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Mo1539 A Novel Metabolic Action for the Multikinase Inhibitor Sorafenib: Targeting Tumor Cells Through Mitochondrial Damage
of HCV patients was determined by qrt-PCR and correlated to viral and host parameters. The effect of IL-10, TGF-β and miR-155 on poly I:C-activated murine Kupffer cells (KC) was assessed In Vitro. NPC were assayed for inflammatory and antiviral cytokines as well as T cell-activating factors. Results: Elevated expression of TGF-β was linked to HCV genotype 1, while hepatic IL-10 expression correlated with HCV titers. TLR3-induced antiviral activity of NPC in a murine HCV-replicon system was potently suppressed by IL-10 or TGF-β. This correlated with decreased TLR3 expression and inhibition of NF-κB and IRF-3 activation in NPC. T cell activation, induced by TLR3-activated NPC, was suppressed by IL-10 and TGF-β which was associated with a down-regulation of CD80 and CD86. In primary KC, miR-155 could be up-regulated by poly I:C. Inhibition of NF-κB by Bay11-7082 impaired TLR3-induced miR-155 expression. The expression levels of miR-155, TNF-α, IL-6 and IFIT1 were elevated after miR-155 mimic treatment compared with controls. Pretreatment with IL-10 or TGF-β potently suppressed TLR3-induced miR-155 expression. The immunosuppressive effects of IL-10 and TGF-β in NPCs could be completely abrogated by treatment with miR-155 mimics. Conclusions: Our data suggest that TLR3-induced innate and adaptive immune responses in the liver are controlled by anti-inflammatory cytokines which in turn is mediated largely through miR-155. This is of relevance for the regulation of local immune responses and may at least partly explain the “IFN-paradox” in the pathogenesis of HCV infection, in particular.
Mo1886 Hispanics With Primary Biliary Cirrhosis Have Increased Risk of Decompensation Jahnavi Naik, Christin Giordano, Cynthia Levy, Amar Mandalia, Paul Martin Background and Aims: Preliminary data has suggested that primary biliary cirrhosis (PBC) may have a more aggressive course in individuals with Hispanic ancestry. We sought to investigate the clinical presentation and progression of PBC in an ethnically diverse population in the South Eastern United States. Methods: We conducted a retrospective study of patients with PBC seen for the first time at the University of Miami between 01/01/2001 and 12/ 31/2009. Follow-up was censored at the time of death or liver transplant. We used Person's chi square or Fisher's exact test to test differences between proportions. Continuous variables were tested using t-test or Wilcoxon's rank sum test as appropriate. Results: 153 patients were included in the study; 136 (89%) were female, 48 (31.4%) Hispanic and 122/142 (86%) had anti-mitochondrial antibodies (AMA), with a mean age at diagnosis 54.5 ± 13 years and a total of 623 person-years of follow-up. Mean dose of ursodeoxycholic acid (UDCA) was 15 ± 6.4 mg/kg/day; 13 patients were not on UDCA. No differences existed with respect to age, gender, AMA status, body mass index, UDCA dose, proportion of overlap cases and frequency of advanced fibrosis between Hispanics and non-Hispanics. Fatigue was present in 55.6% Hispanics vs. 35.3% non-Hispanics (p= 0.03), whereas the proportion of patients with pruritus was similar between groups. Portal hypertension was the initial presentation in only 17 patients, of whom 7 were Hispanic (p=not significant). Hypothyroidism and sicca syndrome were less frequent in Hispanics: 7/48 (14.6%) vs. 32/105 (30.5%; p=0.045), and 0/48 vs. 12/105 (11.4%; p=0.019), respectively. Among 133 patients who had been on UDCA longer than 1 year, Hispanics had a higher serum alkaline phosphatase: median 171 (range 26-1276) vs. 134 (range 11-746) IU/L, p=0.0025. By the end of followup, variceal bleeding had occurred in 25% of Hispanics vs. 9% non-Hispanics (p=0.02; relative risk 2.7, 95% CI 1.2-6.0), and ascites in 31% vs. 15% (p=0.03; relative risk 2.1, 95% CI 1.1-3.9). Twelve (8%) patients had died or undergone liver transplant, 6 in each ethnic group. Conclusion: The present study included a large proportion of Hispanics with PBC. We confirmed more frequent disease progression in Hispanics as evidenced by increased rates of decompensation. Further, Hispanics appear to have a poorer biochemical response to UDCA and may be appropriate candidates for novel therapies.
Mo1538
AASLD Abstracts
Hepatitis B Virus Surface Antigen Suppresses Innate and Adaptive Immune Responses of Murine and Human Liver Cells Min Jiang, Martin Trippler, Ruth Bröring, Lena Poggenpohl, Guido Gerken, Mengji Lu, Joerg F. Schlaak Background: It has recently been shown that Toll-like receptor (TLR) signaling in murine non-parenchymal (NPC) liver cells is suppressed in the presence of Hepatitis B surface antigen (HBsAg). It is not clear, however, whether this is also relevant for adaptive and human immune responses and how this effect is mediated. Methods: Peripheral blood mononuclear cells (PBMCs) from chronic HBV infected patients were stimulated by TLR3 ligands in the absence or presence of autologous serum. Murine Kupffer cells (KC), sinusoidal endothelial cells (LSEC) and hepatocytes as well as human LSEC and hepatocytes were stimulated with TLR3 ligand Poly I:C in the presence or absence of HBsAg. Expression of cytokines and TLR3 was analyzed by quantitative rt-PCR. Mixed lymphocyte reactions (MLR) were performed to study T cell activation induced by TLR-stimulated NPC. Activation of transcription factors was assessed by western blot and reporter gene assays. Results: Activation of T lymphocytes that was induced by TLR3-activated NPC was potently suppressed by HBsAg. This suppressive effect was associated with enhanced IL-10 production that could be reverted by IL-10 antibodies. While Poly I:C-induced TLR3 expression remained unchanged, TLR-induced activation of NFκB, IRF-3 and MAPKs was potently suppressed by HBsAg. TLR3-induced production of IFN-γ was suppressed in PBMC from patients with high HBsAg-levels (p<0.0006) compared to PBMC isolated from patients with low HBsAglevels. In the presence of autologous serum, TLR3 induced IFN-γ production was inhibited in case of HBV-containing serum. Conclusions: These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to establish a persistent infection.
Mo1887 Antibiotic Prophylaxis for Recurrent Bacterial Cholangitis After Orthotopic Liver Transplantation (OLT) for Primary Sclerosing Cholangitis (PSC) Diana Winston, Marshall Kaplan Introduction: Recurrent bacterial cholangitis can be a serious problem in patients who undergo OLT for endstage PSC. We have found that a regimen of rotating antibiotics has been effective in preventing or greatly reducing the frequency and severity of these episodes and now report our experience. Methods: The medical records of patients followed by one of us (MMK) in the Hepatology Clinic of TMC, from January 1, 2000 until May 1, 2010, were reviewed in order to identify patients who had received OLT for PSC. We then identified patients who had had recurrent episodes of fever and rigors consistent with bacterial cholangitis and who had been treated with a regimen of rotating antibiotics. Rotating antibiotics were offered if the following criteria were met: episodes of chills and fever occurred at least once monthly, patients were hospitalized for cholangitis more than twice during the preceding four months, or if more than one episode of blood culture proven bacterial cholangitis occurred during the preceding six months. The antibiotic regimen initially consisted of amoxicillin for one month, then, trimethoprim/ sulfamethoxazole for one month, then ciprofloxacin for one month and then the cycle was repeated. Antibiotics were changed if patients were allergic to or intolerant of an antibiotic. Results: Of 749 patients who had had OLT, 69 patients had PSC. Of these 69, 12 had recurrent episodes of presumed bacterial cholangitis. Despite fevers, chills and frequent positive blood cultures, there was little worsening of serum levels of ALT, AST, alkaline phosphatase, or bilirubin in 7 patients. One patient who had an OLT performed in 1986 for PSC had a second OLT in 2005 because of hepatitis C cirrhosis and was counted twice. Therefore, we describe 13 OLTs in 12 patients (Table 1). The mean (median) duration of follow-up on the antibiotic treatment was 120 (133) months. Nine patients responded fully to the antibiotic regimen and had no further episodes of fever and chills as long as they took the antibiotics. Three patients had a decrease in the frequency of their episodes of fevers and chills. Antibiotics were ineffective in the patient who had had a second OLT for hepatitis C. Five patients had recurrent episodes of chills and fever when they stopped the antibiotics (four of whom were hospitalized for sepsis). All responded again when antibiotics were restarted. Aside from allergies to specific antibiotics, there were few complications. All continue on the antibiotics. Conclusions: Approximately 1/6 of our patients who had OLT for PSC had recurrent episodes of bacterial cholangitis. Rotating antibiotics were effective in preventing these episodes of in 9 of 13 patients and reduced the frequency in 3. The antibiotic regimen was well-tolerated.
Mo1539 A Novel Metabolic Action for the Multikinase Inhibitor Sorafenib: Targeting Tumor Cells Through Mitochondrial Damage Valentina Tesori, Anna C. Piscaglia, Marta Barba, Maria Ausiliatrice Puglisi, Giovambattista Pani, Antonio Gasbarrini BACKGROUND: One of the basis of modern oncology resides on the metabolic alteration of cancer cells, that explicates through an increase of glycolytic pathway, the so called “Warburg Effect”. Glycolysis can be seen as a cancer antioxidant response to allow tumor dissemination protecting cells from ROS-induced senescence and apoptosis. Although the multikinase inhibitor Sorafenib specifically targets tumor cells harboring the V590E mutation of B-RAF, additional mechanisms likely account for the elevated anticancer activity of the drug in tumors lacking this mutation, and for the emergence of chemoresistance. AIM:In the present study a novel metabolic effect of the multikinase inhibitor Sorafenib (SFB) was investigated, alone and in combination with the glycolytic inhibitor 2-deoxyglucose (2dg), in a liver tumor model, the LCSC-2 (Liver Cancer Stem Cells) cell line. In particular we investigated the mitochondrial action of SFB. MATERIAL AND METHODS: Drug toxicity was evaluated through Propidium Iodide (PI) assay. Gene expression was investigated by microarray analysis. Protein expression was evaluated by western blotting. Intracellular ATP level was assessed by chemiluminescence. Mitochondrial activity was assessed by Respirometric Analysis. Mitochondrial potential was measured with the JC-1 probe. RESULTS: We found that in rat hepatocolangiocarcinoma (LCSC-2) cells exposure to Sorafenib was not paralleled by significant inhibition of ERK phosphorylation, but, instead, elicited a raise of intracellular reactive oxygen species and the Ser172 phosphorylation of the AMP-activated protein kinase (AMPK), two events consistent with mitochondrial dysfunction. Accordingly, SFB led to a substantial reduction of oxygen consumption, cellular ATP level and mitochondrial transmembrane potential, and isolated mitochondria from rat liver were depolarized by the drug In Vitro, indicating a direct effect of SFB on the organelle. Interestingly, in keeping with its inhibitory action on mitochondrial respiration, Sorafenib killed much more efficiently LCSC-2 cells, as well as mouse B16F10 melanoma and 293T human kidney carcinoma cells when associated with the glycolysis inhibitor 2-deoxy-glucose. CONCLUSIONS:Taken together our preliminary results identify in mitochondrial damage and generation of ROS a novel modality of cytotoxicity by Sorafenib; moreover, the synergistic action of SFB plus 2DG outline a novel combined therapy to eradicate liver tumors.
AASLD Abstracts
S-990
Mo1886 Hispanics With Primary Biliary Cirrhosis Have Increased Risk of Decompensation Jahnavi Naik, Christin Giordano, Cynthia Levy, Amar Mandalia, Paul Martin Background and Aims: Preliminary data has suggested that primary biliary cirrhosis (PBC) may have a more aggressive course in individuals with Hispanic ancestry. We sought to investigate the clinical presentation and progression of PBC in an ethnically diverse population in the South Eastern United States. Methods: We conducted a retrospective study of patients with PBC seen for the first time at the University of Miami between 01/01/2001 and 12/ 31/2009. Follow-up was censored at the time of death or liver transplant. We used Person's chi square or Fisher's exact test to test differences between proportions. Continuous variables were tested using t-test or Wilcoxon's rank sum test as appropriate. Results: 153 patients were included in the study; 136 (89%) were female, 48 (31.4%) Hispanic and 122/142 (86%) had anti-mitochondrial antibodies (AMA), with a mean age at diagnosis 54.5 ± 13 years and a total of 623 person-years of follow-up. Mean dose of ursodeoxycholic acid (UDCA) was 15 ± 6.4 mg/kg/day; 13 patients were not on UDCA. No differences existed with respect to age, gender, AMA status, body mass index, UDCA dose, proportion of overlap cases and frequency of advanced fibrosis between Hispanics and non-Hispanics. Fatigue was present in 55.6% Hispanics vs. 35.3% non-Hispanics (p= 0.03), whereas the proportion of patients with pruritus was similar between groups. Portal hypertension was the initial presentation in only 17 patients, of whom 7 were Hispanic (p=not significant). Hypothyroidism and sicca syndrome were less frequent in Hispanics: 7/48 (14.6%) vs. 32/105 (30.5%; p=0.045), and 0/48 vs. 12/105 (11.4%; p=0.019), respectively. Among 133 patients who had been on UDCA longer than 1 year, Hispanics had a higher serum alkaline phosphatase: median 171 (range 26-1276) vs. 134 (range 11-746) IU/L, p=0.0025. By the end of followup, variceal bleeding had occurred in 25% of Hispanics vs. 9% non-Hispanics (p=0.02; relative risk 2.7, 95% CI 1.2-6.0), and ascites in 31% vs. 15% (p=0.03; relative risk 2.1, 95% CI 1.1-3.9). Twelve (8%) patients had died or undergone liver transplant, 6 in each ethnic group. Conclusion: The present study included a large proportion of Hispanics with PBC. We confirmed more frequent disease progression in Hispanics as evidenced by increased rates of decompensation. Further, Hispanics appear to have a poorer biochemical response to UDCA and may be appropriate candidates for novel therapies.
Mo1538
AASLD Abstracts
Hepatitis B Virus Surface Antigen Suppresses Innate and Adaptive Immune Responses of Murine and Human Liver Cells Min Jiang, Martin Trippler, Ruth Bröring, Lena Poggenpohl, Guido Gerken, Mengji Lu, Joerg F. Schlaak Background: It has recently been shown that Toll-like receptor (TLR) signaling in murine non-parenchymal (NPC) liver cells is suppressed in the presence of Hepatitis B surface antigen (HBsAg). It is not clear, however, whether this is also relevant for adaptive and human immune responses and how this effect is mediated. Methods: Peripheral blood mononuclear cells (PBMCs) from chronic HBV infected patients were stimulated by TLR3 ligands in the absence or presence of autologous serum. Murine Kupffer cells (KC), sinusoidal endothelial cells (LSEC) and hepatocytes as well as human LSEC and hepatocytes were stimulated with TLR3 ligand Poly I:C in the presence or absence of HBsAg. Expression of cytokines and TLR3 was analyzed by quantitative rt-PCR. Mixed lymphocyte reactions (MLR) were performed to study T cell activation induced by TLR-stimulated NPC. Activation of transcription factors was assessed by western blot and reporter gene assays. Results: Activation of T lymphocytes that was induced by TLR3-activated NPC was potently suppressed by HBsAg. This suppressive effect was associated with enhanced IL-10 production that could be reverted by IL-10 antibodies. While Poly I:C-induced TLR3 expression remained unchanged, TLR-induced activation of NFκB, IRF-3 and MAPKs was potently suppressed by HBsAg. TLR3-induced production of IFN-γ was suppressed in PBMC from patients with high HBsAg-levels (p<0.0006) compared to PBMC isolated from patients with low HBsAglevels. In the presence of autologous serum, TLR3 induced IFN-γ production was inhibited in case of HBV-containing serum. Conclusions: These findings may, at least in part, explain how HBV evades innate and adaptive immune responses to establish a persistent infection.
Mo1887 Antibiotic Prophylaxis for Recurrent Bacterial Cholangitis After Orthotopic Liver Transplantation (OLT) for Primary Sclerosing Cholangitis (PSC) Diana Winston, Marshall Kaplan Introduction: Recurrent bacterial cholangitis can be a serious problem in patients who undergo OLT for endstage PSC. We have found that a regimen of rotating antibiotics has been effective in preventing or greatly reducing the frequency and severity of these episodes and now report our experience. Methods: The medical records of patients followed by one of us (MMK) in the Hepatology Clinic of TMC, from January 1, 2000 until May 1, 2010, were reviewed in order to identify patients who had received OLT for PSC. We then identified patients who had had recurrent episodes of fever and rigors consistent with bacterial cholangitis and who had been treated with a regimen of rotating antibiotics. Rotating antibiotics were offered if the following criteria were met: episodes of chills and fever occurred at least once monthly, patients were hospitalized for cholangitis more than twice during the preceding four months, or if more than one episode of blood culture proven bacterial cholangitis occurred during the preceding six months. The antibiotic regimen initially consisted of amoxicillin for one month, then, trimethoprim/ sulfamethoxazole for one month, then ciprofloxacin for one month and then the cycle was repeated. Antibiotics were changed if patients were allergic to or intolerant of an antibiotic. Results: Of 749 patients who had had OLT, 69 patients had PSC. Of these 69, 12 had recurrent episodes of presumed bacterial cholangitis. Despite fevers, chills and frequent positive blood cultures, there was little worsening of serum levels of ALT, AST, alkaline phosphatase, or bilirubin in 7 patients. One patient who had an OLT performed in 1986 for PSC had a second OLT in 2005 because of hepatitis C cirrhosis and was counted twice. Therefore, we describe 13 OLTs in 12 patients (Table 1). The mean (median) duration of follow-up on the antibiotic treatment was 120 (133) months. Nine patients responded fully to the antibiotic regimen and had no further episodes of fever and chills as long as they took the antibiotics. Three patients had a decrease in the frequency of their episodes of fevers and chills. Antibiotics were ineffective in the patient who had had a second OLT for hepatitis C. Five patients had recurrent episodes of chills and fever when they stopped the antibiotics (four of whom were hospitalized for sepsis). All responded again when antibiotics were restarted. Aside from allergies to specific antibiotics, there were few complications. All continue on the antibiotics. Conclusions: Approximately 1/6 of our patients who had OLT for PSC had recurrent episodes of bacterial cholangitis. Rotating antibiotics were effective in preventing these episodes of in 9 of 13 patients and reduced the frequency in 3. The antibiotic regimen was well-tolerated.
Mo1539 A Novel Metabolic Action for the Multikinase Inhibitor Sorafenib: Targeting Tumor Cells Through Mitochondrial Damage Valentina Tesori, Anna C. Piscaglia, Marta Barba, Maria Ausiliatrice Puglisi, Giovambattista Pani, Antonio Gasbarrini BACKGROUND: One of the basis of modern oncology resides on the metabolic alteration of cancer cells, that explicates through an increase of glycolytic pathway, the so called “Warburg Effect”. Glycolysis can be seen as a cancer antioxidant response to allow tumor dissemination protecting cells from ROS-induced senescence and apoptosis. Although the multikinase inhibitor Sorafenib specifically targets tumor cells harboring the V590E mutation of B-RAF, additional mechanisms likely account for the elevated anticancer activity of the drug in tumors lacking this mutation, and for the emergence of chemoresistance. AIM:In the present study a novel metabolic effect of the multikinase inhibitor Sorafenib (SFB) was investigated, alone and in combination with the glycolytic inhibitor 2-deoxyglucose (2dg), in a liver tumor model, the LCSC-2 (Liver Cancer Stem Cells) cell line. In particular we investigated the mitochondrial action of SFB. MATERIAL AND METHODS: Drug toxicity was evaluated through Propidium Iodide (PI) assay. Gene expression was investigated by microarray analysis. Protein expression was evaluated by western blotting. Intracellular ATP level was assessed by chemiluminescence. Mitochondrial activity was assessed by Respirometric Analysis. Mitochondrial potential was measured with the JC-1 probe. RESULTS: We found that in rat hepatocolangiocarcinoma (LCSC-2) cells exposure to Sorafenib was not paralleled by significant inhibition of ERK phosphorylation, but, instead, elicited a raise of intracellular reactive oxygen species and the Ser172 phosphorylation of the AMP-activated protein kinase (AMPK), two events consistent with mitochondrial dysfunction. Accordingly, SFB led to a substantial reduction of oxygen consumption, cellular ATP level and mitochondrial transmembrane potential, and isolated mitochondria from rat liver were depolarized by the drug In Vitro, indicating a direct effect of SFB on the organelle. Interestingly, in keeping with its inhibitory action on mitochondrial respiration, Sorafenib killed much more efficiently LCSC-2 cells, as well as mouse B16F10 melanoma and 293T human kidney carcinoma cells when associated with the glycolysis inhibitor 2-deoxy-glucose. CONCLUSIONS:Taken together our preliminary results identify in mitochondrial damage and generation of ROS a novel modality of cytotoxicity by Sorafenib; moreover, the synergistic action of SFB plus 2DG outline a novel combined therapy to eradicate liver tumors.
AASLD Abstracts
S-990