- Email: [email protected]
Sa1703 High Levels of Non-Clonal Serum Immunoglobulin Free Light Chains As a Biomarker of Hepatobiliary Disorders
(MCD) diet for 8 weeks. Development of liver injury in both models was followed by intraperitoneal administration of GL or vehicle control two times a week for the last 4 weeks on the diet in MCD mice. The phenotypes of hepatic and splenic CD4+T cells and the preparation of splenic CD4+T, CD4+CD25+ and CD4+CD25-T cells were used by flow cytometry. The detection of serum PPAR- γ and T-cell cytokines were measured by ELISA. T-cell proliferation assay was tested by the thymidine method and cell apoptosis was analyzed using Annexin V/Dead Cell Apoptosis Kit. Purified splenic CD4+CD25-T cells from MCD-fed mice or GL treated MCD-fed mice were collected and co-cultured with splenic CD4+CD25-T cells from control mice. Furthermore, specific inhibitor or agonist of PPAR- γ was also added along with GL treatment to observe whether the effects of GL were dependent on PPAR- γ signaling or not. Results: GL alleviated MCD-induced liver injury by decreases in serum ALT and AST levels to near to normalization in a duration-dependent and a dose-dependent manners. GL also reduced hepatic inflammatory cell infiltrates, hepatic lipid overloading and the NAFLD activity in MCD-fed mice. GL altered the proportions of hepatic and splenic CD4+T cells and cytokines secretion and in MCD-fed mice, which showed the prevalence of Tregs and decreased proportions of Th17 cells. GL promoted the apoptosis of hepatic and splenic Th1 and Th17 cells but relatively inhibited which of Tregs. GL enhanced the level of serum PPARγ, which correlated with the enhanced proportions of serum CD4+CD25+T cells. In vitro experiment, PPAR γ signaling participated in the GL-induced proliferation of splenic nTregs. GL enhanced the inhibitory effect of splenic nTregs on Teffs, which was dependent on PPAR-γ. Furthermore, GL promoted the production of iTregs and altered the proliferation, apoptosis and cytokines secretion of iTregs. Conclusion: GL, which has excellent anti-inflammatory characteristics, ameliorates hepatic injury and inhibits hepatic lipidosiss, and may be capable of alleviating the progression of NASH. The therapeutic effects of GL partly contribute the enhanced induction and modulation of Tregs, and PPARγ signaling may be evolved in the modulatory mechanism of GL.
Sa1703 High Levels of Non-Clonal Serum Immunoglobulin Free Light Chains As a Biomarker of Hepatobiliary Disorders Alina M. Allen, W. Ray Kim, Lewis R. Roberts, Jordan Rosedahl, Angela Dispenzieri, Robert A. Kyle, Vincent S. Rajkumar, Terry M. Therneau
Sa1705 Interleukin-33-Dependent Innate Lymphoid Cells (Ilc2) Are Important Mediators of Liver Fibrosis Tamar Mchedlidze, Maximilian J. Waldner, Markus F. Neurath, Stefan J. Wirtz
Background: While monoclonal immunoglobulin free light chains (FLC) is a well-established biomarker for disease burden of multiple myeloma, non-clonal FLC levels have been proposed as a surrogate indicator for immune senescence in patients with or without hematologic disorders. Previous studies have demonstrated that serum FLC levels are associated with overall mortality in the general population. Aim: To explore the potential role of serum FLC levels as a biomarker of liver disease. Methods: The sum of κ and λ FLC (∑ FLC) was determined for a population sample of Olmsted County, Minnesota residents age > 50 years as of 1/1/1995. Subjects who were still residents in the county on 4/1/2009 were identified. The diagnostic indices of the Rochester Epidemiology Project, a well-established community record linkage system, were queried to extract all ICD codes generated from medical care between 2004- 2009. The ICD codes were summarized for analysis using Clinical Classification Software (CCS) codes. The relative diagnostic intensity, expressed as standardized incidence ratio (SIR) of each CCS code was compared between subjects above the 90th percentile of FLC and all other participants, after adjustment for age and sex. Cox regression analysis was used to determine the mortality hazard associated with high FLC levels. Results: FLC levels were available on 15,797 subjects; 9,551 of them were still Olmsted County residents on 4/1/2009. At the time of the blood draw, the median age of the subjects was 65 (50-99) years. Women accounted for 55% of the sample. The median ∑FLC was 2.8 (0.09-48.2mg/dL), with subjects with the highest value (top decile, 10%) having levels ≥ 4.71 mg/dL. After a median follow-up duration of 11.8 (0-14.3) years, the median age of the subjects was 74 (64-106) years. As shown in the Table, 4,254 County residents had at least one hepatobiliary diagnosis. In 69% of cases, a blood sample in which ∑FLC was measured preceded the diagnosis of a hepatobiliary disorder, with a median gap of 3.77 years. ∑FLC in the top decile was associated with a 13.5-fold increase in the incidence of hepatobiliary malignancy, a 5.5-fold increase in hepatitis, a 2.0-fold increase in biliary disease and a 2.6 fold increase in other liver disease. Moreover, within two hepatobiliary disorder categories, ∑FLC in the top decile was associated with increased hazard of death after adjusting for age and sex: 2.3 and 1.5 fold for patients who develop hepatobiliary malignancy and hepatitis, respectively. Conclusion: Non-clonal elevation of ∑FLC in subjects is predictive of future development of a variety of hepatobiliary diagnoses as well as mortality in those with hepatobiliary malignancy and hepatitis. FLC may have a role as a predictive biomarker of liver and biliary disease, especially liver and bile duct cancers.
Liver fibrosis is a widespread consequence of chronic liver diseases and thus a major cause of mortality and morbidity worldwide. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we report that hepatic expression of the IL-1 related cytokine interleukin33 (IL-33) was both required and sufficient for severe hepatic fibrosis in vivo in mice. We have demonstrated for the first time that the profibrotic effects of IL-33 were related to activation and expansion of a population of liver resident innate lymphoid cells (ILC2). We identified ILC2-derived Interleukin-13 (IL-13), acting through type-II IL-4 receptordependent signaling via the transcription factor STAT6 and hepatic stellate-cell activation, as a critical downstream cytokine of IL-33-dependent pathologic tissue remodeling and fibrosis. Our data reveal key immunological networks implicated in hepatic fibrosis and support the concept of modulation of IL-33 bioactivity for therapeutic purposes. Sa1706 Fine Mapping of the Ncan Locus: Evidence for Interaction Between Rs228603 Genotype, Liver Steatosis, and Locus mRNA Levels Alexis Gorden, Rongze Yang, Christopher D. Still, Glenn Gerhard, Alan R. Shuldiner INTRODUCTION The NCAN single nucleotide polymorphism (SNP) rs2228603 has been associated with non-alcoholic fatty liver disease (NAFLD). Because NCAN is not highly expressed in liver tissue, we sought to fine map the locus by examining liver mRNA levels of genes at this locus and their association with liver steatosis and rs2228603 genotype. METHODS Liver biopsies from 1,092 bariatric surgery patients were graded for steatosis and DNA from these patients were genotyped for SNP rs228603. Sixty-nine of these patients were selected and grouped according to rs2228603 genotype and liver steatosis grade: grade 0 and CC genotype (n=22), grade 0 and CT genotype (n=19), grade 3 and CC genotype (n=16), and grade 3 and CT genotype (n=12). RNA was extracted from the liver samples and RT-qPCR was performed to measure mRNA expression levels of six NCAN locus genes: GATAD2A, HAPLN4, NDUFA13, RFXANK, SF4, TM6SF2. RESULTS In CC genotype patients without NAFLD, GATAD2A mRNA levels were higher than in CT genotype patients with NAFLD (p=3x10-3). Among CC genotype patients, GATAD2A mRNA levels were lower in those with NAFLD compared to those without NAFLD (p=5.7x10-7). Furthermore, among CT genotype patients, GATAD2A mRNA levels were higher in NAFLD patients compared to those without NAFLD (p=8.5x10-4). There was no significant difference in TM6SF2 mRNA levels between CC genotype patients without NAFLD and CT genotype patients with NAFLD (p=0.058). However, among CC genotype patients, TM6SF2 mRNA levels were lower in those with NAFLD compared to those without NAFLD (p=3.0x10-3). Among CT genotype patients, there was no difference in TM6SF2 mRNA level between the NAFLD patients and the patients without NAFLD (p=0.10). For HAPLN4, NDUFA13, RFXANK, and SF4, there were no differences in mRNA levels between the 4 subgroups of patients. CONCLUSION The results suggest that NCAN SNP rs2228603 genotype may affect the way in which GATAD2A and TM6SF2 levels are associated with liver steatosis. For GATAD2A, lower levels were associated with steatosis among patients with the CC genotype, while higher gene expression levels were associated with steatosis among patients with the CT genotype. Similarly, lower TM6SF2 expression levels were associated with steatosis among patients with the CC genotype; there was only a trend toward statistical significance for the relationship higher expression levels and steatosis among patients with the CT genotype. These gene expression studies identify GATAD2A and TM6SF2 as potential contributors to liver steatosis, but they do not explain the underlying mechanism(s) whereby they promote or inhibit hepatic lipid accumulation. Functional studies will help to elucidate the role of these two genes in NAFLD pathogenesis.
Sa1704 Glycyrrhizin Alleviates Steatosis and Induces the Production of Regulatory T Cells (Tregs) That Attenuates Nonalcoholic Steatohepatitis (NASH) Progression Yun Liu, Wei Jiang Background: NASH has been confirmed as the rate-limiting step of NAFLD progress to cirrhosis. Recently, high-fat diet (HFD)-induced steatosis was associated with the depletion of hepatic Tregs. Glycyrrhizin (GL), a natural triterpene in clinical treatment of patients with chronic liver disease, can significantly reduce free fatty acid (FFA)/HFD-induced hepatic lipotoxicity. Methods: For induction of NASH, mice were fed a methionin-choline-deficient
S-955
AASLD Abstracts
AASLD Abstracts
transmitted diseases. Those with serologic antibody positivity at the time of presentation underwent confirmatory testing. Pts with and without suspected FPAs underwent routine serologic monitoring including liver associated tests and antibody testing. Only positive antibodies prior to interferon use were included to avoid errors. These pts were then evaluated to see if they lost these antibodies, and were included as the "false antibody" (FA) group. The control group consisted of patients being followed at the clinical center for acute hepatitis C. Pts were routinely followed and treated for acute hepatitis as appropriate. Results: 24 pts with acute hepatitis were evaluated (46% males, 54% white, 92% HCV, 4% HBV, 4% CMV). Of the 24 pts, 7 pts (29%) had FPAs (71% HCV, 14% HBV, 14% CMV). FPAs included HIV (1), HTLV (1), anti-smooth muscle (1), rheumatoid factor (2), RPR (1), and hepatitis E IgM (1). No pts with FPAs were infected via occupational exposure compared to 53% of the control group (p=0.009). Transaminase levels and HCV RNA quantitative measurements (in those with acute HCV infection) did not significantly differ between pts with and without FPAs. After initial detection, FPAs became undetectable after a median of 77 days (range 15 to 448). At the time of diagnosis, pts with FPAs had significantly higher mean IgM levels compared to those without FPAs (326 vs 147 mg/dL, p=0.002). However, at the time of FPA resolution mean IgM levels were no longer significant different between groups. Pts with FPA also had higher ESR levels at the time of diagnosis compared to those without FPAs (32 vs 22 mm/hr, p=0.03). Conclusions: Serologic detection of FPAs during acute viral hepatitis is likely associated higher viral inoculum as well as higher IgM levels and nonspecific markers of inflammation. This suggests a different mechanism for FPAs in acute and chronic viral hepatitis, one that may be inflammatory mediated in acute viral hepatitis. This has both mechanistic and clinical implications and should be evaluated further.
Sa1703 High Levels of Non-Clonal Serum Immunoglobulin Free Light Chains As a Biomarker of Hepatobiliary Disorders Alina M. Allen, W. Ray Kim, Lewis R. Roberts, Jordan Rosedahl, Angela Dispenzieri, Robert A. Kyle, Vincent S. Rajkumar, Terry M. Therneau
Sa1705 Interleukin-33-Dependent Innate Lymphoid Cells (Ilc2) Are Important Mediators of Liver Fibrosis Tamar Mchedlidze, Maximilian J. Waldner, Markus F. Neurath, Stefan J. Wirtz
Background: While monoclonal immunoglobulin free light chains (FLC) is a well-established biomarker for disease burden of multiple myeloma, non-clonal FLC levels have been proposed as a surrogate indicator for immune senescence in patients with or without hematologic disorders. Previous studies have demonstrated that serum FLC levels are associated with overall mortality in the general population. Aim: To explore the potential role of serum FLC levels as a biomarker of liver disease. Methods: The sum of κ and λ FLC (∑ FLC) was determined for a population sample of Olmsted County, Minnesota residents age > 50 years as of 1/1/1995. Subjects who were still residents in the county on 4/1/2009 were identified. The diagnostic indices of the Rochester Epidemiology Project, a well-established community record linkage system, were queried to extract all ICD codes generated from medical care between 2004- 2009. The ICD codes were summarized for analysis using Clinical Classification Software (CCS) codes. The relative diagnostic intensity, expressed as standardized incidence ratio (SIR) of each CCS code was compared between subjects above the 90th percentile of FLC and all other participants, after adjustment for age and sex. Cox regression analysis was used to determine the mortality hazard associated with high FLC levels. Results: FLC levels were available on 15,797 subjects; 9,551 of them were still Olmsted County residents on 4/1/2009. At the time of the blood draw, the median age of the subjects was 65 (50-99) years. Women accounted for 55% of the sample. The median ∑FLC was 2.8 (0.09-48.2mg/dL), with subjects with the highest value (top decile, 10%) having levels ≥ 4.71 mg/dL. After a median follow-up duration of 11.8 (0-14.3) years, the median age of the subjects was 74 (64-106) years. As shown in the Table, 4,254 County residents had at least one hepatobiliary diagnosis. In 69% of cases, a blood sample in which ∑FLC was measured preceded the diagnosis of a hepatobiliary disorder, with a median gap of 3.77 years. ∑FLC in the top decile was associated with a 13.5-fold increase in the incidence of hepatobiliary malignancy, a 5.5-fold increase in hepatitis, a 2.0-fold increase in biliary disease and a 2.6 fold increase in other liver disease. Moreover, within two hepatobiliary disorder categories, ∑FLC in the top decile was associated with increased hazard of death after adjusting for age and sex: 2.3 and 1.5 fold for patients who develop hepatobiliary malignancy and hepatitis, respectively. Conclusion: Non-clonal elevation of ∑FLC in subjects is predictive of future development of a variety of hepatobiliary diagnoses as well as mortality in those with hepatobiliary malignancy and hepatitis. FLC may have a role as a predictive biomarker of liver and biliary disease, especially liver and bile duct cancers.
Liver fibrosis is a widespread consequence of chronic liver diseases and thus a major cause of mortality and morbidity worldwide. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we report that hepatic expression of the IL-1 related cytokine interleukin33 (IL-33) was both required and sufficient for severe hepatic fibrosis in vivo in mice. We have demonstrated for the first time that the profibrotic effects of IL-33 were related to activation and expansion of a population of liver resident innate lymphoid cells (ILC2). We identified ILC2-derived Interleukin-13 (IL-13), acting through type-II IL-4 receptordependent signaling via the transcription factor STAT6 and hepatic stellate-cell activation, as a critical downstream cytokine of IL-33-dependent pathologic tissue remodeling and fibrosis. Our data reveal key immunological networks implicated in hepatic fibrosis and support the concept of modulation of IL-33 bioactivity for therapeutic purposes. Sa1706 Fine Mapping of the Ncan Locus: Evidence for Interaction Between Rs228603 Genotype, Liver Steatosis, and Locus mRNA Levels Alexis Gorden, Rongze Yang, Christopher D. Still, Glenn Gerhard, Alan R. Shuldiner INTRODUCTION The NCAN single nucleotide polymorphism (SNP) rs2228603 has been associated with non-alcoholic fatty liver disease (NAFLD). Because NCAN is not highly expressed in liver tissue, we sought to fine map the locus by examining liver mRNA levels of genes at this locus and their association with liver steatosis and rs2228603 genotype. METHODS Liver biopsies from 1,092 bariatric surgery patients were graded for steatosis and DNA from these patients were genotyped for SNP rs228603. Sixty-nine of these patients were selected and grouped according to rs2228603 genotype and liver steatosis grade: grade 0 and CC genotype (n=22), grade 0 and CT genotype (n=19), grade 3 and CC genotype (n=16), and grade 3 and CT genotype (n=12). RNA was extracted from the liver samples and RT-qPCR was performed to measure mRNA expression levels of six NCAN locus genes: GATAD2A, HAPLN4, NDUFA13, RFXANK, SF4, TM6SF2. RESULTS In CC genotype patients without NAFLD, GATAD2A mRNA levels were higher than in CT genotype patients with NAFLD (p=3x10-3). Among CC genotype patients, GATAD2A mRNA levels were lower in those with NAFLD compared to those without NAFLD (p=5.7x10-7). Furthermore, among CT genotype patients, GATAD2A mRNA levels were higher in NAFLD patients compared to those without NAFLD (p=8.5x10-4). There was no significant difference in TM6SF2 mRNA levels between CC genotype patients without NAFLD and CT genotype patients with NAFLD (p=0.058). However, among CC genotype patients, TM6SF2 mRNA levels were lower in those with NAFLD compared to those without NAFLD (p=3.0x10-3). Among CT genotype patients, there was no difference in TM6SF2 mRNA level between the NAFLD patients and the patients without NAFLD (p=0.10). For HAPLN4, NDUFA13, RFXANK, and SF4, there were no differences in mRNA levels between the 4 subgroups of patients. CONCLUSION The results suggest that NCAN SNP rs2228603 genotype may affect the way in which GATAD2A and TM6SF2 levels are associated with liver steatosis. For GATAD2A, lower levels were associated with steatosis among patients with the CC genotype, while higher gene expression levels were associated with steatosis among patients with the CT genotype. Similarly, lower TM6SF2 expression levels were associated with steatosis among patients with the CC genotype; there was only a trend toward statistical significance for the relationship higher expression levels and steatosis among patients with the CT genotype. These gene expression studies identify GATAD2A and TM6SF2 as potential contributors to liver steatosis, but they do not explain the underlying mechanism(s) whereby they promote or inhibit hepatic lipid accumulation. Functional studies will help to elucidate the role of these two genes in NAFLD pathogenesis.
Sa1704 Glycyrrhizin Alleviates Steatosis and Induces the Production of Regulatory T Cells (Tregs) That Attenuates Nonalcoholic Steatohepatitis (NASH) Progression Yun Liu, Wei Jiang Background: NASH has been confirmed as the rate-limiting step of NAFLD progress to cirrhosis. Recently, high-fat diet (HFD)-induced steatosis was associated with the depletion of hepatic Tregs. Glycyrrhizin (GL), a natural triterpene in clinical treatment of patients with chronic liver disease, can significantly reduce free fatty acid (FFA)/HFD-induced hepatic lipotoxicity. Methods: For induction of NASH, mice were fed a methionin-choline-deficient
S-955
AASLD Abstracts
AASLD Abstracts
transmitted diseases. Those with serologic antibody positivity at the time of presentation underwent confirmatory testing. Pts with and without suspected FPAs underwent routine serologic monitoring including liver associated tests and antibody testing. Only positive antibodies prior to interferon use were included to avoid errors. These pts were then evaluated to see if they lost these antibodies, and were included as the "false antibody" (FA) group. The control group consisted of patients being followed at the clinical center for acute hepatitis C. Pts were routinely followed and treated for acute hepatitis as appropriate. Results: 24 pts with acute hepatitis were evaluated (46% males, 54% white, 92% HCV, 4% HBV, 4% CMV). Of the 24 pts, 7 pts (29%) had FPAs (71% HCV, 14% HBV, 14% CMV). FPAs included HIV (1), HTLV (1), anti-smooth muscle (1), rheumatoid factor (2), RPR (1), and hepatitis E IgM (1). No pts with FPAs were infected via occupational exposure compared to 53% of the control group (p=0.009). Transaminase levels and HCV RNA quantitative measurements (in those with acute HCV infection) did not significantly differ between pts with and without FPAs. After initial detection, FPAs became undetectable after a median of 77 days (range 15 to 448). At the time of diagnosis, pts with FPAs had significantly higher mean IgM levels compared to those without FPAs (326 vs 147 mg/dL, p=0.002). However, at the time of FPA resolution mean IgM levels were no longer significant different between groups. Pts with FPA also had higher ESR levels at the time of diagnosis compared to those without FPAs (32 vs 22 mm/hr, p=0.03). Conclusions: Serologic detection of FPAs during acute viral hepatitis is likely associated higher viral inoculum as well as higher IgM levels and nonspecific markers of inflammation. This suggests a different mechanism for FPAs in acute and chronic viral hepatitis, one that may be inflammatory mediated in acute viral hepatitis. This has both mechanistic and clinical implications and should be evaluated further.