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Tu1823 Early Activation of Peripheral Monocytes With Hallmarks of M1 and M2 Monocytic Cells in Excessive Alcohol Drinkers
causes behind significantly high charges submitted by proprietary hospitals to CMS and uninsured patients.
unresponsive SAH, with severity score criteria (DF > 32, ABCI > 12, Lille >0.45, MELD > 25) Exclusion: hepatitis A, B, C, GI bleed, sepsis, HIV Group A MMF 500 mg bid with Pentoxifylline (PTX) 400 mg and Vit D orally for 30 days. Group B Prednisolone 30 mg plus Vit D and PTX 400 mg orally 30 days. Group C Prednisolone plus MMF and PTX orally for 30 days. Results: See table Conclusion: Preliminary data suggests MMF is well tolerated, safe, and lowers MELD and inflammatory parameters. A larger randomized trial needs to elucidate the potential role of MMF in ASAH. Results
AASLD Abstracts
Tu1823 Early Activation of Peripheral Monocytes With Hallmarks of M1 and M2 Monocytic Cells in Excessive Alcohol Drinkers Crystal Walline, Janice Blum, Suthat Liangpunsakul Background: Chronic, excessive drinking can lead to the development of immune dysfunction. Following chronic alcohol exposure, human monocytes display enhanced sensitivity to bacterial LPS (lipopolysaccharides) as measured by increased cytokine secretion ex vivo. However, early biomarkers to directly detect the activation status of circulating monocytes and inflammation following chronic alcohol consumption in the absence of alcoholic liver disease (ALD) have not been well studied. Our aim is to investigate biomarkers of early immune activation from circulating peripheral blood monocytes in subjects with chronic alcohol intake in the absence of ALD. Methods: 22 chronic excessive drinkers (ED, mean age 37 years/69% men/77% Caucasian) were enrolled from Fairbanks Alcohol Treatment Center. 20 non-excessive drinkers (NED, mean age 31 years/85% men/65% Caucasian) were recruited from Roudebush VAMC. Alcohol intake was considered as categorical variable (ED/NED) using NIAAA criteria. Time Line Follow-Back (TLFB) was used to quantify the amount of alcohol consumed in the past 30 days before enrollment. Anthropometric and laboratory measurements were obtained. Peripheral blood derived CD14+ monocytes (PBM) were isolated from each subject to determine the genes associated with monocyte differentiation. In addition, serum cytokines and LPS were also measured. Results: Serum LPS concentrations were significantly higher in ED compared to NED (0.26±0.07 vs. 0.13±0.03 EU/ml, p<0.05). No differences in the levels of circulating IL-6 and IL-10 were observed. Interestingly, the relative levels of gene transcripts (RQ) for Il6 (an M1-polarizing cytokine) and Il10 (an M2-polarizing cytokine) were significantly higher in PBM from ED compared to NED (Il6: 4.3, 95% CI [2.31, 8.28] vs. 1.0, 95% CI [0.433, 2.24] p < 0.01. Il10: 2.25, 95% CI [1.66, 5.06] vs. 1.0, 95% CI [0.809, 2.23] p < 0.05). However, no differences in other M1 (Il1b, Tnf and Ccl2) and M2 monocyte differentiation markers (Mrc1 and Cd163) were detected. Conclusions: Early monitoring of peripheral blood monocyte mRNA transcripts, notably Il6 and Il10, could identify individuals with excessive alcohol drinking with early immune activation in the absence of ALD.
Tu1821 Regulation of the Extrinsic Apoptotic Pathway by MicroRNA-21 in Alcoholic Liver Injury Morgan Quezada, Kelly McDaniel, Yuyan Han, Heather L. Francis, Shannon Glaser, Julie Venter, Gianfranco Alpini, Fanyin Meng Background: microRNAs are endogenous molecules which constitute a new class of negative regulators of gene expression. New evidence identifies miR-21 as a regulatory master-switch during liver injuries. The current study aimed to characterize the functional role of miR-21 and its downstream pro-apoptotic target genes during alcoholic-induced hepatitis. Methods: miRNA expression was evaluated by qPCR array and Taqman miRNA assay in ethanol treated normal human hepatocytes (N-Hep), hepatic stellate cells (HSC) and HepG2 HCC cells as well as 5 weeks chronic alcohol-fed mouse liver specimens and control liver tissue,. Cell proliferation, survival and the transformation index were quantitated using commercial assays. The anti-miR-21 effect in ALD mice was verified by specific Vivo-Morpholino treatment. Results: The overexpression of miR-21 and miR-34a along with the altered ultrasound liver images, increased ALT level and pathology score was seen in the liver tissues from mice administered 5% (v/v) ethanol (EtOH) after five weeks. The expression of miR-21 was also increased in ethanol-treated N-Heps and HSCs with a higher apoptotic rate compared with that of controls. In N-Heps, HSCs, and HepG2 cells transfected with pre-miR-21 or anti-miR-21 inhibitor, cell survival was significantly changed compared to cells transfected with controls consisting of altered dosage and duration of ethanol treatment. After alcohol exposure, the levels of miR-21 significantly increased by approximately 1.6-fold and 2.2fold in N-Heps and HSCs, respectively. Meanwhile, one week long-term ethanol treatment induced a 2.1-fold and 2.5-fold increase in miR-21 expression in N-Heps and HSCs respectively, which is higher than that in transfected cells. The anti-apoptotic effect of miR-21 was verified in EtOH-treated hepatocytes and HSCs by Caspase-3/7 Assay as well as flow cytometric analysis. The combined analysis revealed that the well-characterized regulator genes of apoptosis in liver biology, FASLG (Fas ligand, TNF superfamily, member 6) and DR5 (Death Receptor 5), are the direct targets of miR-21. The silencing effects on miR-21 by specific Vivo-Morpholino were verified in mice liver tissues by Taqman real-time PCR analysis. Treatment of ethanol exposed mice with miR-21 Vivo-Morpholino increased hepatic FASLG and DR5 expression. Furthermore, the serum ALT level was significantly increased after miR-21 inhibition, along with enhanced α-smooth muscle actin and Sirius red staining in ethanol-exposed mouse liver sections. Conclusion: Our findings identify a previously unrecognized mechanism for direct regulation of FASLG and DR5, involving miR-21 in ALD. The data presented here may have direct application to the future translational research for an improved diagnosis and treatment of ALD patients.
Tu1824 Efficacy and Safety of Baclofen for Alcohol Abstinence: A Meta-Analysis of Randomized Placebo Controlled Trials Habeeb Salameh, Maen M. Masadeh, Sonam Jaggi, Ashwani K. Singal Purpose: Alcohol dependence (AD) affects nearly 10% of people both in the US. Data on efficacy and safety of baclofen for alcohol abstinence (AA) in AD patients is controversial. We performed a meta-analysis of randomized studies to examine benefits and safety of baclofen in AD. Methods: Electronic databases (1980-2013) were searched to include randomized controlled trials (RCT) comparing baclofen and placebo in achieving and maintaining AA in AD patients. Study outcomes were AA proportion, AA rate, relapse rate, and mean change in obsessive compulsive drinking score (OCDS), Pennsylvania alcohol craving score (PACS), depression score and anxiety score. Pooled data on categorical variables was reported as odds ratios (OR) with 95% confidence interval (CI) and continuous variables as differences in means (pretreatment - post treatment) with 95% CI. Data were considered significant if the 95% CI on OR did not cross 1 and on mean difference did not cross 0. Random effects model was used for analyzing the pooled data. I2 and Egger's tests were used for assessing heterogeneity and publication bias respectively P value <0.1 was considered significant. Results: Five RCT were included; 2 in cirrhosis (N=108) and 3 studies excluded cirrhosis patients (N=150). RCT also differed for patient demographics and baclofen dose. Treatment duration was 12 weeks in all except one study which treated patients for only 30 days. Pooled data favored baclofen over placebo for AA proportion (76% vs. 29%; 7.5 [3.5-16.2]; P<0.0001), AA rate (4.5 [1.3-16]; P=0.02), AA duration (5.5 [4.7-6.3]; P<0.0001) and relapse rate (24% vs. 52%; 0.29 [0.12-0.74]; P=0.01). While there was significant improvement in the mean OCDS in the baclofen group (0.61 [0.23-0.99]; P=0.002), there was no significant improvement in the mean change of depression score (0.43 [-0.38 - 1.24]; P=0.30), anxiety score (2.35 [-2.1 - 6.8]; P=0.30) or PACS (0.014 [-0.43-0.45]; P=0.95). There were no differences in adverse effects with proportion of patients dropping out of the study for safety
Tu1822 Mycophenolate Mofetil (MMF) in Steroid Non-Responsive Acute Severe Alcoholic Hepatitis (Asah): A Sequentially Randomised Open Label Prospective Clinical (Mash Trial) Patrick Basu, Niraj J. Shah, Mark Aloysius, Robert S. Brown Objective: Severe alcoholic hepatitis (SAH) is a clinical challenge with high mortality. Steroids remain the SOC and are often continued even when initial response is poor. Mycophenolate mofetil (MMF) is an immunosuppressive that reduces B and T lymphocytic recruitment and immune injury. This pilot study evaluates the safety and efficacy of MMF in steroid unresponsive SAH. Methods: Eligibility: Thirty consecutive patients aged 25-60 diagnosed with steroid
AASLD Abstracts
S-1006
unresponsive SAH, with severity score criteria (DF > 32, ABCI > 12, Lille >0.45, MELD > 25) Exclusion: hepatitis A, B, C, GI bleed, sepsis, HIV Group A MMF 500 mg bid with Pentoxifylline (PTX) 400 mg and Vit D orally for 30 days. Group B Prednisolone 30 mg plus Vit D and PTX 400 mg orally 30 days. Group C Prednisolone plus MMF and PTX orally for 30 days. Results: See table Conclusion: Preliminary data suggests MMF is well tolerated, safe, and lowers MELD and inflammatory parameters. A larger randomized trial needs to elucidate the potential role of MMF in ASAH. Results
AASLD Abstracts
Tu1823 Early Activation of Peripheral Monocytes With Hallmarks of M1 and M2 Monocytic Cells in Excessive Alcohol Drinkers Crystal Walline, Janice Blum, Suthat Liangpunsakul Background: Chronic, excessive drinking can lead to the development of immune dysfunction. Following chronic alcohol exposure, human monocytes display enhanced sensitivity to bacterial LPS (lipopolysaccharides) as measured by increased cytokine secretion ex vivo. However, early biomarkers to directly detect the activation status of circulating monocytes and inflammation following chronic alcohol consumption in the absence of alcoholic liver disease (ALD) have not been well studied. Our aim is to investigate biomarkers of early immune activation from circulating peripheral blood monocytes in subjects with chronic alcohol intake in the absence of ALD. Methods: 22 chronic excessive drinkers (ED, mean age 37 years/69% men/77% Caucasian) were enrolled from Fairbanks Alcohol Treatment Center. 20 non-excessive drinkers (NED, mean age 31 years/85% men/65% Caucasian) were recruited from Roudebush VAMC. Alcohol intake was considered as categorical variable (ED/NED) using NIAAA criteria. Time Line Follow-Back (TLFB) was used to quantify the amount of alcohol consumed in the past 30 days before enrollment. Anthropometric and laboratory measurements were obtained. Peripheral blood derived CD14+ monocytes (PBM) were isolated from each subject to determine the genes associated with monocyte differentiation. In addition, serum cytokines and LPS were also measured. Results: Serum LPS concentrations were significantly higher in ED compared to NED (0.26±0.07 vs. 0.13±0.03 EU/ml, p<0.05). No differences in the levels of circulating IL-6 and IL-10 were observed. Interestingly, the relative levels of gene transcripts (RQ) for Il6 (an M1-polarizing cytokine) and Il10 (an M2-polarizing cytokine) were significantly higher in PBM from ED compared to NED (Il6: 4.3, 95% CI [2.31, 8.28] vs. 1.0, 95% CI [0.433, 2.24] p < 0.01. Il10: 2.25, 95% CI [1.66, 5.06] vs. 1.0, 95% CI [0.809, 2.23] p < 0.05). However, no differences in other M1 (Il1b, Tnf and Ccl2) and M2 monocyte differentiation markers (Mrc1 and Cd163) were detected. Conclusions: Early monitoring of peripheral blood monocyte mRNA transcripts, notably Il6 and Il10, could identify individuals with excessive alcohol drinking with early immune activation in the absence of ALD.
Tu1821 Regulation of the Extrinsic Apoptotic Pathway by MicroRNA-21 in Alcoholic Liver Injury Morgan Quezada, Kelly McDaniel, Yuyan Han, Heather L. Francis, Shannon Glaser, Julie Venter, Gianfranco Alpini, Fanyin Meng Background: microRNAs are endogenous molecules which constitute a new class of negative regulators of gene expression. New evidence identifies miR-21 as a regulatory master-switch during liver injuries. The current study aimed to characterize the functional role of miR-21 and its downstream pro-apoptotic target genes during alcoholic-induced hepatitis. Methods: miRNA expression was evaluated by qPCR array and Taqman miRNA assay in ethanol treated normal human hepatocytes (N-Hep), hepatic stellate cells (HSC) and HepG2 HCC cells as well as 5 weeks chronic alcohol-fed mouse liver specimens and control liver tissue,. Cell proliferation, survival and the transformation index were quantitated using commercial assays. The anti-miR-21 effect in ALD mice was verified by specific Vivo-Morpholino treatment. Results: The overexpression of miR-21 and miR-34a along with the altered ultrasound liver images, increased ALT level and pathology score was seen in the liver tissues from mice administered 5% (v/v) ethanol (EtOH) after five weeks. The expression of miR-21 was also increased in ethanol-treated N-Heps and HSCs with a higher apoptotic rate compared with that of controls. In N-Heps, HSCs, and HepG2 cells transfected with pre-miR-21 or anti-miR-21 inhibitor, cell survival was significantly changed compared to cells transfected with controls consisting of altered dosage and duration of ethanol treatment. After alcohol exposure, the levels of miR-21 significantly increased by approximately 1.6-fold and 2.2fold in N-Heps and HSCs, respectively. Meanwhile, one week long-term ethanol treatment induced a 2.1-fold and 2.5-fold increase in miR-21 expression in N-Heps and HSCs respectively, which is higher than that in transfected cells. The anti-apoptotic effect of miR-21 was verified in EtOH-treated hepatocytes and HSCs by Caspase-3/7 Assay as well as flow cytometric analysis. The combined analysis revealed that the well-characterized regulator genes of apoptosis in liver biology, FASLG (Fas ligand, TNF superfamily, member 6) and DR5 (Death Receptor 5), are the direct targets of miR-21. The silencing effects on miR-21 by specific Vivo-Morpholino were verified in mice liver tissues by Taqman real-time PCR analysis. Treatment of ethanol exposed mice with miR-21 Vivo-Morpholino increased hepatic FASLG and DR5 expression. Furthermore, the serum ALT level was significantly increased after miR-21 inhibition, along with enhanced α-smooth muscle actin and Sirius red staining in ethanol-exposed mouse liver sections. Conclusion: Our findings identify a previously unrecognized mechanism for direct regulation of FASLG and DR5, involving miR-21 in ALD. The data presented here may have direct application to the future translational research for an improved diagnosis and treatment of ALD patients.
Tu1824 Efficacy and Safety of Baclofen for Alcohol Abstinence: A Meta-Analysis of Randomized Placebo Controlled Trials Habeeb Salameh, Maen M. Masadeh, Sonam Jaggi, Ashwani K. Singal Purpose: Alcohol dependence (AD) affects nearly 10% of people both in the US. Data on efficacy and safety of baclofen for alcohol abstinence (AA) in AD patients is controversial. We performed a meta-analysis of randomized studies to examine benefits and safety of baclofen in AD. Methods: Electronic databases (1980-2013) were searched to include randomized controlled trials (RCT) comparing baclofen and placebo in achieving and maintaining AA in AD patients. Study outcomes were AA proportion, AA rate, relapse rate, and mean change in obsessive compulsive drinking score (OCDS), Pennsylvania alcohol craving score (PACS), depression score and anxiety score. Pooled data on categorical variables was reported as odds ratios (OR) with 95% confidence interval (CI) and continuous variables as differences in means (pretreatment - post treatment) with 95% CI. Data were considered significant if the 95% CI on OR did not cross 1 and on mean difference did not cross 0. Random effects model was used for analyzing the pooled data. I2 and Egger's tests were used for assessing heterogeneity and publication bias respectively P value <0.1 was considered significant. Results: Five RCT were included; 2 in cirrhosis (N=108) and 3 studies excluded cirrhosis patients (N=150). RCT also differed for patient demographics and baclofen dose. Treatment duration was 12 weeks in all except one study which treated patients for only 30 days. Pooled data favored baclofen over placebo for AA proportion (76% vs. 29%; 7.5 [3.5-16.2]; P<0.0001), AA rate (4.5 [1.3-16]; P=0.02), AA duration (5.5 [4.7-6.3]; P<0.0001) and relapse rate (24% vs. 52%; 0.29 [0.12-0.74]; P=0.01). While there was significant improvement in the mean OCDS in the baclofen group (0.61 [0.23-0.99]; P=0.002), there was no significant improvement in the mean change of depression score (0.43 [-0.38 - 1.24]; P=0.30), anxiety score (2.35 [-2.1 - 6.8]; P=0.30) or PACS (0.014 [-0.43-0.45]; P=0.95). There were no differences in adverse effects with proportion of patients dropping out of the study for safety
Tu1822 Mycophenolate Mofetil (MMF) in Steroid Non-Responsive Acute Severe Alcoholic Hepatitis (Asah): A Sequentially Randomised Open Label Prospective Clinical (Mash Trial) Patrick Basu, Niraj J. Shah, Mark Aloysius, Robert S. Brown Objective: Severe alcoholic hepatitis (SAH) is a clinical challenge with high mortality. Steroids remain the SOC and are often continued even when initial response is poor. Mycophenolate mofetil (MMF) is an immunosuppressive that reduces B and T lymphocytic recruitment and immune injury. This pilot study evaluates the safety and efficacy of MMF in steroid unresponsive SAH. Methods: Eligibility: Thirty consecutive patients aged 25-60 diagnosed with steroid
AASLD Abstracts
S-1006