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Enhanced Expression of Toll-Like Receptor 4 and MyD88 is Associated With Disease Progression in Human Nonalcoholic Fatty Liver Disease
Tu1696
represents a step forward and will serve as a platform for studies to enable the further optimization of terminal differentiation of IPS derived hepatocyte-like cells.
AASLD Abstracts
Enhanced Expression of Toll-Like Receptor 4 and MyD88 is Associated With Disease Progression in Human Nonalcoholic Fatty Liver Disease Tadanobu Nagaya, Naoki Tanaka, Takefumi Kimura, Michiharu Komatsu, Eiji Tanaka
Tu1884
Background/Aims: Activation of Toll-like receptors (TLRs) and their downstream genes, such as tumor necrosis factor-alpha (TNFα) and interleukin-1beta (IL1β), are known to be implicated in various liver diseases such as alcoholic liver disease and viral hepatitis. Recent studies using knockout mice reveal a major contribution of TLRs to the pathogenesis of nonalcoholic steatohepatitis (NASH). However, their roles in human NASH development have not been clarified. As such, we aimed to investigate the expression of TLRs in nonalcoholic fatty liver disease (NAFLD) using human liver samples. Methods: Ninety-eight NAFLD patients who underwent percutaneous liver biopsy in Shinshu University Hospital from 2006 to 2009 were enrolled. They were diagnosed as having simple steatosis (SS, n = 15), mild NASH (fibrosis stage 1-2, n = 59), or advanced NASH (fibrosis stage 3-4, n = 24). Liver biopsy specimens from healthy liver transplantation donors (n = 6) were also used as a control. Total RNA was extracted from biopsied liver tissues, and the mRNA levels of TLR1-9 and the related genes were assessed by quantitative polymerase chain reaction. The relationship between the mRNA levels and fibrosis stage were also examined. Results: Among TLRs, the mRNA levels of TLR4 only were significantly higher in the advanced NASH group than in the normal group and mild NASH group (P = 0.034 and 0.041, respectively). Additionally, MyD88 mRNA levels were significantly increased in the mild NASH group compared with the normal group (P = 0.032). Hepatic TLR4 levels were positively correlated with those of TNFα (r = 0.531, P < 0.001) and IL1β (r = 0.383, P < 0.019), and the MyD88 levels were also correlated with TNFα (r = 0.438, P = 0.001). Finally, the mRNA levels of TLR4 and MyD88 demonstrated significant positive correlations with fibrosis stage (r = 0.329 and 0.470, respectively; both P < 0.001). Conclusion: Increased expression of TLR4/ MyD88 may be associated with the progression of human NAFLD.
Cell Microparticles: A Novel Marker of Hepatic Inflammation and Progression in Patients With HCV or NASH Miroslaw Kornek, Michael C. Lynch, Mark A. Exley, Nezam Afdhal, Detlef Schuppan Introduction: The shedding of microparticles (MP) was shown for several cell types during activation or apoptosis In Vitro and In Vivo besides their presence in human body fluids. It has been unclear if such MP might have diagnostic or therapeutic relevance in human hepatic diseases as hepatitis C (HCV) or NASH. We therefore isolated and characterized circulating inflammatory cell MP from the plasma or serum of patients with hepatitis C (HCV) or NASH, and correlated their levels with histological and serological parameters of disease activity. Methods: MP were obtained from human blood plasma or serum samples as the fraction sedimenting between 10,000 and 100,000g and characterized/quantified by FACS analysis for the T cell markers CD3, CD4, CD8 and CD25. In addition, the presence and quantity of platelet-, monocyte /macrophage/dendritic cell- and neutrophil-derived MP was investigated by FACS for CD14, CD15 and CD41, respectively. The invariant NKT markers Vα24 and Vβ11 were used for iNKT cell-derived MP. All stainings were done in conjunction with the MP marker Annexin V. Correlation between different parameters was performed using Person's rank test (CI=95%). Results: Circulating CD3+ MP were readily detectable in human blood plasma or serum after purification by differential centrifugation. No MP profile difference was observed between plasma or serum samples from the same subject. 85% of the MP were positive for CD25, indicating that they derived from activated T cells. ALT levels in HCV (n=33) and NASH (n=49) correlated with increased levels of circulating CD8+/Annexin V double positive MP (r=0.58 (p=0.0006) and r=0.44 (p=0.031), respectively). In NASH, CD4+ MP were modest elevated compared to healthy controls, but did not correlate with ALT (r=0.09 (p=0.578)). Circulating CD8+ MP correlated significantly with histological inflammation and fibrosis in HCV (r=0.54 (p=0.007) and r=0.49 (p=0.017), respectively), while CD8+, CD14+ and iNKT MP correlated strongly with the histological NAS score (r=0.83 (p<0.0001), r=0.91 (p<0.0001) and r=0.87 (p<0.0001), respectively). Conclusions: 1. T cell and iNKT cell MP are detectable as a part of the circulating MP population in human blood plasma and serum 2. CD3+ MP are most likely released by activated T cells and reflect the CD8+ dominated pattern of hepatic inflammation in active HCV and NASH. 3. In NASH, CD14+ and iNKT cell MP showed a strong correlation with the NAS score. 4. MP profiling is a novel tool to noninvasively assess the extent and characteristics of hepatic inflammation in chronic liver diseases.
Tu1697 Significant Increase in CYP24A1 mRNA Level After 1,25-Dihydroxyvitamin D3 Treatment of Hepatocellular Carcinoma Cell Lines Evelin Horváth, Peter Lakatos, Bernadett Balla, Janos P. Kosa, Ilona Kovalszky, Ferenc Szalay Background and aims: The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-D3) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast and prostate cancers. The role of 1,25-D3 in the development of the hepatocellular carcinoma has not yet been established. The aim of the present study was to examine the gene expression of the vitamin D inactivating CYP24A1 as well as the activating 1-alpha-hydroxylase (CYP27B1) enzyme after 1,25-D3 treatment in hepatocellular carcinoma cell lines. Methods: HepG2 and HUHneo (Dept. of Molecular Virology, Univ. of Heidelberg) cells were treated with 4 nM concentrations of 1,25-D3 in Optimem medium and were incubated in 24-well plates in two parallels for treatment duration of 30, 60, 120 minutes, 5, 8, 10, 12, 14, 24, 26, 28 hours. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred ng of total RNA was reversetranscribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time PCR. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. Results: CYP24A1 mRNA expression was significantly (p<0.001) increased in response to 1,25-D3 administration in a time-dependent manner in both cell lines. In HepG2 cell line the CYP24A1 mRNA expression showed 5300-fold elevation reaching the maximum value at 8 hours. In the HUHneo cells the increase was 152-fold of the baseline level, and the curve reached the maximum at 14 hours. There was no change in CYP27B1 gene activity of the cells treated with 1,25-D3. The activating effect of vitamin D3 on CYP24 mRNA expression was at 28 hours still detectable. Conclusions: Our novel data raise the possibility that the significant increase in the CYP24A1 gene expression of hepatocellular carcinoma cell lines following vitamin D administration may stimulate the inactivation of 1,25-D3., thus “protecting” the tumor cells against the anti-cancer effects of 1,25-D3. This is the first report on the role of vitamin D3 on the CYP24A1 mRNA transcription in hepatocellular carcinoma cells In Vitro.
Tu1885 Are There Different Values of Liver Stiffness Evaluated by Means of Transient Elastography in Patients With HBV or HCV Chronic Hepatitis ? Roxana Sirli, Ioan Sporea, Diana Nicolita, Simona Bota, Alina Popescu, Mirela Danila Introduction: Liver biopsy (LB) is still considered to be the gold standard for fibrosis severity in chronic hepatitis. Liver stiffness (LS) measurement by means of Transient Elastography (TE) has been accepted for fibrosis assessment in HCV hepatitis. The aim of our study was to assess the LS values in patients with HBV chronic hepatitis and to compare them with those in patients with chronic HCV hepatitis. Patients and methods: The study included 171 patients with HBV, and 407 patients with HCV chronic hepatitis, in which LS was measured (FibroScan®-Echosens®) and LB was performed, in the same session (assessed according to the METAVIR score). In each patient 10 valid LS measurements were performed and a median value was calculated and only evaluations with at least 60% success rate and IQR<30% were considered reliable. Results: Reliable LS measurements were obtained in 166/171 (97.1%) HBV patients and in 394/407 (96.8%) HCV patients. A significant direct correlation of LS measurements with fibrosis was found in HCV patients (Spearman's r= 0.605, P<0.0001), as well as in HBV patients (r=0.392, P<0.0001). Conclusion: In our group, the mean values of LS in patients with chronic B hepatitis were similar to the ones in patients with chronic C hepatitis, for the same stage of fibrosis. LS had better predictive value in HCV than in HBV patients. Table 1: Mean LS values for different stages of fibrosis in HBV vs. HCV patients
Tu1698 Human IPS Cells Differentiate Into Hepatocyte-Like Cells With the Phenotypic and Functional Characteristics of Hepatocytes Robert Schwartz, Sangeeta Bhatia In the United States, 3 million Americans live with cirrhosis and 31,000 Americans die each year from acute and chronic causes of liver disease while 12,000 die yearly waiting for an organ for transplantation. Human embryonic stem (hES) cells and induced pluripotent stem cells (iPS) derived hepatocyte-like cells would enable cell based therapeutics, the study of the mechanisms of human disease and human development as well as provide a platform for pharmacology and toxicology drug screening. We used a protocol that has previously been shown to enable hES and iPS cells differentiation in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells not only with the morphologic and phenotypic characteristics of hepatocytes but with the functional characteristics as well. The percentage of albumin positive cells at day 20 of the differentiation was 84% and increased to 91% by day 25. At day 20, the iPS derived hepatocyte-cell cells have been shown to have functional activity as they secrete urea (~44μg/million cells/day), alpha-1-antitrypsin (~2 μg/million cells/day), and albumin (~4 μg/million cells/day) and have phase 1/2 cytochrome P450 inducible expression and activity and bile canalicular transport. At day 20, forty percent of the iPS derived hepatocyte-like cells were asialoglycoprotein receptor positive. 99% of asialoglycoprotein receptor positive cells were albumin positive by flow cytometry and these cells were magnetically isolated with positive selection. The asialoglycoprotein receptor positive fraction was enriched for liver-specific genes and cytochrome P450 expression and can be micropatterned using microscale technologies. This micropatterned system
AASLD Abstracts
Table 2: LS predictive value for the severity of fibrosis, in HBV vs. HCV patients
S-978
represents a step forward and will serve as a platform for studies to enable the further optimization of terminal differentiation of IPS derived hepatocyte-like cells.
AASLD Abstracts
Enhanced Expression of Toll-Like Receptor 4 and MyD88 is Associated With Disease Progression in Human Nonalcoholic Fatty Liver Disease Tadanobu Nagaya, Naoki Tanaka, Takefumi Kimura, Michiharu Komatsu, Eiji Tanaka
Tu1884
Background/Aims: Activation of Toll-like receptors (TLRs) and their downstream genes, such as tumor necrosis factor-alpha (TNFα) and interleukin-1beta (IL1β), are known to be implicated in various liver diseases such as alcoholic liver disease and viral hepatitis. Recent studies using knockout mice reveal a major contribution of TLRs to the pathogenesis of nonalcoholic steatohepatitis (NASH). However, their roles in human NASH development have not been clarified. As such, we aimed to investigate the expression of TLRs in nonalcoholic fatty liver disease (NAFLD) using human liver samples. Methods: Ninety-eight NAFLD patients who underwent percutaneous liver biopsy in Shinshu University Hospital from 2006 to 2009 were enrolled. They were diagnosed as having simple steatosis (SS, n = 15), mild NASH (fibrosis stage 1-2, n = 59), or advanced NASH (fibrosis stage 3-4, n = 24). Liver biopsy specimens from healthy liver transplantation donors (n = 6) were also used as a control. Total RNA was extracted from biopsied liver tissues, and the mRNA levels of TLR1-9 and the related genes were assessed by quantitative polymerase chain reaction. The relationship between the mRNA levels and fibrosis stage were also examined. Results: Among TLRs, the mRNA levels of TLR4 only were significantly higher in the advanced NASH group than in the normal group and mild NASH group (P = 0.034 and 0.041, respectively). Additionally, MyD88 mRNA levels were significantly increased in the mild NASH group compared with the normal group (P = 0.032). Hepatic TLR4 levels were positively correlated with those of TNFα (r = 0.531, P < 0.001) and IL1β (r = 0.383, P < 0.019), and the MyD88 levels were also correlated with TNFα (r = 0.438, P = 0.001). Finally, the mRNA levels of TLR4 and MyD88 demonstrated significant positive correlations with fibrosis stage (r = 0.329 and 0.470, respectively; both P < 0.001). Conclusion: Increased expression of TLR4/ MyD88 may be associated with the progression of human NAFLD.
Cell Microparticles: A Novel Marker of Hepatic Inflammation and Progression in Patients With HCV or NASH Miroslaw Kornek, Michael C. Lynch, Mark A. Exley, Nezam Afdhal, Detlef Schuppan Introduction: The shedding of microparticles (MP) was shown for several cell types during activation or apoptosis In Vitro and In Vivo besides their presence in human body fluids. It has been unclear if such MP might have diagnostic or therapeutic relevance in human hepatic diseases as hepatitis C (HCV) or NASH. We therefore isolated and characterized circulating inflammatory cell MP from the plasma or serum of patients with hepatitis C (HCV) or NASH, and correlated their levels with histological and serological parameters of disease activity. Methods: MP were obtained from human blood plasma or serum samples as the fraction sedimenting between 10,000 and 100,000g and characterized/quantified by FACS analysis for the T cell markers CD3, CD4, CD8 and CD25. In addition, the presence and quantity of platelet-, monocyte /macrophage/dendritic cell- and neutrophil-derived MP was investigated by FACS for CD14, CD15 and CD41, respectively. The invariant NKT markers Vα24 and Vβ11 were used for iNKT cell-derived MP. All stainings were done in conjunction with the MP marker Annexin V. Correlation between different parameters was performed using Person's rank test (CI=95%). Results: Circulating CD3+ MP were readily detectable in human blood plasma or serum after purification by differential centrifugation. No MP profile difference was observed between plasma or serum samples from the same subject. 85% of the MP were positive for CD25, indicating that they derived from activated T cells. ALT levels in HCV (n=33) and NASH (n=49) correlated with increased levels of circulating CD8+/Annexin V double positive MP (r=0.58 (p=0.0006) and r=0.44 (p=0.031), respectively). In NASH, CD4+ MP were modest elevated compared to healthy controls, but did not correlate with ALT (r=0.09 (p=0.578)). Circulating CD8+ MP correlated significantly with histological inflammation and fibrosis in HCV (r=0.54 (p=0.007) and r=0.49 (p=0.017), respectively), while CD8+, CD14+ and iNKT MP correlated strongly with the histological NAS score (r=0.83 (p<0.0001), r=0.91 (p<0.0001) and r=0.87 (p<0.0001), respectively). Conclusions: 1. T cell and iNKT cell MP are detectable as a part of the circulating MP population in human blood plasma and serum 2. CD3+ MP are most likely released by activated T cells and reflect the CD8+ dominated pattern of hepatic inflammation in active HCV and NASH. 3. In NASH, CD14+ and iNKT cell MP showed a strong correlation with the NAS score. 4. MP profiling is a novel tool to noninvasively assess the extent and characteristics of hepatic inflammation in chronic liver diseases.
Tu1697 Significant Increase in CYP24A1 mRNA Level After 1,25-Dihydroxyvitamin D3 Treatment of Hepatocellular Carcinoma Cell Lines Evelin Horváth, Peter Lakatos, Bernadett Balla, Janos P. Kosa, Ilona Kovalszky, Ferenc Szalay Background and aims: The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-D3) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast and prostate cancers. The role of 1,25-D3 in the development of the hepatocellular carcinoma has not yet been established. The aim of the present study was to examine the gene expression of the vitamin D inactivating CYP24A1 as well as the activating 1-alpha-hydroxylase (CYP27B1) enzyme after 1,25-D3 treatment in hepatocellular carcinoma cell lines. Methods: HepG2 and HUHneo (Dept. of Molecular Virology, Univ. of Heidelberg) cells were treated with 4 nM concentrations of 1,25-D3 in Optimem medium and were incubated in 24-well plates in two parallels for treatment duration of 30, 60, 120 minutes, 5, 8, 10, 12, 14, 24, 26, 28 hours. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred ng of total RNA was reversetranscribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time PCR. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. Results: CYP24A1 mRNA expression was significantly (p<0.001) increased in response to 1,25-D3 administration in a time-dependent manner in both cell lines. In HepG2 cell line the CYP24A1 mRNA expression showed 5300-fold elevation reaching the maximum value at 8 hours. In the HUHneo cells the increase was 152-fold of the baseline level, and the curve reached the maximum at 14 hours. There was no change in CYP27B1 gene activity of the cells treated with 1,25-D3. The activating effect of vitamin D3 on CYP24 mRNA expression was at 28 hours still detectable. Conclusions: Our novel data raise the possibility that the significant increase in the CYP24A1 gene expression of hepatocellular carcinoma cell lines following vitamin D administration may stimulate the inactivation of 1,25-D3., thus “protecting” the tumor cells against the anti-cancer effects of 1,25-D3. This is the first report on the role of vitamin D3 on the CYP24A1 mRNA transcription in hepatocellular carcinoma cells In Vitro.
Tu1885 Are There Different Values of Liver Stiffness Evaluated by Means of Transient Elastography in Patients With HBV or HCV Chronic Hepatitis ? Roxana Sirli, Ioan Sporea, Diana Nicolita, Simona Bota, Alina Popescu, Mirela Danila Introduction: Liver biopsy (LB) is still considered to be the gold standard for fibrosis severity in chronic hepatitis. Liver stiffness (LS) measurement by means of Transient Elastography (TE) has been accepted for fibrosis assessment in HCV hepatitis. The aim of our study was to assess the LS values in patients with HBV chronic hepatitis and to compare them with those in patients with chronic HCV hepatitis. Patients and methods: The study included 171 patients with HBV, and 407 patients with HCV chronic hepatitis, in which LS was measured (FibroScan®-Echosens®) and LB was performed, in the same session (assessed according to the METAVIR score). In each patient 10 valid LS measurements were performed and a median value was calculated and only evaluations with at least 60% success rate and IQR<30% were considered reliable. Results: Reliable LS measurements were obtained in 166/171 (97.1%) HBV patients and in 394/407 (96.8%) HCV patients. A significant direct correlation of LS measurements with fibrosis was found in HCV patients (Spearman's r= 0.605, P<0.0001), as well as in HBV patients (r=0.392, P<0.0001). Conclusion: In our group, the mean values of LS in patients with chronic B hepatitis were similar to the ones in patients with chronic C hepatitis, for the same stage of fibrosis. LS had better predictive value in HCV than in HBV patients. Table 1: Mean LS values for different stages of fibrosis in HBV vs. HCV patients
Tu1698 Human IPS Cells Differentiate Into Hepatocyte-Like Cells With the Phenotypic and Functional Characteristics of Hepatocytes Robert Schwartz, Sangeeta Bhatia In the United States, 3 million Americans live with cirrhosis and 31,000 Americans die each year from acute and chronic causes of liver disease while 12,000 die yearly waiting for an organ for transplantation. Human embryonic stem (hES) cells and induced pluripotent stem cells (iPS) derived hepatocyte-like cells would enable cell based therapeutics, the study of the mechanisms of human disease and human development as well as provide a platform for pharmacology and toxicology drug screening. We used a protocol that has previously been shown to enable hES and iPS cells differentiation in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells not only with the morphologic and phenotypic characteristics of hepatocytes but with the functional characteristics as well. The percentage of albumin positive cells at day 20 of the differentiation was 84% and increased to 91% by day 25. At day 20, the iPS derived hepatocyte-cell cells have been shown to have functional activity as they secrete urea (~44μg/million cells/day), alpha-1-antitrypsin (~2 μg/million cells/day), and albumin (~4 μg/million cells/day) and have phase 1/2 cytochrome P450 inducible expression and activity and bile canalicular transport. At day 20, forty percent of the iPS derived hepatocyte-like cells were asialoglycoprotein receptor positive. 99% of asialoglycoprotein receptor positive cells were albumin positive by flow cytometry and these cells were magnetically isolated with positive selection. The asialoglycoprotein receptor positive fraction was enriched for liver-specific genes and cytochrome P450 expression and can be micropatterned using microscale technologies. This micropatterned system
AASLD Abstracts
Table 2: LS predictive value for the severity of fibrosis, in HBV vs. HCV patients
S-978