- Email: [email protected]
117 SPONTANEOUS DECREASE IN LIVER INJURY DOES NOT STRONGLY PREDICT EVOLUTION OF PATIENTS WITH SEVERE ALCOHOLIC HEPATITIS TREATED WITH CORTICOSTEROIDS
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NK NKG2D 1000
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Figure 1. S52
Alcoholic Cirrhosis
Healthy Controls
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Alcoholic Cirrhosis
Healthy Controls
Alcoholic Cirrhosis
Healthy Controls
Figure 2.
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Background and Aims: Progressive hepatic injury and susceptibility to infection are hallmarks in the dismal prognosis of alcoholic hepatitis (AH). The hepatic lymphocytes are particularly rich in natural killer (NK), NKT and NK-like CD8+ T cells. NKG2D is a central activation receptor expressed on these cytotoxic cells. Receptor ligands are induced in response to cellular stress, injury as well as infections and activate the cytotoxic cells to kill target cells. As products of cell damage are known to hold immunogenic potential, defective clearing of injured cells may lead to chronic inflammation. Dysfunctional cytotoxic cells may, thus, participate in the sustained immune activation in AH and also in the decreased resistance towards infections. Our aim was, therefore, to evaluate the frequency and activational state of NK, NKT and NK-like CD8+ T cells in patients with severe AH. Methods: We analysed blood samples from 20 consecutive patients with severe AH. As control group, we included 10 patients with stable alcoholic cirrhosis (AC) and 10 healthy controls (HC). The expression of NKG2D by flow cytometry is presented as median fluorescence intensity. Results: In AH patients compared with HC, there was no difference in the frequency of NK cells (CD3− CD56+ ) (median±IQR: 10.4%±12.4 vs. 14.0%±10.1) or NKT cells (CD3+ CD56+ ) (2.9%±3.9 vs. 2.0%±6.4), yet the frequency of NK-like CD8+ T cells (CD3+ CD56+ CD8++ ) was halved (27.0%±29.9 vs. 56.6±28.1, p = 0.005). Despite little difference in cell frequencies, the expression of NKG2D was clearly lower in NK cells of AH patients compared with both AC (903±584 vs. 1031±176, p = 0.05) and HC (1179±356, p = 0.02) and the same was seen in NKT cells compared with both control groups (903±656 vs. 1417±450, p = 0.01; 1296±386, p = 0.03). A similar but not significant tendency was evident also for NK-like CD8+ T cells (Figures 1–3).
NK-like CD8+ T cells NKG2D 1000 1500 2000
116 DYSFUNCTIONAL NK, NKT AND NK-LIKE CD8+ T CELLS IN ALCOHOLIC HEPATITIS S. Støy1 , A. Dige1 , T.D. Sandahl1 , M. Hokland2 , H. Vilstrup1 . 1 Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital, 2 Institute of Biomedicine, Aarhus University, Aarhus, Denmark E-mail: [email protected]
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alcohol, low BMI/high alcohol and high BMI/high alcohol groups respectively. Only the combination of high BMI and high alcohol reached significance (table and figure). Conclusions: The combination of high alcohol intake and high BMI interact synergistically to produce an increased risk of CLD. Alcohol may be the more significant contributing factor. Further work is required to define thresholds for each risk factor that independently and in combination increase risk of CLD.
2500
ORAL PRESENTATIONS
Alcoholic Hepatitis
Figure 3.
Conclusion: The functionality of NK, NKT and NK-like CD8+ T cells has never previously been investigated in AH. Our results, however, suggest dysfunctional activation of these cells in AH, which is likely to contribute towards both the sustained inflammatory state and the patients’ susceptibility to infections. 117 SPONTANEOUS DECREASE IN LIVER INJURY DOES NOT STRONGLY PREDICT EVOLUTION OF PATIENTS WITH SEVERE ALCOHOLIC HEPATITIS TREATED WITH CORTICOSTEROIDS A. Louvet1,2 , F. Artru1,2 , M. Colin1 , G. Lassailly1,2 , P. Deltenre1,3 , V. Canva-Delcambre1 , S. Dharancy1,2 , P. Mathurin1,2 . 1 Service des Maladies de l’Appareil Digestif, Hˆ opital Huriez, 2 Unit´e INSERM 995, 3 Lille, France; Service d’H´epato-Gastroent´erologie, Hˆ opital de Jolimont, Haine-Saint-Paul, Belgium E-mail: [email protected] Early improvement in liver function under treatment with corticosteroids for severe alcoholic hepatitis is related to survival. One can suppose that spontaneous improvement before therapy may predict outcome. Aims: To evaluate 1. spontaneous improvement in liver function from the admission to corticosteroid treatment 2. the impact of such improvement on survival and therapeutic response.
Journal of Hepatology 2013 vol. 58 | S45–S61
ORAL PRESENTATIONS Methods: Modification in liver parameters was prospectively registered from the admission up to the beginning of corticosteroids and thereafter. Results: Characteristics of the 339 included patients at admission were: age 49.6 years, male 58.6%, ascites 73.9%, encephalopathy 24.6%, bilirubin 162 mg/l, Maddrey function 59.8. After infection screening and liver biopsy, corticosteroids were started after 7 days (median). Both bilirubin [evolution of 0 mg/l (95% CI: −1 to 4.4)] and DF (0.31, 95% CI: 0–1.32) remained stable from admission to corticosteroid onset. Thus, 50% of patients decreased their bilirubin and DF, so-called spontaneous responders (SR), in contrary to spontaneous non-responders (SNR): – 16 vs. +5.5 mg/l, p < 0.001 and −7.6 vs. +6.9, p < 0.001. SR and SNR were not different for DF at admission: 57.3 vs. 61.1, p = 0.3. On overall patients after 7 days of corticosteroids, bilirubin decreased by 22 mg/l (15.2–30), Lille model was 0.34 (0.27–0.39) and 61.4% of patients were responders to corticosteroids (Lille model <0.45). Survival was higher in SR: 69.7±3.7 vs. 55.8±4.1%, p = 0.0006. Leukocytes, encephalopathy, ascites, MELD score, spontaneous response and Lille model predicted 6-month survival in univariate analysis. In multivariate analysis, ascites (RR 2.12, p = 0.007), MELD score (RR 1.03, p = 0.03) and Lille model (RR 44.5, p < 0.001) were independent prognostic factors, whereas encephalopathy (p = 0.13), leukocytes (p = 0.46) and spontaneous response (p = 0.08) were not. After 7 days of corticosteroids, SR and SNR decreased their bilirubin level: −23.9 vs. −26.9 mg/l, p = 0.14. SR were more frequently responders to corticosteroids (72.8 vs. 52.7%, p < 0.001) and had lower liver injury at corticosteroid onset: DF 51.9 vs. 67.6, p < 0.001. Conclusion: Spontaneous improvement is observed in 50% of patients with severe alcoholic hepatitis but does not predict survival. Probability of response to corticosteroids is the highest predictor of outcome and is more frequent in spontaneous responders. 118 METABOLIC FATTY-LIVER DISEASE INCREASES THE RISK OF HEPATOCELLULAR CARCINOMA IN PATIENTS WITH ALCOHOLIC CIRRHOSIS LISTED FOR LIVER TRANSPLANTATION R. Pais, P. Lebray, G. Rousseau, M. Rudler, D. Thabut, F. Charlotte, L. Bonyhay, O. Lucidarme, L. Hannoun, T. Poynard, J.-C. Vaillant, V. Ratziu, for the FLIP Consortium. Hopital Pitie Salpetriere, Paris, France E-mail: [email protected] Fatty liver and alcohol are risk factors for HCC but their synergism in alcoholic cirrhosis is unknown. Aim: To analyze the impact of fatty liver and coexistent metabolic risk factors (MRF) on HCC development in patients with alcoholic cirrhosis listed for liver transplantation (LT). Methods: One-hundred patients transplanted for alcoholic ESLD were studied for: length of abstinence before LT, steatosis (≥10% on the native liver) and previously diagnosed MRF (overweight/obesity or type-2 diabetes) in relation to the presence of HCC. Results: Indications for LT were: 77% decompensated cirrhosis without HCC, 23% HCC. Mean age was 55 yrs, mean abstinence 2.2 yrs (≥6 months in 71%). HCC was not confirmed in 3 patients and incidentally found on liver explant in eight patients; thus 28 patients had HCC. Thirty-two patients had neither steatosis nor MRF (NONE); 24 steatosis alone (STEAT ); 20 steatosis coexisting with MRF (STEAT+MRF); 24 MRF but no steatosis (MRF). Mean age increased gradually between these categories. Patients in the MRF and STEAT+MRF groups were older than those in the NONE and STEAT groups (56.8 vs. 53.7 yrs, respectively, p < 0.04) but had similar length of abstinence (≥6 months in 80% vs. 64%, p = 0.12). HCC patients were older (57.4 vs. 54.2 yrs, p = 0.05), have been more frequently overweight (54% vs. 14%, p < 0.001) or diabetic (43% vs.
22%, p < 0.04) than those without HCC. There was no difference in disease duration, length of abstinence, waiting time before LT or proportion of steatosis between HCC and non-HCC. However, the proportion of HCC was highest among the STEAT+MRF group (50%) and declined to 6% in the NONE group. Only 13% of patients in the NONE/STEAT groups had HCC vs. 48% in the MRF/STEAT+MRF groups (p = 0.0001). Results were similar in patients with >6 months abstinence. After adjusting for sex, age and alcohol, history of overweight/obesity or diabetes remained significantly associated with HCC (p < 0.01 and <0.04, respectively). Conclusion: The risk of HCC was significantly increased by previous exposure to MRF and concurrent steatosis, even in patients with >6 months alcohol abstinence. Concurrent metabolic fatty-liver disease could be a carcinogenic cofactor in alcoholic cirrhosis. 119 ROLE OF ChREBP IN LIVER ALCOHOLIC STEATOSIS DEVELOPMENT S. Marmier1 , R. Dentin1 , S. Loterstazjn2 , C. Postic1 . 1 Endocrinology Metabolism and Cancer, Institut Cochin, INSERM (IMR-S1016), CNRS (UMR 8104), Paris, 2 Hˆ opital Henri Mondor Inserm U99, Cr´eteil, France E-mail: [email protected] Background and Aims: Carbohydrate Responsive Element Binding Protein (ChREBP) is a key transcription factor involved in the control of hepatic lipogenesis under physiological and physiopathological conditions. Ethanol-induced fatty liver is a worldwide health problem without effective therapeutic method. In the current study, we investigated the contribution of ChREBP to alcohol-induced steatosis in a binge-drinking model. Methods: C57BL/6J male mice were fasted for 4 hours before receiving 33% (vol/vol) ethanol at a total accumulating dose of 3.5 g/kg body weight by 3 equally divided gavages in 30 minutes intervals. Control mice received the same volume of dextrinmaltose (DM) in order to reach similar caloric value. Ten hours after the first gavage, mice were sacrificed. To determine the specific role of ChREBP, a shRNA directed against ChREBP (shChREBP), was injected 5 days before the binge drinking protocol. In parallel, primary cultures of mouse hepatocytes incubated with 50 mM ethanol in the presence or not of the shChREBP were performed. Results: Binge-drinking markedly raised circulating ethanol concentrations. In liver of ethanol-treated mice, a significant decrease of the NAD/NADH ratio was observed suggesting that the activity of NAD-dependent deacetylases, such as SIRT1, was reduced. Indeed we observed that global and ADH (Alcohol Dehydrogenase) acetylation, the first enzyme which catalyses ethanol oxidation, was significantly induced under binge-drinking conditions. We also observed that ChREBP acetylation as well as its recruitment on its target genes was significantly increased leading to hepatic steatosis development with a 3-fold increase in triglycerides (TG) concentrations compared to DM-treated mice. Experiments performed in primary cultures of mouse hepatocytes confirmed that ethanol-induced TG accumulation could be prevented by ChREBP silencing. In vivo ChREBP knockdown, by reducing lipogenic gene expression, significantly prevented alcoholinduced TG accumulation. Interestingly, under binge-drinking conditions, the expression of SIRT1, a direct negative target of ChREBP, was re-induced upon ChREBP knockdown. As a consequence, global and ADH acetylation was decreased suggesting that ethanol oxidation may be reduced under ChREBP inhibition. In conclusion: Our results reveal the contribution of ChREBP to alcohol-induced hepatic steatosis under binge drinking conditions and underline the importance of ChREBP in controlling SIRT1 expression in liver.
Journal of Hepatology 2013 vol. 58 | S45–S61
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Figure 1. S52
Alcoholic Cirrhosis
Healthy Controls
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Alcoholic Hepatitis
Alcoholic Cirrhosis
Healthy Controls
Alcoholic Cirrhosis
Healthy Controls
Figure 2.
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Background and Aims: Progressive hepatic injury and susceptibility to infection are hallmarks in the dismal prognosis of alcoholic hepatitis (AH). The hepatic lymphocytes are particularly rich in natural killer (NK), NKT and NK-like CD8+ T cells. NKG2D is a central activation receptor expressed on these cytotoxic cells. Receptor ligands are induced in response to cellular stress, injury as well as infections and activate the cytotoxic cells to kill target cells. As products of cell damage are known to hold immunogenic potential, defective clearing of injured cells may lead to chronic inflammation. Dysfunctional cytotoxic cells may, thus, participate in the sustained immune activation in AH and also in the decreased resistance towards infections. Our aim was, therefore, to evaluate the frequency and activational state of NK, NKT and NK-like CD8+ T cells in patients with severe AH. Methods: We analysed blood samples from 20 consecutive patients with severe AH. As control group, we included 10 patients with stable alcoholic cirrhosis (AC) and 10 healthy controls (HC). The expression of NKG2D by flow cytometry is presented as median fluorescence intensity. Results: In AH patients compared with HC, there was no difference in the frequency of NK cells (CD3− CD56+ ) (median±IQR: 10.4%±12.4 vs. 14.0%±10.1) or NKT cells (CD3+ CD56+ ) (2.9%±3.9 vs. 2.0%±6.4), yet the frequency of NK-like CD8+ T cells (CD3+ CD56+ CD8++ ) was halved (27.0%±29.9 vs. 56.6±28.1, p = 0.005). Despite little difference in cell frequencies, the expression of NKG2D was clearly lower in NK cells of AH patients compared with both AC (903±584 vs. 1031±176, p = 0.05) and HC (1179±356, p = 0.02) and the same was seen in NKT cells compared with both control groups (903±656 vs. 1417±450, p = 0.01; 1296±386, p = 0.03). A similar but not significant tendency was evident also for NK-like CD8+ T cells (Figures 1–3).
NK-like CD8+ T cells NKG2D 1000 1500 2000
116 DYSFUNCTIONAL NK, NKT AND NK-LIKE CD8+ T CELLS IN ALCOHOLIC HEPATITIS S. Støy1 , A. Dige1 , T.D. Sandahl1 , M. Hokland2 , H. Vilstrup1 . 1 Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital, 2 Institute of Biomedicine, Aarhus University, Aarhus, Denmark E-mail: [email protected]
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alcohol, low BMI/high alcohol and high BMI/high alcohol groups respectively. Only the combination of high BMI and high alcohol reached significance (table and figure). Conclusions: The combination of high alcohol intake and high BMI interact synergistically to produce an increased risk of CLD. Alcohol may be the more significant contributing factor. Further work is required to define thresholds for each risk factor that independently and in combination increase risk of CLD.
2500
ORAL PRESENTATIONS
Alcoholic Hepatitis
Figure 3.
Conclusion: The functionality of NK, NKT and NK-like CD8+ T cells has never previously been investigated in AH. Our results, however, suggest dysfunctional activation of these cells in AH, which is likely to contribute towards both the sustained inflammatory state and the patients’ susceptibility to infections. 117 SPONTANEOUS DECREASE IN LIVER INJURY DOES NOT STRONGLY PREDICT EVOLUTION OF PATIENTS WITH SEVERE ALCOHOLIC HEPATITIS TREATED WITH CORTICOSTEROIDS A. Louvet1,2 , F. Artru1,2 , M. Colin1 , G. Lassailly1,2 , P. Deltenre1,3 , V. Canva-Delcambre1 , S. Dharancy1,2 , P. Mathurin1,2 . 1 Service des Maladies de l’Appareil Digestif, Hˆ opital Huriez, 2 Unit´e INSERM 995, 3 Lille, France; Service d’H´epato-Gastroent´erologie, Hˆ opital de Jolimont, Haine-Saint-Paul, Belgium E-mail: [email protected] Early improvement in liver function under treatment with corticosteroids for severe alcoholic hepatitis is related to survival. One can suppose that spontaneous improvement before therapy may predict outcome. Aims: To evaluate 1. spontaneous improvement in liver function from the admission to corticosteroid treatment 2. the impact of such improvement on survival and therapeutic response.
Journal of Hepatology 2013 vol. 58 | S45–S61
ORAL PRESENTATIONS Methods: Modification in liver parameters was prospectively registered from the admission up to the beginning of corticosteroids and thereafter. Results: Characteristics of the 339 included patients at admission were: age 49.6 years, male 58.6%, ascites 73.9%, encephalopathy 24.6%, bilirubin 162 mg/l, Maddrey function 59.8. After infection screening and liver biopsy, corticosteroids were started after 7 days (median). Both bilirubin [evolution of 0 mg/l (95% CI: −1 to 4.4)] and DF (0.31, 95% CI: 0–1.32) remained stable from admission to corticosteroid onset. Thus, 50% of patients decreased their bilirubin and DF, so-called spontaneous responders (SR), in contrary to spontaneous non-responders (SNR): – 16 vs. +5.5 mg/l, p < 0.001 and −7.6 vs. +6.9, p < 0.001. SR and SNR were not different for DF at admission: 57.3 vs. 61.1, p = 0.3. On overall patients after 7 days of corticosteroids, bilirubin decreased by 22 mg/l (15.2–30), Lille model was 0.34 (0.27–0.39) and 61.4% of patients were responders to corticosteroids (Lille model <0.45). Survival was higher in SR: 69.7±3.7 vs. 55.8±4.1%, p = 0.0006. Leukocytes, encephalopathy, ascites, MELD score, spontaneous response and Lille model predicted 6-month survival in univariate analysis. In multivariate analysis, ascites (RR 2.12, p = 0.007), MELD score (RR 1.03, p = 0.03) and Lille model (RR 44.5, p < 0.001) were independent prognostic factors, whereas encephalopathy (p = 0.13), leukocytes (p = 0.46) and spontaneous response (p = 0.08) were not. After 7 days of corticosteroids, SR and SNR decreased their bilirubin level: −23.9 vs. −26.9 mg/l, p = 0.14. SR were more frequently responders to corticosteroids (72.8 vs. 52.7%, p < 0.001) and had lower liver injury at corticosteroid onset: DF 51.9 vs. 67.6, p < 0.001. Conclusion: Spontaneous improvement is observed in 50% of patients with severe alcoholic hepatitis but does not predict survival. Probability of response to corticosteroids is the highest predictor of outcome and is more frequent in spontaneous responders. 118 METABOLIC FATTY-LIVER DISEASE INCREASES THE RISK OF HEPATOCELLULAR CARCINOMA IN PATIENTS WITH ALCOHOLIC CIRRHOSIS LISTED FOR LIVER TRANSPLANTATION R. Pais, P. Lebray, G. Rousseau, M. Rudler, D. Thabut, F. Charlotte, L. Bonyhay, O. Lucidarme, L. Hannoun, T. Poynard, J.-C. Vaillant, V. Ratziu, for the FLIP Consortium. Hopital Pitie Salpetriere, Paris, France E-mail: [email protected] Fatty liver and alcohol are risk factors for HCC but their synergism in alcoholic cirrhosis is unknown. Aim: To analyze the impact of fatty liver and coexistent metabolic risk factors (MRF) on HCC development in patients with alcoholic cirrhosis listed for liver transplantation (LT). Methods: One-hundred patients transplanted for alcoholic ESLD were studied for: length of abstinence before LT, steatosis (≥10% on the native liver) and previously diagnosed MRF (overweight/obesity or type-2 diabetes) in relation to the presence of HCC. Results: Indications for LT were: 77% decompensated cirrhosis without HCC, 23% HCC. Mean age was 55 yrs, mean abstinence 2.2 yrs (≥6 months in 71%). HCC was not confirmed in 3 patients and incidentally found on liver explant in eight patients; thus 28 patients had HCC. Thirty-two patients had neither steatosis nor MRF (NONE); 24 steatosis alone (STEAT ); 20 steatosis coexisting with MRF (STEAT+MRF); 24 MRF but no steatosis (MRF). Mean age increased gradually between these categories. Patients in the MRF and STEAT+MRF groups were older than those in the NONE and STEAT groups (56.8 vs. 53.7 yrs, respectively, p < 0.04) but had similar length of abstinence (≥6 months in 80% vs. 64%, p = 0.12). HCC patients were older (57.4 vs. 54.2 yrs, p = 0.05), have been more frequently overweight (54% vs. 14%, p < 0.001) or diabetic (43% vs.
22%, p < 0.04) than those without HCC. There was no difference in disease duration, length of abstinence, waiting time before LT or proportion of steatosis between HCC and non-HCC. However, the proportion of HCC was highest among the STEAT+MRF group (50%) and declined to 6% in the NONE group. Only 13% of patients in the NONE/STEAT groups had HCC vs. 48% in the MRF/STEAT+MRF groups (p = 0.0001). Results were similar in patients with >6 months abstinence. After adjusting for sex, age and alcohol, history of overweight/obesity or diabetes remained significantly associated with HCC (p < 0.01 and <0.04, respectively). Conclusion: The risk of HCC was significantly increased by previous exposure to MRF and concurrent steatosis, even in patients with >6 months alcohol abstinence. Concurrent metabolic fatty-liver disease could be a carcinogenic cofactor in alcoholic cirrhosis. 119 ROLE OF ChREBP IN LIVER ALCOHOLIC STEATOSIS DEVELOPMENT S. Marmier1 , R. Dentin1 , S. Loterstazjn2 , C. Postic1 . 1 Endocrinology Metabolism and Cancer, Institut Cochin, INSERM (IMR-S1016), CNRS (UMR 8104), Paris, 2 Hˆ opital Henri Mondor Inserm U99, Cr´eteil, France E-mail: [email protected] Background and Aims: Carbohydrate Responsive Element Binding Protein (ChREBP) is a key transcription factor involved in the control of hepatic lipogenesis under physiological and physiopathological conditions. Ethanol-induced fatty liver is a worldwide health problem without effective therapeutic method. In the current study, we investigated the contribution of ChREBP to alcohol-induced steatosis in a binge-drinking model. Methods: C57BL/6J male mice were fasted for 4 hours before receiving 33% (vol/vol) ethanol at a total accumulating dose of 3.5 g/kg body weight by 3 equally divided gavages in 30 minutes intervals. Control mice received the same volume of dextrinmaltose (DM) in order to reach similar caloric value. Ten hours after the first gavage, mice were sacrificed. To determine the specific role of ChREBP, a shRNA directed against ChREBP (shChREBP), was injected 5 days before the binge drinking protocol. In parallel, primary cultures of mouse hepatocytes incubated with 50 mM ethanol in the presence or not of the shChREBP were performed. Results: Binge-drinking markedly raised circulating ethanol concentrations. In liver of ethanol-treated mice, a significant decrease of the NAD/NADH ratio was observed suggesting that the activity of NAD-dependent deacetylases, such as SIRT1, was reduced. Indeed we observed that global and ADH (Alcohol Dehydrogenase) acetylation, the first enzyme which catalyses ethanol oxidation, was significantly induced under binge-drinking conditions. We also observed that ChREBP acetylation as well as its recruitment on its target genes was significantly increased leading to hepatic steatosis development with a 3-fold increase in triglycerides (TG) concentrations compared to DM-treated mice. Experiments performed in primary cultures of mouse hepatocytes confirmed that ethanol-induced TG accumulation could be prevented by ChREBP silencing. In vivo ChREBP knockdown, by reducing lipogenic gene expression, significantly prevented alcoholinduced TG accumulation. Interestingly, under binge-drinking conditions, the expression of SIRT1, a direct negative target of ChREBP, was re-induced upon ChREBP knockdown. As a consequence, global and ADH acetylation was decreased suggesting that ethanol oxidation may be reduced under ChREBP inhibition. In conclusion: Our results reveal the contribution of ChREBP to alcohol-induced hepatic steatosis under binge drinking conditions and underline the importance of ChREBP in controlling SIRT1 expression in liver.
Journal of Hepatology 2013 vol. 58 | S45–S61
S53