the intestinal glutamine transporter ATB 0 were measured in cultured intestinal Caco-2 cells. Cells were treated with TGF- (0 - 50 ng/ml), inhibitors of the mitogen-activated protein kinase (MAPK) MEK1 (PD 98059, 0 - 50 M), the MAPK p38 (SB 203580, 0-10 M), as well as actinomycin-D (0 - 1 M) and cycloheximide (0 - 20 M). Data were analyzed by ANOVA. Results: Continuous incubation with IGF-2 stimulated glutamine transport activity in Caco-2 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 12 hours) resulted in a 50% increase in transport activity (0.93 ⫾ 0.20 nmole/mg protein/minute in TGF- vs. 0.61 ⫾ 0.17 nmole/mg protein/minute in control) and a 1.8-fold increase of glutamine transporter ATB 0 mRNA levels. TGF- stimulated transport activity by increasing transport maximal capacity (Vmax 9.21 ⫾ 0.51 nmole/mg protein/minute in TGF- vs. 5.91 ⫾ 0.34 nmole/mg protein/minute in control) without affecting the transport affinity (Km 290 ⫾ 40 M glutamine in TGF- vs 305 ⫾ 34 M glutamine in controls, p ⫽ NS). This TGF- induced glutamine transport activity was individually attenuated by inhibitors of transcription (actinomycin-D), transla-
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
Following transplantation, VE-cadherin became undetectable until P.O day 7 in isografts and P.O.day 5 in allografts at which time it became detectable at low levels on endothelial cells. Beta-1 integrin showed a pattern similar to VE-cadherin, except that it became barely detectable in either iso or allografts after transplantation. ET-1 did not show any detectable changes in either allo or isografts after transplantation. Conclusion: VE-cadherin and Beta-1 integrin expression on endothelial cells is diminished after cardiac transplantation, while ET-1 expression continues after transplantation. These data provide molecular insights into the loss of vascular endothelial integrity in cardiac allografts.
GASTROINTESTINAL/NUTRITION PARALLEL SESSION I 53. Adverse Effects of Total Parenteral Nutrition (TPN) on Small Intestine and Immune System: TPN vs. Lack of Enteral Nutrition. B. E. Wildhaber, M.D., H. Yang, M.D., D. H. Teitelbaum, M.D. University of Michigan. Introduction: TPN leads to phenotypic and functional changes in the mucosal intraepithelial lymphocytes (IEL). Such IEL changes may cause a loss of epithelial barrier function and lead to an bacterial translocation (BT). This study addresses if these changes were due to a lack of enteral feeding, or to the TPN, itself. Methods: Mice received either oral feeding (Control), TPN alone (TPN), or TPN plus oral feeding (TPN ⫹ Food). Mice were sacrificed at 7 days, and bacteriological cultures from spleen, liver and mesenteric lymph nodes were obtained. Positive cultures were considered consistent with BT. The intestine was harvested, IEL isolated, stained for lymphocyte markers, and phenotypic distribution was analyzed by flow cytometry. IEL mRNA cytokine expression was assessed by RT-PCR. Apoptosis was histochemically detected by TUNEL staining. Results are expressed as mean ⫾ SD, or mean (range). Statistics used ANOVA, P ⬍ 0.05 being significant. Results: BT significantly (P ⬍ 0.05) increased in the TPN group (53%) compared to Control (9%) and TPN ⫹ Food (14%) groups. TPN also resulted in a significant (P ⬍ 0.01) increase of epithelial cell apoptosis: TPN 7.6 ⫾ 1.1% versus Control 2.9 ⫾ 1.1%, and TPN ⫹ Food 2.1 ⫾ 0.3%. Height of the villus/crypt complex was significantly (P ⬍ 0.05) decreased in TPN mice (315 ⫾ 16 m) compared to Control (431 ⫾ 27 m), and TPN ⫹ Food (421 ⫾ 26 m) groups. Tables show other results (*P ⬍ 0.05 TPN vs. other 2 groups):
TABLE—ABSTRACT 53 Group
CD4⫹CD8⫺
CD8␣ ⫹ thymus-dependent
CD8⫹CD44⫹ mature
Control TPN TPN ⫹ Food
4.7% (3.9–6.1) 0.6% (0.2–0.7)* 2.5% (1.5–2.2)
4.8% (3.0–6.5) 0.4% (0.1–0.8)* 7.4% (5.6–8.6)
29% (28–37) 10% (1.0–18)* 40% (38–46)
Cytokines
IL-2
IL-4
INF-␥
TNF-␣
Control TPN TPN ⫹ Food
0.32 ⫾ 0.2 0.13 ⫾ 0.1* 0.31 ⫾ 0.2
0.10 ⫾ 0.1 0.73 ⫾ 0.4* 0.11 ⫾ 0.1
0.15 ⫾ 0.1 0.29 ⫾ 0.1* 0.14 ⫾ 0.0
0.34 ⫾ 0.1 0.55 ⫾ 0.1* 0.33 ⫾ 0.1
Conclusions: This study demonstrates that the major factor responsible for TPN-induced BT and IEL changes is the lack of enteral feeding, and not the administration of the PN solution.
54. Carbon Monoxide (co) Downregulates Hepatic Inducible Nitric Oxide Synthase (inos) by a Mitogen-Activated Protein Kinase (mapk) Dependent Mechanism. E. L. Marderstein, M.D., Z. Guo, M.D., L. Sonis, B.S., L. Shao, B.S., K. Reid, M.D., B. Bucher, B.S., D. A. Geller, M.D. University of Pittsburgh Medical Center, Pittsburgh, PA. Background: Low dose CO is non-toxic and has been shown to possess potent anti-inflammatory properties. We hypothesize that CO would decrease cytokine-stimulated iNOS induction in rat hepatocytes. Methods: Primary hepatocytes from Sprague-Dawley rats were exposed to combinations of cytokines (TNF␣, IL-1, IF␥) in the presence or absence of CO pre- and co- treatment at 250 ppm. Protein was harvested for Western blotting at 0, 1 and 6 hours after treatment. Greiss reaction was used to quantify NO production and crystal violet staining was used to measure cell viability. Experiments were performed 2-3 times each and t-test was used to assess significance between groups. Results: CO exposure at 250 ppm did not decrease hepatocyte viability measured by crystal violet staining at 24 hours. Pretreatment with CO resulted in a statistically significant decrease in cytokine mixture (TNF␣ 500 U/ml, IL-1 200 U/ml, IF␥ 100 U/ml) stimulated hepatocyte NO production measured by Greiss reaction (37.8 ⫾ 7.4 to 20.8 ⫾ 5.7 M, p ⬍ 0.05). Western blot 6 hours after stimulation resulted in a similar decrease in iNOS protein in the CO exposed hepatocytes. Pretreatment with CO upregulated mitogen activated protein kinase phosphatase-1 (MKP-1) protein and decreased TNF␣ induced c-Jun N-terminal kinase (JNK) activation measured by Western blot. Conclusions: CO pretreatment decreased cytokine stimulated hepatocyte iNOS protein synthesis and NO production. A possible mechanism for this effect is the up-regulation of MKP-1 protein and subsequent decrease in cytokine stimulated JNK activation. The study provides a new mechanism for the anti-inflammatory effects of CO and validates the importance of JNK activation in pro-inflammatory signaling. 55. Acute Pancreatitis Induces FasL Gene Expression and Upregulates Apoptotic Pathways in the Liver. S. F. Gallagher, M.D., J. Yang, M.D., P. K. Epling-Burnette, Ph.D., W. R. Gower, Jr., Ph.D., F. Bai, M.D., K. Baksh, K. Haines, B.A., J. Norman, M. M. Murr, M.D. University of South Florida. Background: Liver injury is of prognostic importance in acute pancreatitis. We previously demonstrated that Kupffer cell-derived cytokines mediate liver injury. Aim: To determine the role of Fas Ligand (FasL) in liver injury during acute pancreatitis. Methods: CCK was used to induce pancreatitis in mice; serum FasL (ELISA), AST, and liver FasL, p38-MAPK and Caspase-3 (western) were measured. Rat Kupffer cells were treated with elastase (1U/ml) to mimic acute pancreatitis; FasL and its receptor (Fas) (western) and FasL mRNA (RT-PCR) were determined. Apoptosis was measured by flow cytometry. Immunoblots were done in triplicates and quantified with densitometry. Results: CCK-induced pancreatitis increased serum AST and FasL, upregulated liver FasL (1315 ⫾ 111, vs. 310 ⫾ 164, p ⫽ 0.002 vs. sham), and induced phosphorylation of p38-MAPK (p ⬍ 0.01 vs. sham) and cleavage of Caspase-3 (p ⬍ 0.04 vs. sham); all of which were attenuated by pre-treatment with the Kupffer cell inhibitor, Gadolinium (all p ⬍ 0.003). In-vitro, elastase induced a timedependent increase in Kupffer cell FasL protein (FasL⫽ 512 ⫾ 53 vs. 170 ⫾ 40, p ⫽ 0.01, 2hrs vs. control), a 100-fold increase in FasL mRNA and upregulated FasL receptor (Fas) (Figure; E: elastase, G: Gadolinium, ⬘: min). Gadolinium significantly attenuated the elastase-induced increase in FasL and FasL mRNA (FasL⫽ 230 ⫾ 20 vs. 512 ⫾ 53, p ⫽ 0.01, Gadolinium vs. elastase) but had little effect on Fas (receptor). The medium of elastase-primed Kupffer cells induced apoptosis in fresh rat hepatocytes (29 ⫾ 1 vs. 16 ⫾ 1%; vs. control, p ⬍ 0.001). Conclusions: Acute pancreatitis induced liver injury and hepatocyte death, upregulated FasL production and activated hepatic p38-MAPK and Caspase-3. Utilizing Gadolinium,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS Kupffer cell-derived FasL production was significantly attenuated both in-vivo and in-vitro. Similar to other immune cells, FasL receptor (Fas) was upregulated within Kupffer cells suggesting that FasL may auto-regulate its production by inducing its originator-cell death. The ability to manipulate interactions between Kupffer cells and hepatocytes may have important therapeutic implications.
56. Interference of Hepatitis B Virus Gene Expression and Replication by siRNA. D. Yang, M.D., Ph.D., B. Sun, Ph.D., Z. Zhang, M.D., Y. Tian, M.D., X. Lin, Ph.D., X. Feng, Ph.D. Tongji Medical College, Wuhan, China (D. Yang, Z. Zhang, Y. Tian), Baylor College of Medicine (B. Sun, X. Lin, X. Feng). Introduction: Hepatitis B virus (HBV) is a public health problem in the world. HBV-infected carriers, over 300 million people worldwide, are at high risk of developing end-stage liver diseases such as cirrhosis and have also dramatically increased the risk of liver cancer. Current treatments for HBV infections are not efficient. The enormous disease burden caused by the HBV infections makes the development of novel therapies critical. Molecular therapies for HBV can be directed at reducing viral load by interfering with the life cycle of the viruses. Objective: To explore the potential use of RNA interference (siRNA) as a therapeutic approach to inhibit HBV viral gene expression and replication. Methods: We first chose the target sequences for siRNA along the coding region of S antigen of HBV adw strain (HBsAg). A selected target sequence contained AA leader followed 19-nt. Double-stranded RNA oligos containing the 19-nt with added TT at 3⬘ ends were synthesized. DNA constructs containing the siRNA were also made in a vector called pSUPERretro. Effects of these siRNA or DNA construct on the expression of HBsAg were examined by anti-HBsAg Western blot on cell lysates of a variety of cell lines. Results: We have designed a series of small interfering RNA (siRNA) targeted to various regions of HBV genome and first tested the efficacy of these siRNAs in suppression of HBV gene expression. Three of four siRNAs directed to the coding region of HBsAg (siHBsAg) were very effective in knocking down the expression of co-transfected HBsAg in a variety of cell lines, including hepatocellular carcinoma HepG2 and Hep3B cells as well as nonliver cell lines such as HeLa and 293T cells. siHBsAg-mediated gene suppression is in a dose-dependent manner with 1 nM of siHBsAg1 being sufficient to inhibit HBsAg accumulation in cells. We are currently testing the ability of these siRNAs in inhibiting HBV viral replication in cell lines carrying whole HBV genome and animal models. Conclusion: siRNA-directed gene silencing is effective in inhibiting HBV gene expression, suggesting that it can be potentially used as a successful therapeutic approach to the prevention and treatment of HBV in the foreseeable future. 57. Glutamine Stimulates Amino Acid Transport During Ischemia-Reperfusion in Human Intestinal Epithelial Cells. M. Wasa, M.D., H. S. Wang, M.D., Y. Shimizu, M.D., M. Fukuzawa, M.D. Osaka University Department of Pediatric Surgery, Osaka, Japan. Introduction: The potential mechanism of intestinal ischemiareperfusion (I/R) injury includes oxygen-derived toxic free radicals. We tested the hypothesis that glutamine (Gln) increases intracellular glutathione, a protective substrate against oxidative stress, by stimulating membrane amino acid transport during I/R using hu-
257
man intestinal cell line Caco-2. Methods: Ischemic conditions were obtained by combining both hypoxic (1%O 2-5%CO 2-94% N 2) and nutrient-deprived (PBS) conditions. After two hours of ischemia, reoxygenation (5%CO 2-95% air) was initiated and the culture medium was changed to PBS or amino acid solution (A.A.) with or without Gln (2 mM) (reperfusion). After ischemia and four hours of reperfusion, transport of 3H-Gln and 3H-glutamate was assayed and intracellular glutathione was measured. The expression of glutamine transporter (system ATB o) was analyzed by using RT-PCR. Data (mean ⫾ SD) were analyzed by ANOVA. Results: Ischemia decreased Gln and glutamate transport compared with control by a mechanism that down-regulates the expression of system ATB o mRNA. After reperfusion, Gln and glutamate transport in the PBS and A.A. without Gln groups decreased significantly compared with control (p ⬍ 0.01), whereas glutamine supplementation increased Gln transport to the levels found in control (p ⬍ 0.01) and partially increased glutamate transport (p ⬍ 0.01) (Fig. 1). Gln significantly increased intracellular glutathione compared with the PBS and A.A. without Gln groups (p ⬍ 0.05) (Fig. 2). Conclusions: These results suggest that Gln would be protective against intestinal I/R injury through preservation of intracellular amino acid essential for glutathione synthesis and energy production.
58. Regulation of Intestinal Glutamine Absorption by Transforming Growth Factor-Beta. Q. Meng, M.D., W. W. Souba, M.D., S.C.D., M. J. Epler, M.D., A. M. Karinch, Ph.D., C. Lin, Ph.D., T. C. Vary, Ph.D., M. Pan, M.D., Ph.D. Penn State College of Medicine. Introduction: Transforming growth factor-beta (TGF-) plays a central role in regulating intestinal epithelial cell proliferation, migration and wound repair but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulation mechanisms and intracellular signaling pathways involved in the regulation of TGF- on glutamine transport in cultured intestinal cells. Methods: [ 3H]L-glutamine (50 M) transport activity and mRNA levels for the intestinal glutamine transporter ATB 0 were measured in cultured intestinal Caco-2 cells. Cells were treated with TGF- (0 - 50 ng/ml), inhibitors of the mitogen-activated protein kinase (MAPK) MEK1 (PD 98059, 0 - 50 M), the MAPK p38 (SB 203580, 0-10 M), as well as actinomycin-D (0 - 1 M) and cycloheximide (0 - 20 M). Data were analyzed by ANOVA. Results: Continuous incubation with IGF-2 stimulated glutamine transport activity in Caco-2 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 12 hours) resulted in a 50% increase in transport activity (0.93 ⫾ 0.20 nmole/mg protein/minute in TGF- vs. 0.61 ⫾ 0.17 nmole/mg protein/minute in control) and a 1.8-fold increase of glutamine transporter ATB 0 mRNA levels. TGF- stimulated transport activity by increasing transport maximal capacity (Vmax 9.21 ⫾ 0.51 nmole/mg protein/minute in TGF- vs. 5.91 ⫾ 0.34 nmole/mg protein/minute in control) without affecting the transport affinity (Km 290 ⫾ 40 M glutamine in TGF- vs 305 ⫾ 34 M glutamine in controls, p ⫽ NS). This TGF- induced glutamine transport activity was individually attenuated by inhibitors of transcription (actinomycin-D), transla-