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Activated Complement Components Target Hepatobiliary System in Experimental Biliary Atresia
483
AASLD Abstracts
Activated Complement Components Target Hepatobiliary System in Experimental Biliary Atresia Janet Pfister, Jorge A. Bezerra, Pranavkumar Shivakumar Biliary atresia (BA) is characterized by a rapid inflammatory obstruction of bile ducts in newborn children. While cellular components of innate immunity involving NK cells are required for extrahepatic bile duct (EHBD) injury in experimental models, activation of the fluid-phase complement components remain unexplored. Based on the identification of complement genes following a genome-wide gene-expression analysis, we hypothesized that activated complement components target hepatobiliary injury in experimental biliary atresia. Methods: To test the hypothesis, we determined the expression of genes of complement activation pathways by real-time PCR in a mouse model of rotavirus (RRV)-induced biliary atresia and control animals. We used ELISA to quantify serum levels of Complement 3 (C3) and the anaphylatoxins, C3a and C5a. Immunofluorescence was used in conjunction with a cholangiocyte marker, pan-cytokeratin, to determine the deposition and localization of activated complement component C3b/iC3b/C3c (C3b) within the livers and EHBDs of control and RRV-challenged mice. Fluorescent-conjugated secondary antibodies were used to visualize the signals with DAPI staining the nuclei. Total number of C3b-positive fluorescent pixels was determined using Image-Pro Plus software. Results: Validating transcriptional footprint by real-time PCR, we found increased expression of C1qa, C1qb, C1qc, C4a, C4b, C6 and Cfb in bile ducts from virus-challenged mice at days 3, 7 and 14 after infection. Following RRV infection, serum levels of C3 initially decreased at days 3 (238.0±18.0μg/ ml; control: 493.0±38.5μg/ml; P<0.01) and 7 (202.0±54.0μg/ml; control: 743.0±19.5μg/ ml; P<0.001) and increased at day 14-post infection (1561.0±36.0μg/ml; control: 943.0±2.0μg/ml; P<0.001). Exploring whether lower levels of C3 were due to its active utilization, we found increased levels of C3a at day 7 (2567.7±53.0ng/ml; control: 2225.0±102.0ng/ml; P<0.01) and C5a at days 3 (324.0±7.5ng/ml; control: 280.0±13.0ng/ ml; P<0.01) and 7 (898.3±25.8ng/ml; control: 325.7±8.6ng/ml; P<0.001) post infection. Immunofluorescence revealed a unique localization of C3b signals to peribiliary glands in control EHBDs while in RRV-infected ducts, immunoreactive C3b signals co-localized with injured duct epithelium at early time-points, to areas of intense ductular injury at day 7, and fibrosis at day 14 after infection. In the livers, deposited C3b correlated with extent of injury, localized to portal tracts and to centrilobular regions of tissue injury but not in viable parenchyma. Conclusions: In experimental BA, complement activation 1) generated C3a and C5a capable of inducing inflammatory circuits, and 2) deposited C3b specifically localizing to injured intra- and extrahepatic bile ducts. Targeting complement activation may thus be a viable approach to limit hepatobiliary injury in biliary atresia.
485 Alanine Aminotransferase (ALT) Elevation in Obese Children and Adolescents: A Predictor of Insulin Resistance and Possible Precursor to Metabolic Syndrome Emily J. Rothbaum Perito, Robert H. Lustig, Nathan M. Bass Introduction: ALT is recommended as initial screening for non-alcoholic fatty liver disease (NAFLD) in children. Schwimmer et al. (2010) identified the 95th percentile (%tile) of ALT in the NHANES sample of healthy children as 25.8 U/L in boys and 22.1 U/L in girls. ALT values of 40-60 U/L commonly trigger further NAFLD workup but may miss a significant proportion of NAFLD in children. Methods: Baseline data from children ages 2-18 years presenting to a multidisciplinary clinic for obesity was analyzed. Using t-tests and multivariate logistic regression, we tested the hypothesis that ALT above the NHANES 95th%tile predicted insulin resistance and components of the metabolic syndrome. Results: Analysis included 449 obese children with mean age 11.8 years (SD 3.9). Subjects with ALT>108 U/L for girls or >118 U/L for boys, known liver disease, on medications associated with hepatic steatosis, or with incomplete data were excluded. 63% of boys and 47% of girls had ALT>95th%ile. Components of metabolic syndrome associated with ALT> 95th%ile differed by gender (Table 1). In multivariate analysis accounting for gender, age, BMI z-score, Latino ethnicity and African-American race, ALT>95th%ile remained an independent predictor of insulin resistance, as measured by HOMA-IR>4.3 (OR 2.6, p<0.0005, 95% CI 1.6-4.2). Age and BMI z-score were also statistically significant predictors of insulin resistance in this model (Age: OR 1.4, p<0.0005, 95% CI 1.3-1.5. BMI z-score: OR 3.2, p<0.0005, 95% CI 1.95.3). Latinos with insulin resistance had higher mean ALT than African-Americans with insulin resistance (40 vs. 27, p=0.004) with no significant differences in BMI z-scores. Among children without insulin resistance, Latinos still had higher mean ALT than AfricanAmericans (28 vs. 21, p=0.007) but no difference in BMI z-scores. ALT>95%ile did not independently predict metabolic syndrome or its other components in multivariate models. Conclusions: In this population at high NAFLD risk, ALT>95th%ile is associated with insulin resistance in all age and ethnicity groups examined. ALT is a common but imperfect proxy for NAFLD in patients without other liver disease. Longitudinal studies of obese children are needed to better characterize cutoffs for ALT elevation, to examine racial/ethnic differences, and to investigate whether NAFLD and insulin resistance may develop as precursors to metabolic syndrome in children. Components of metabolic syndrome in obese children, with ALT>NHANES 95th percentile as NAFLD proxy*
484 Hedgehog Signaling is Innately Active During Infancy and Causes Ductular Dysmorphogenesis by Priming Ductular Cells to Arrest Epithelial Differentiation in Response to Different Types of Biliary Injury Alessia Omenetti, Lee M. Bass, Cynthia D. Guy, Peter F. Whitington, Anna Mae Diehl Background Accumulation of immature ductular cells and stromal elements occurs transiently during hepatobiliary development. Growth of the intrahepatic biliary system then continues after birth, reaching full maturation only at adolescence. Hedgehog (Hh) signaling is known to play key roles in development and adult tissue remodeling, but its significance in early childhood has never been explored. When overly active, Hh signaling usually inhibits epithelial differentiation and causes Hh-responsive cells to retain more mesenchymal features. We hypothesised that Hh signaling may be innately active in the process of organogenesis during infancy, priming ductular cells to arrest epithelial differentiation in response to different types of biliary injury, resulting in ductular dysmorphogenesis. Methods Whole liver tissue mRNA and immunostained liver sections from non diseased (ND) children (N= 6) were compared to those of adult ND subjects (N=5) or age-matched patients with different diseases for which ductular paucity and dysmorphogenesis is notable: biliary atresia (BA, N=8) Alagille Syndrome (AGS, N=5), Progressive Intrahepatic Familial Cholestasis (PFIC) type 1 (FIC1, N=7) and 2 (BSEP, N=6) (median age 20 months). QRTPCR was performed to test differential expression in Hh ligands/target genes and mesenchymal markers. The significance of gene expression differences was evaluated by Wilcoxon rank-sum test; correlation and linear regression analyses were performed to evaluate covariances. Immunohistochemistry (IHC) was done to evaluate the cells producing and responsive to Hh signaling. CoIHC of Hh target gene and immature epithelial (KT7, AE1/AE3) or mesenchymal (Vimentin, Fibroblast-specific protein-FSP1) markers was assessed to define the maturation state of Hhresponsive cells. Results ND children displayed more Hh-responsive ductular cells than ND adults. Livers of children with all 4 types of biliary disease exhibited striking upregulation of Shh ligand, Hh target genes, and mesenchymal markers compared to ND agematched controls. Covariance analyses between the Hh target genes and Vimentin (Table 1) revealed differential patterns among groups, with strongest Hh-correlated Vimentin expression in more fibrogenic diseases. In all diseases, double-IHC constantly revealed that Hhresponsive ductular cells retained both immature epithelial and mesenchimal features. Such cells often appeared as oval cell-like cells; occasional myofibroblast-like cells expressing epithelial keratins were also noted. Conclusions Neonatal livers are enriched with Hh responsive ductular cells. Hence, Hh ligands which are produced in response to diverse causes of biliary injury trigger a self-perpetuating stimulus that arrests ductular epithelial maturation, promotes accumulation of dysmorphic, immature ductular structures, and causes progressive biliary fibrosis. Table 1
AASLD Abstracts
* Measurements from initial clinic visit, fasting samples. Mean (SD) unless otherwise indicated.**P-values refer to t-test for age, BMI, SBP, DBP, glucose; Mann-Whitney for insulin, HOMA-IR, triglycerides; Chi-squared for Latino, insulin resistance, and metabolic syndrome.***”NS” p-values>0.05. ****Includes 124 females, 198 males with complete data.
S-892
AASLD Abstracts
Activated Complement Components Target Hepatobiliary System in Experimental Biliary Atresia Janet Pfister, Jorge A. Bezerra, Pranavkumar Shivakumar Biliary atresia (BA) is characterized by a rapid inflammatory obstruction of bile ducts in newborn children. While cellular components of innate immunity involving NK cells are required for extrahepatic bile duct (EHBD) injury in experimental models, activation of the fluid-phase complement components remain unexplored. Based on the identification of complement genes following a genome-wide gene-expression analysis, we hypothesized that activated complement components target hepatobiliary injury in experimental biliary atresia. Methods: To test the hypothesis, we determined the expression of genes of complement activation pathways by real-time PCR in a mouse model of rotavirus (RRV)-induced biliary atresia and control animals. We used ELISA to quantify serum levels of Complement 3 (C3) and the anaphylatoxins, C3a and C5a. Immunofluorescence was used in conjunction with a cholangiocyte marker, pan-cytokeratin, to determine the deposition and localization of activated complement component C3b/iC3b/C3c (C3b) within the livers and EHBDs of control and RRV-challenged mice. Fluorescent-conjugated secondary antibodies were used to visualize the signals with DAPI staining the nuclei. Total number of C3b-positive fluorescent pixels was determined using Image-Pro Plus software. Results: Validating transcriptional footprint by real-time PCR, we found increased expression of C1qa, C1qb, C1qc, C4a, C4b, C6 and Cfb in bile ducts from virus-challenged mice at days 3, 7 and 14 after infection. Following RRV infection, serum levels of C3 initially decreased at days 3 (238.0±18.0μg/ ml; control: 493.0±38.5μg/ml; P<0.01) and 7 (202.0±54.0μg/ml; control: 743.0±19.5μg/ ml; P<0.001) and increased at day 14-post infection (1561.0±36.0μg/ml; control: 943.0±2.0μg/ml; P<0.001). Exploring whether lower levels of C3 were due to its active utilization, we found increased levels of C3a at day 7 (2567.7±53.0ng/ml; control: 2225.0±102.0ng/ml; P<0.01) and C5a at days 3 (324.0±7.5ng/ml; control: 280.0±13.0ng/ ml; P<0.01) and 7 (898.3±25.8ng/ml; control: 325.7±8.6ng/ml; P<0.001) post infection. Immunofluorescence revealed a unique localization of C3b signals to peribiliary glands in control EHBDs while in RRV-infected ducts, immunoreactive C3b signals co-localized with injured duct epithelium at early time-points, to areas of intense ductular injury at day 7, and fibrosis at day 14 after infection. In the livers, deposited C3b correlated with extent of injury, localized to portal tracts and to centrilobular regions of tissue injury but not in viable parenchyma. Conclusions: In experimental BA, complement activation 1) generated C3a and C5a capable of inducing inflammatory circuits, and 2) deposited C3b specifically localizing to injured intra- and extrahepatic bile ducts. Targeting complement activation may thus be a viable approach to limit hepatobiliary injury in biliary atresia.
485 Alanine Aminotransferase (ALT) Elevation in Obese Children and Adolescents: A Predictor of Insulin Resistance and Possible Precursor to Metabolic Syndrome Emily J. Rothbaum Perito, Robert H. Lustig, Nathan M. Bass Introduction: ALT is recommended as initial screening for non-alcoholic fatty liver disease (NAFLD) in children. Schwimmer et al. (2010) identified the 95th percentile (%tile) of ALT in the NHANES sample of healthy children as 25.8 U/L in boys and 22.1 U/L in girls. ALT values of 40-60 U/L commonly trigger further NAFLD workup but may miss a significant proportion of NAFLD in children. Methods: Baseline data from children ages 2-18 years presenting to a multidisciplinary clinic for obesity was analyzed. Using t-tests and multivariate logistic regression, we tested the hypothesis that ALT above the NHANES 95th%tile predicted insulin resistance and components of the metabolic syndrome. Results: Analysis included 449 obese children with mean age 11.8 years (SD 3.9). Subjects with ALT>108 U/L for girls or >118 U/L for boys, known liver disease, on medications associated with hepatic steatosis, or with incomplete data were excluded. 63% of boys and 47% of girls had ALT>95th%ile. Components of metabolic syndrome associated with ALT> 95th%ile differed by gender (Table 1). In multivariate analysis accounting for gender, age, BMI z-score, Latino ethnicity and African-American race, ALT>95th%ile remained an independent predictor of insulin resistance, as measured by HOMA-IR>4.3 (OR 2.6, p<0.0005, 95% CI 1.6-4.2). Age and BMI z-score were also statistically significant predictors of insulin resistance in this model (Age: OR 1.4, p<0.0005, 95% CI 1.3-1.5. BMI z-score: OR 3.2, p<0.0005, 95% CI 1.95.3). Latinos with insulin resistance had higher mean ALT than African-Americans with insulin resistance (40 vs. 27, p=0.004) with no significant differences in BMI z-scores. Among children without insulin resistance, Latinos still had higher mean ALT than AfricanAmericans (28 vs. 21, p=0.007) but no difference in BMI z-scores. ALT>95%ile did not independently predict metabolic syndrome or its other components in multivariate models. Conclusions: In this population at high NAFLD risk, ALT>95th%ile is associated with insulin resistance in all age and ethnicity groups examined. ALT is a common but imperfect proxy for NAFLD in patients without other liver disease. Longitudinal studies of obese children are needed to better characterize cutoffs for ALT elevation, to examine racial/ethnic differences, and to investigate whether NAFLD and insulin resistance may develop as precursors to metabolic syndrome in children. Components of metabolic syndrome in obese children, with ALT>NHANES 95th percentile as NAFLD proxy*
484 Hedgehog Signaling is Innately Active During Infancy and Causes Ductular Dysmorphogenesis by Priming Ductular Cells to Arrest Epithelial Differentiation in Response to Different Types of Biliary Injury Alessia Omenetti, Lee M. Bass, Cynthia D. Guy, Peter F. Whitington, Anna Mae Diehl Background Accumulation of immature ductular cells and stromal elements occurs transiently during hepatobiliary development. Growth of the intrahepatic biliary system then continues after birth, reaching full maturation only at adolescence. Hedgehog (Hh) signaling is known to play key roles in development and adult tissue remodeling, but its significance in early childhood has never been explored. When overly active, Hh signaling usually inhibits epithelial differentiation and causes Hh-responsive cells to retain more mesenchymal features. We hypothesised that Hh signaling may be innately active in the process of organogenesis during infancy, priming ductular cells to arrest epithelial differentiation in response to different types of biliary injury, resulting in ductular dysmorphogenesis. Methods Whole liver tissue mRNA and immunostained liver sections from non diseased (ND) children (N= 6) were compared to those of adult ND subjects (N=5) or age-matched patients with different diseases for which ductular paucity and dysmorphogenesis is notable: biliary atresia (BA, N=8) Alagille Syndrome (AGS, N=5), Progressive Intrahepatic Familial Cholestasis (PFIC) type 1 (FIC1, N=7) and 2 (BSEP, N=6) (median age 20 months). QRTPCR was performed to test differential expression in Hh ligands/target genes and mesenchymal markers. The significance of gene expression differences was evaluated by Wilcoxon rank-sum test; correlation and linear regression analyses were performed to evaluate covariances. Immunohistochemistry (IHC) was done to evaluate the cells producing and responsive to Hh signaling. CoIHC of Hh target gene and immature epithelial (KT7, AE1/AE3) or mesenchymal (Vimentin, Fibroblast-specific protein-FSP1) markers was assessed to define the maturation state of Hhresponsive cells. Results ND children displayed more Hh-responsive ductular cells than ND adults. Livers of children with all 4 types of biliary disease exhibited striking upregulation of Shh ligand, Hh target genes, and mesenchymal markers compared to ND agematched controls. Covariance analyses between the Hh target genes and Vimentin (Table 1) revealed differential patterns among groups, with strongest Hh-correlated Vimentin expression in more fibrogenic diseases. In all diseases, double-IHC constantly revealed that Hhresponsive ductular cells retained both immature epithelial and mesenchimal features. Such cells often appeared as oval cell-like cells; occasional myofibroblast-like cells expressing epithelial keratins were also noted. Conclusions Neonatal livers are enriched with Hh responsive ductular cells. Hence, Hh ligands which are produced in response to diverse causes of biliary injury trigger a self-perpetuating stimulus that arrests ductular epithelial maturation, promotes accumulation of dysmorphic, immature ductular structures, and causes progressive biliary fibrosis. Table 1
AASLD Abstracts
* Measurements from initial clinic visit, fasting samples. Mean (SD) unless otherwise indicated.**P-values refer to t-test for age, BMI, SBP, DBP, glucose; Mann-Whitney for insulin, HOMA-IR, triglycerides; Chi-squared for Latino, insulin resistance, and metabolic syndrome.***”NS” p-values>0.05. ****Includes 124 females, 198 males with complete data.
S-892