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Loss of Autophagy-Enhanced Acetaminophen Hepatotoxicity is Mediated by JNK Activation
towards elevated transaminases (ALT 60 ± 38.8 IU/L, AST 74.7 ± 45.4 IU/L) it failed to reach statistical significance when compared to the HBV-DNA negative population (ALT 41.3 ± 37.2 IU/L; p=0.6, AST 43 ± 31.7 IU/L; p=0.06). No significant clinical or biochemical surrogate marker for occult HBV infection could be identified. Presence of anti-HBs antibodies did not rule out OHBV infection. Liver biopsy in the 12 OHBV cases showed varying stages of chronic hepatitis (HAI score9.7 ± 2.7; range 5-15) even in those with a normal transaminases and no radiological evidence of chronic liver disease. One OHBV patient developed fulminant hepatitis after initiation of HAART. Sequence analysis for amino acid substitution in the HBV surface gene region revealed substitutions at position T114S, F134Y/I, Y161F and T118V. However, the common immune escape mutant G145R was not detected in our study population. Conclusion: Histopathological evidence of liver fibrosis highlights the clinical importance of diagnosing OHBV. ‘Surface gene’ mutations are common but no single mutation explains the pathogenesis. In the absence of any surrogate markers, HBV DNA testing should be performed in all HIV patients who are HBsAg negative irrespective of antiHBs status. In view of possible HBV related immune reconstitution syndrome, OHBV should be ruled out in all HIV patients prior to HAART initiation.
Tu1680 Markers for Bile Ductular Reaction Are Associated With Hepatic iNOS Expression and Hepatic Fibrosis in Post-Transplant Hepatitis C Virus (HCV) Infection Katherine M. Johnson, Marla Z. Wolfert, Grace Guzman, Scott Cotler Background: Hepatitis C viral proteins upregulate inducible nitric oxide synthase (iNOS) leading to nitrosatative stress. There are conflicting data regarding whether iNOS expression is associated with HCV-related liver injury. Bile ductular reaction occurs in acute and chronic liver injury and has been associated with hepatic fibrosis. The aim of this study was to assess the relationship among hepatic iNOS expression, bile ductular reaction, and fibrosis progression in patients with HCV infected liver transplant recipients. Methods: Subjects consisted of 28 hepatitis C infected liver transplant recipients who had liver biopsy specimens available from 58 protocol liver biopsies. A liver pathologist, masked to clinical data, assessed inflammation and fibrosis for each biopsy by Batts and Ludwig criteria. Advanced fibrosis was defined as F3-F4. Standard immunohistochemistry was performed on paraffin embedded 4 micron thick tissue sections using an anti-iNOS antibody (Abcam, Cambridge, MA) and cytokeratin markers CK7 and CK19 (Dako, Carpinteria, (CA)) to identify bile ductular reaction. Slides were scanned and the percent of cells with cytoplasmic iNOS immunostaining was quantified by image analysis using Aperio (Vista, CA) digital pathology software. Staining for bile ductular reaction by CK7 and CK19 was categorized as present or absent. Results: The subjects' mean age was 50 + 10 years and 64% were male. Thirty-six percent were non-Hispanic White, 21% African American, and 36% were African American. Hepatic iNOS expression was greater in cases with evidence of bile ductular reaction by staining for CK19 (p=0.006) and by staining for CK7 (p=0.049). The intensity of iNOS staining was greatest in cases positive for both CK19 and CK7 (p=0.01). Women had higher degrees of staining for iNOS than men (p=0.036). There was no difference in iNOS expression as a function of donor or recipient age, or ethnicity. Staining for iNOS was not associated with degree of hepatic fibrosis or rate of fibrosis progression. CK19 expression was correlated with piecemeal necrosis (p=0.034) and advanced fibrosis (p=0.006). There were trends toward associations between CK7 staining and portal inflammation (p=0.088) and advanced fibrosis (p=0.094). Conclusions: Hepatic iNOS expression in liver tissue was associated with staining for CK19 and CK7, providing a link between bile ductular reaction and nitrosatative stress. Markers for bile ductular reaction correlated with hepatic fibrosis, implicating bile ductular reaction in HCV-related fibrosis post-transplant. iNOS expression was not associated with fibrosis, suggesting that nitrosatative stress is not a major factor in HCV progression in liver transplant recipients.
Mo1656
AASLD Abstracts
Liver Stiffness Measurements in Inactive Hbs AG Carriers Manuel Schendl, Sabrina Wimmer, Johanna Willeboer, Ivo Graziadei, Wolfgang Vogel, Robert O. Koch Introduction: Transient elastography (FibroScan®) is a noninvasive method proposed for the assessment of liver fibrosis in patients with chronic liver diseases by measuring liver stiffness. It can be easily performed in the outpatient clinic with immediate results and good reproducibility. FibroScan® is validated for the diagnosis of significant fibrosis and cirrhosis in chronic hepatitis B. Among these patients inactive HBsAg carriers present the largest group. Although the normally fortunately benign prognosis of inactive carriers, patients are at increased risk of reactivation and progression to fibrosis. Therefore the goal of this study was to evaluate liver stiffness (LS) in inactive HBsAg carriers. Material and methods: Between March 2008 and May 2010 liver stiffness measurements employing FibroScan® was performed in a total of 93 inactive HBsAg carriers. 49 patients were male and 44 female with a mean age of 43.1 years. The results of the female patients were compared to those of the male patients. Furthermore, the correlation between liver stiffness and clinical, demographic and laboratory findings was studied. Results: FibroScan® measurements were successfully performed in 96.8% of cases. Non valid measurements were mainly caused due to obesity. The mean value of liver stiffness inactive HBsAg carriers was 5.1 ± 2.14 kPa (2.2-13.0 kPa). So inactive HbsAg carriers showed similar values compared with healthy subjects. LS values were significantly lower in women than in men (4.6 ± 2.17 vs. 5.6 ± 1.98, p=0.001). Significant correlation was demonstrated between LS and the degree of hepatic steatosis (p= 0.004) as well as between LS and BMI (p=0.003). Patients with hepatic steatosis or increased BMI showed significantly higher LS values than those without. Furthermore, regression analysis showed that the duration of infection had a significant influence on LS (p=0.032). No influence on LS was observed for age, co morbidities, DNA-levels and laboratory parameters. Conclusion: FibroScan® is an excellent tool for monitoring inactive HbsAg carriers and for the early detection of transient reactivations. Inactive HBsAg carriers display similar LS values compared with healthy subjects. A significant correlation with LS measured by transient elastography was found with the stage of hepatic steatosis as well as the BMI.
Tu1681 Severe Hepatotoxicity in a Patient Treated With Hedgehog Inhibitor: First Case Report Bertha E. Sanchez, Layla Hajjafar Introduction: In the United States, drug-induced liver injury (DILI) is now the leading cause of fulminant liver failure among patients referred for liver transplant. DILI is mainly a diagnosis of exclusion and mandates a temporal relationship between drug initiation and development of the abnormal liver panel. Phase I clinical trials testing the novel hedgehog inhibitor GDC-0449 in advanced basal cell carcinoma (BCC) showed promising results for a neoplasm with short-term survival and no standard treatment. We report on a case of severe hepatotoxicity in a patient treated with hedgehog inhibitor for advanced BCC. Case Description: An 83-year-old Caucasian female with advanced BCC on her face was enrolled in a phase II trial testing GDC-0449 in advanced BCC and started taking daily oral doses of GDC-0449. One week later she developed nausea and emesis, and 3 weeks subsequently was hospitalized. Her medical history was significant for hypertension, atrial fibrillation and COPD, with no prior evidence of abnormal liver function testing. The patient was taking lasix, digoxin and theophylline. Social history revealed moderate daily alcohol use. Physical exam showed an unresponsive patient with icteric sclera and jaundice. Liver tests revealed a cholestatic pattern of hepatotoxicity (Table. 1). Ferritin, ceruloplasmin, α-1 antitrypsin and digoxin concentrations were normal. Serology for hepatitis A, B and C as well as CMV and EBV were negative. Anti-nuclear, anti-smooth muscle, antimitochondrial and liverkidney microsomal antibodies were negative. Abdominal CT, HIDA scan, ERCP and MRCP were normal. Liver biopsy showed nonspecific cholestasis with portal fibrosis; no steatosis was noted (Fig 1). Supportive care was administered during her hospitalization with improvement in both liver function and mental status. She had complete normalization of her liver profile six months following discontinuation of hedgehog inhibitor, but a liver ultrasound described an undulated liver contour concerning for cirrhosis. Discussion: In our patient, the temporal relationship and exclusion of other causes of liver failure favored the diagnosis of GDC-0449 induced liver injury. In addition, a risk factor predisposition for developing DILI in patients with chronic alcohol use was established in other agents and it might have contributed in this case. Nevertheless, the patient did not have evidence of chronic liver injury secondary to alcohol use on biopsy. Most cases of DILI are identified in the postmarketing phase after thousands of people have been exposed; therefore, the evidence of DILI in a phase II clinical trial of GDC-0449 should warrant actions to prevent and/or minimize the risk to others. An exhaustive evaluation when selecting patients for further trials should include history of alcohol use, co-administration of other medications and baseline liver enzymes.
Tu1679 Loss of Autophagy-Enhanced Acetaminophen Hepatotoxicity is Mediated by JNK Activation Yuki Igusa, Shunhei Yamashina, Kousuke Izumi, Hiroo Fukada, Akira Uchiyama, Kazuyoshi Kon, Kenichi Ikejima, Sumio Watanabe Background: Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis. Previously we reported that loss of autophagy enhances acetaminophen-induced liver injury in an In Vivo model. Recently, it was shown that c-jun N-terminal kinase (JNK), which is an important component of cellular stress response, promotes acetaminophen-induced liver injury. Here, we investigate if JNK activation is linked to loss of autophagy-enhanced acetaminophen hepatotoxicity. Methods: Hepatocytes were isolated from liver specific autophagy deficient mice (C57BL/6 AtgF/F: mx1-cre) and wild type mice (C57BL/6 Atg7F/F). Hepatocytes were cultured in media with or without 10mM acetaminophen. Moreover, some hepatocytes were treated with JNK inhibitor SP600123 and APAP. Activation of JNK was evaluated by western blot analysis. Mitochondrial membrane depolarization in cultured hepatocytes after APAP treatment was evaluated by fluorescent microscopy using JC-1 dye. Cell death was evaluated by flow cytometer using Alexa Fluor 488-conjugated annexin V and propidium iodide. Results: APAP treatment increased JNK activation (phosphorylation) in hepatocytes isolated from WT mice. On the other hand, autophagy deficiency enhanced APAP-induced JNK activation. Flow cytometric analysis revealed that cell death was caused by APAP treatment in 21.23% of hepatocytes isolated from wild type mouse (apoptotic cells 4.3%, necrotic cells 16.93%). Autophagy deficiency enhanced cell death to 42.1% of hepatocytes after APAP treatment (apoptotic cells 12.66%, necrotic cells 29.44%). Treatment with SP600125 suppressed APAP-induced cell death to 15.36% in hepatocytes isolated from autophagy-deficient mice; however, APAP-induced hepatocyte toxicity was not changed by addition of SP600125 in wild type hepatocytes. APAP treatment strongly induced mitochondrial membrane depolarization in autophagy deficient hepatocytes but not wild type hepatocytes. Interestingly, treatment with SP600125 blunted APAP-induced mitochondrial membrane depolarization in autophagy deficient hepatocytes. Conclusion: Loss of autophagy enhanced both activation of JNK and cell death in hepatocytes after APAP treatment. Moreover, JNK inhibitor suppressed APAP-induced mitochondrial membrane depolarization and cell death in autophagy-deficient hepatocytes. Our results indicated that JNK activation is essential for loss of autophagy-enhanced acetaminophen hepatotoxicity
AASLD Abstracts
S-974
Tu1680 Markers for Bile Ductular Reaction Are Associated With Hepatic iNOS Expression and Hepatic Fibrosis in Post-Transplant Hepatitis C Virus (HCV) Infection Katherine M. Johnson, Marla Z. Wolfert, Grace Guzman, Scott Cotler Background: Hepatitis C viral proteins upregulate inducible nitric oxide synthase (iNOS) leading to nitrosatative stress. There are conflicting data regarding whether iNOS expression is associated with HCV-related liver injury. Bile ductular reaction occurs in acute and chronic liver injury and has been associated with hepatic fibrosis. The aim of this study was to assess the relationship among hepatic iNOS expression, bile ductular reaction, and fibrosis progression in patients with HCV infected liver transplant recipients. Methods: Subjects consisted of 28 hepatitis C infected liver transplant recipients who had liver biopsy specimens available from 58 protocol liver biopsies. A liver pathologist, masked to clinical data, assessed inflammation and fibrosis for each biopsy by Batts and Ludwig criteria. Advanced fibrosis was defined as F3-F4. Standard immunohistochemistry was performed on paraffin embedded 4 micron thick tissue sections using an anti-iNOS antibody (Abcam, Cambridge, MA) and cytokeratin markers CK7 and CK19 (Dako, Carpinteria, (CA)) to identify bile ductular reaction. Slides were scanned and the percent of cells with cytoplasmic iNOS immunostaining was quantified by image analysis using Aperio (Vista, CA) digital pathology software. Staining for bile ductular reaction by CK7 and CK19 was categorized as present or absent. Results: The subjects' mean age was 50 + 10 years and 64% were male. Thirty-six percent were non-Hispanic White, 21% African American, and 36% were African American. Hepatic iNOS expression was greater in cases with evidence of bile ductular reaction by staining for CK19 (p=0.006) and by staining for CK7 (p=0.049). The intensity of iNOS staining was greatest in cases positive for both CK19 and CK7 (p=0.01). Women had higher degrees of staining for iNOS than men (p=0.036). There was no difference in iNOS expression as a function of donor or recipient age, or ethnicity. Staining for iNOS was not associated with degree of hepatic fibrosis or rate of fibrosis progression. CK19 expression was correlated with piecemeal necrosis (p=0.034) and advanced fibrosis (p=0.006). There were trends toward associations between CK7 staining and portal inflammation (p=0.088) and advanced fibrosis (p=0.094). Conclusions: Hepatic iNOS expression in liver tissue was associated with staining for CK19 and CK7, providing a link between bile ductular reaction and nitrosatative stress. Markers for bile ductular reaction correlated with hepatic fibrosis, implicating bile ductular reaction in HCV-related fibrosis post-transplant. iNOS expression was not associated with fibrosis, suggesting that nitrosatative stress is not a major factor in HCV progression in liver transplant recipients.
Mo1656
AASLD Abstracts
Liver Stiffness Measurements in Inactive Hbs AG Carriers Manuel Schendl, Sabrina Wimmer, Johanna Willeboer, Ivo Graziadei, Wolfgang Vogel, Robert O. Koch Introduction: Transient elastography (FibroScan®) is a noninvasive method proposed for the assessment of liver fibrosis in patients with chronic liver diseases by measuring liver stiffness. It can be easily performed in the outpatient clinic with immediate results and good reproducibility. FibroScan® is validated for the diagnosis of significant fibrosis and cirrhosis in chronic hepatitis B. Among these patients inactive HBsAg carriers present the largest group. Although the normally fortunately benign prognosis of inactive carriers, patients are at increased risk of reactivation and progression to fibrosis. Therefore the goal of this study was to evaluate liver stiffness (LS) in inactive HBsAg carriers. Material and methods: Between March 2008 and May 2010 liver stiffness measurements employing FibroScan® was performed in a total of 93 inactive HBsAg carriers. 49 patients were male and 44 female with a mean age of 43.1 years. The results of the female patients were compared to those of the male patients. Furthermore, the correlation between liver stiffness and clinical, demographic and laboratory findings was studied. Results: FibroScan® measurements were successfully performed in 96.8% of cases. Non valid measurements were mainly caused due to obesity. The mean value of liver stiffness inactive HBsAg carriers was 5.1 ± 2.14 kPa (2.2-13.0 kPa). So inactive HbsAg carriers showed similar values compared with healthy subjects. LS values were significantly lower in women than in men (4.6 ± 2.17 vs. 5.6 ± 1.98, p=0.001). Significant correlation was demonstrated between LS and the degree of hepatic steatosis (p= 0.004) as well as between LS and BMI (p=0.003). Patients with hepatic steatosis or increased BMI showed significantly higher LS values than those without. Furthermore, regression analysis showed that the duration of infection had a significant influence on LS (p=0.032). No influence on LS was observed for age, co morbidities, DNA-levels and laboratory parameters. Conclusion: FibroScan® is an excellent tool for monitoring inactive HbsAg carriers and for the early detection of transient reactivations. Inactive HBsAg carriers display similar LS values compared with healthy subjects. A significant correlation with LS measured by transient elastography was found with the stage of hepatic steatosis as well as the BMI.
Tu1681 Severe Hepatotoxicity in a Patient Treated With Hedgehog Inhibitor: First Case Report Bertha E. Sanchez, Layla Hajjafar Introduction: In the United States, drug-induced liver injury (DILI) is now the leading cause of fulminant liver failure among patients referred for liver transplant. DILI is mainly a diagnosis of exclusion and mandates a temporal relationship between drug initiation and development of the abnormal liver panel. Phase I clinical trials testing the novel hedgehog inhibitor GDC-0449 in advanced basal cell carcinoma (BCC) showed promising results for a neoplasm with short-term survival and no standard treatment. We report on a case of severe hepatotoxicity in a patient treated with hedgehog inhibitor for advanced BCC. Case Description: An 83-year-old Caucasian female with advanced BCC on her face was enrolled in a phase II trial testing GDC-0449 in advanced BCC and started taking daily oral doses of GDC-0449. One week later she developed nausea and emesis, and 3 weeks subsequently was hospitalized. Her medical history was significant for hypertension, atrial fibrillation and COPD, with no prior evidence of abnormal liver function testing. The patient was taking lasix, digoxin and theophylline. Social history revealed moderate daily alcohol use. Physical exam showed an unresponsive patient with icteric sclera and jaundice. Liver tests revealed a cholestatic pattern of hepatotoxicity (Table. 1). Ferritin, ceruloplasmin, α-1 antitrypsin and digoxin concentrations were normal. Serology for hepatitis A, B and C as well as CMV and EBV were negative. Anti-nuclear, anti-smooth muscle, antimitochondrial and liverkidney microsomal antibodies were negative. Abdominal CT, HIDA scan, ERCP and MRCP were normal. Liver biopsy showed nonspecific cholestasis with portal fibrosis; no steatosis was noted (Fig 1). Supportive care was administered during her hospitalization with improvement in both liver function and mental status. She had complete normalization of her liver profile six months following discontinuation of hedgehog inhibitor, but a liver ultrasound described an undulated liver contour concerning for cirrhosis. Discussion: In our patient, the temporal relationship and exclusion of other causes of liver failure favored the diagnosis of GDC-0449 induced liver injury. In addition, a risk factor predisposition for developing DILI in patients with chronic alcohol use was established in other agents and it might have contributed in this case. Nevertheless, the patient did not have evidence of chronic liver injury secondary to alcohol use on biopsy. Most cases of DILI are identified in the postmarketing phase after thousands of people have been exposed; therefore, the evidence of DILI in a phase II clinical trial of GDC-0449 should warrant actions to prevent and/or minimize the risk to others. An exhaustive evaluation when selecting patients for further trials should include history of alcohol use, co-administration of other medications and baseline liver enzymes.
Tu1679 Loss of Autophagy-Enhanced Acetaminophen Hepatotoxicity is Mediated by JNK Activation Yuki Igusa, Shunhei Yamashina, Kousuke Izumi, Hiroo Fukada, Akira Uchiyama, Kazuyoshi Kon, Kenichi Ikejima, Sumio Watanabe Background: Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis. Previously we reported that loss of autophagy enhances acetaminophen-induced liver injury in an In Vivo model. Recently, it was shown that c-jun N-terminal kinase (JNK), which is an important component of cellular stress response, promotes acetaminophen-induced liver injury. Here, we investigate if JNK activation is linked to loss of autophagy-enhanced acetaminophen hepatotoxicity. Methods: Hepatocytes were isolated from liver specific autophagy deficient mice (C57BL/6 AtgF/F: mx1-cre) and wild type mice (C57BL/6 Atg7F/F). Hepatocytes were cultured in media with or without 10mM acetaminophen. Moreover, some hepatocytes were treated with JNK inhibitor SP600123 and APAP. Activation of JNK was evaluated by western blot analysis. Mitochondrial membrane depolarization in cultured hepatocytes after APAP treatment was evaluated by fluorescent microscopy using JC-1 dye. Cell death was evaluated by flow cytometer using Alexa Fluor 488-conjugated annexin V and propidium iodide. Results: APAP treatment increased JNK activation (phosphorylation) in hepatocytes isolated from WT mice. On the other hand, autophagy deficiency enhanced APAP-induced JNK activation. Flow cytometric analysis revealed that cell death was caused by APAP treatment in 21.23% of hepatocytes isolated from wild type mouse (apoptotic cells 4.3%, necrotic cells 16.93%). Autophagy deficiency enhanced cell death to 42.1% of hepatocytes after APAP treatment (apoptotic cells 12.66%, necrotic cells 29.44%). Treatment with SP600125 suppressed APAP-induced cell death to 15.36% in hepatocytes isolated from autophagy-deficient mice; however, APAP-induced hepatocyte toxicity was not changed by addition of SP600125 in wild type hepatocytes. APAP treatment strongly induced mitochondrial membrane depolarization in autophagy deficient hepatocytes but not wild type hepatocytes. Interestingly, treatment with SP600125 blunted APAP-induced mitochondrial membrane depolarization in autophagy deficient hepatocytes. Conclusion: Loss of autophagy enhanced both activation of JNK and cell death in hepatocytes after APAP treatment. Moreover, JNK inhibitor suppressed APAP-induced mitochondrial membrane depolarization and cell death in autophagy-deficient hepatocytes. Our results indicated that JNK activation is essential for loss of autophagy-enhanced acetaminophen hepatotoxicity
AASLD Abstracts
S-974