Oligoclonal immunoglobulin repertoire in biliary remnants of biliary atresia.

Biliary atresia (BA) is a neonatal cholestatic liver disease that is the leading cause of pediatric liver transplantation, however, the mechanism of disease remains unknown. There are two major forms of BA: isolated BA (iBA) comprises the majority of cases and is thought to result from an aberrant immune response to an environmental trigger, whereas syndromic BA (BASM) has associated malformations and is thought to arise from a congenital insult. To determine whether B cells in BA biliary remnants are antigen driven, we examined the immunoglobulin (Ig) repertoire of diseased tissue from each BA group. Deep sequencing of the Ig chain DNA was performed on iBA and BASM biliary remnants and lymph nodes obtained from the Childhood Liver Disease Research Network (ChiLDReN) repository. Statistical analysis of the Ig repertoire provided measures of Ig clonality and the Ig phenotype. Our data demonstrate that B cells infiltrate diseased iBA and BASM biliary remnant tissue. The Ig repertoires of iBA and BASM disease groups were oligoclonal supporting a role for an antigen-driven immune response in both sub-types. These findings shift the current understanding of BA and suggest a role for antigen stimulation in early iBA and BASM disease pathogenesis.

Outcomes of surgical management of familial intrahepatic cholestasis 1 and bile salt export protein deficiencies.

Progressive familial intrahepatic cholestasis (PFIC) with normal circulating gamma-glutamyl transpeptidase levels can result from mutations in the ATP8B1 gene (encoding familial intrahepatic cholestasis 1 [FIC1] deficiency) or the ABCB11 gene (bile salt export protein [BSEP] deficiency). We investigated the outcomes of partial external biliary diversion, ileal exclusion, and liver transplantation in these two conditions. We conducted a retrospective multicenter study of 42 patients with FIC1 deficiency (FIC1 patients) and 60 patients with BSEP deficiency (BSEP patients) who had undergone one or more surgical procedures (57 diversions, 6 exclusions, and 57 transplants). For surgeries performed prior to transplantation, BSEP patients were divided into two groups, BSEP-common (bearing common missense mutations D482G or E297G, with likely residual function) and BSEP-other. We evaluated clinical and biochemical outcomes in these patients. Overall, diversion improved biochemical parameters, pruritus, and growth, with substantial variation in individual response. BSEP-common or FIC1 patients survived longer after diversion without developing cirrhosis, being listed for or undergoing liver transplantation, or dying, compared to BSEP-other patients. Transplantation resolved cholestasis in all groups. However, FIC1 patients commonly developed hepatic steatosis, diarrhea, and/or pancreatic disease after transplant accompanied by biochemical abnormalities and often had continued poor growth. In BSEP patients with impaired growth, this generally improved after transplantation. Conclusion: Diversion can improve clinical and biochemical status in FIC1 and BSEP deficiencies, but outcomes differ depending on genetic etiology. For many patients, particularly BSEP-other, diversion is not a permanent solution and transplantation is required. Although transplantation resolves cholestasis in patients with FIC1 and BSEP deficiencies, the overall outcome remains unsatisfactory in many FIC1 patients; this is mainly due to extrahepatic manifestations. (Hepatology Communications 2018;2:515-528).

Indeterminate pediatric acute liver failure is uniquely characterized by a CD103+ CD8+ T-cell infiltrate.

The cause of pediatric acute liver failure (PALF) is unknown in up to 40% of cases. Evidence suggests that aberrant immune system activation may play a role. We hypothesized that indeterminate PALF cases would exhibit a unique pattern of hepatic inflammation. This was a retrospective and prospective study of PALF cases due to indeterminate (iPALF), autoimmune hepatitis, or known diagnosis (dPALF) etiology. Liver tissue sections were stained with immunohistochemical markers for cytotoxic T-cells (cluster of differentiation 8 [CD8]), perforin, and tissue resident memory T-cells (CD103) and scored as minimal, moderate, or dense. Lymphocytes were isolated from liver tissue for T-cell receptor beta sequencing and flow-cytometric studies. Thirty-three iPALF, 9 autoimmune hepatitis, and 14 dPALF cases were included. Dense hepatic infiltrates of CD8+ T-cells were found in 27 (82%) iPALF cases compared to 1 (7%) dPALF case (P < 0.0001). Perforin staining was dense or moderate in 19 (73%) of 26 iPALF cases compared to minimal in all 7 dPALF cases (P = 0.004); 16 (62%) of 26 iPALF cases had dense CD103 staining compared to none of the 6 dPALF cases (P = 0.001). T-cell receptor beta sequencing of iPALF cases demonstrated increased clonality compared to dPALF and control cases. Flow cytometry and immunohistochemistry revealed that iPALF intrahepatic leukocytes were predominantly tissue resident memory CD8+ T-cells. CONCLUSION: Indeterminate PALF is characterized by a dense CD8+ T-cell hepatic infiltrate consistent with expansion of a tissue resident memory T-cell phenotype; CD8+ T-cells are a biomarker of immune dysregulation in iPALF and may be used to better identify and define this group. (Hepatology 2018).

The Effects of Gestational Alloimmune Liver Disease on Fetal and Infant Morbidity and Mortality.

OBJECTIVES: To evaluate pregnancy outcomes in pedigrees of neonatal hemochromatosis to determine the spectrum of gestational alloimmune liver disease (GALD) in a large cohort. STUDY DESIGN: We prospectively collected data from women with a prior offspring with proven neonatal hemochromatosis between 1997 and 2015 and analyzed pregnancy outcomes. RESULTS: The pedigrees from 150 women included 350 gestations with outcomes potentially related to GALD. There were 105 live-born infants without liver disease, 157 live-born infants with liver failure, and 88 fetal losses. Fetal loss occurred in 25% of total gestations. Ninety-seven pedigrees contained a single affected offspring, whereas 53 contained multiple affected offspring. Analysis of these 53 pedigrees yielded a per-pregnancy repeat occurrence rate of 95%. Notably, the first poor outcome occurred in the first pregnancy in 60% of pedigrees. Outcomes of the 157 live-born infants with liver failure were poor: 18% survived, 82% died. Of the 134 live-born infants with treatment data, 20 received intravenous immunoglobulin with or without double-volume exchange transfusion of which 9 (45%) survived; 14 infants (10%) received a liver transplant of which 6 (43%) survived. CONCLUSIONS: GALD is a significant cause of both fetal loss and neonatal mortality with a high rate of disease recurrence in untreated pregnancies at risk. Poor outcomes related to GALD commonly occur in the first gestation, necessitating a high index of suspicion to diagnose this disorder at first presentation.

Hepatic Histology and Morphometric Measurements in Idiopathic Extrahepatic Portal Vein Thrombosis in Children, Correlated to Clinical Outcome of Meso-Rex Bypass.

OBJECTIVE: The aim of this study was to correlate clinical, histologic, and morphometric features of the liver in children with extrahepatic portal vein thrombosis (EHPVT), with surgical outcome after Meso-Rex bypass (MRB). BACKGROUND: Idiopathic EHPVT, a significant cause of portal hypertension, is surgically corrected by MRB. Correlation of histologic and morphometric features of the liver with outcome has not been reported in children. METHODS: We retrospectively reviewed clinical and intraoperative data of 45 children with idiopathic EHPVT. Liver samples were obtained at the time of MRB. Morphometric measurements of portal tract structures were performed and correlated with surgical outcome. Median follow-up was 3.65 years after surgery (range 1.5 to 10 years). RESULTS: Thirty-seven (82.2%) children had successful MRB. There was no association between age, sex, and suture material with surgical outcome. Average patient age was higher in patients with postoperative complications (P = NS). Portal fibrosis, bridging, parenchymal nodules, portal inflammation, hepatocellular swelling, steatosis, dilatation of portal lymphatics, and periductal fibrosis did not show a significant difference between the 2 groups. Portal vein and bile duct area index were significantly smaller in the unsuccessful group (P = 0.004 and 0.003, respectively). A portal vein area index <0.08 had a lower chance of successful surgical outcome. Hepatic artery area index was not significantly different. Measured intraoperative portal blood inflow was the only significant clinical factor affecting surgical outcome (P = 0.0003). CONCLUSIONS: Low portal vein area index and intraoperative portal blood inflow may be negative prognostic factors for MRB outcome in children with idiopathic EHPVT. Average patient age was higher, although not statistically significant, in patients with postoperative complications.

Antenatal Treatment with Intravenous Immunoglobulin to Prevent Gestational Alloimmune Liver Disease: Comparative Effectiveness of 14-Week versus 18-Week Initiation.

BACKGROUND: Antenatal therapy with high-dose intravenous immunoglobulin (IVIG) may prevent gestational alloimmune liver disease (GALD). OBJECTIVE: The objective of this study was to determine the effectiveness of this approach in a large cohort of women at risk for poor pregnancy outcome due to GALD. METHODS: Women with a history of affected offspring were provided antenatal IVIG treatment and data were acquired prospectively from 1997 to 2015. The outcomes of treated pregnancies were compared to those of untreated pregnancies, and the effectiveness of starting at 14 weeks was compared to that of starting at 18 weeks. RESULTS: A total of 188 treated pregnancies in 151 women were analyzed. Only 30% (n = 105) of untreated gestations resulted in healthy offspring as compared to 94% (n = 177) of treated pregnancies (p < 0.0001). Treated gestations of both the 14-week (n = 108) and the 18-week (n = 80) start cohort showed a decreased rate of fetal loss relative to untreated gestations (p < 0.0001). Equivalent outcomes were recorded in the 18-week versus the 14-week start cohort (p > 0.05). Few adverse events or complications of antenatal therapy were recorded. CONCLUSION: Antenatal therapy with high-dose IVIG initiated at either 18 or 14 gestational weeks effectively prevents poor outcome of pregnancies at risk for GALD.

In Children With Nonalcoholic Fatty Liver Disease, Zone 1 Steatosis Is Associated With Advanced Fibrosis.

BACKGROUND & AIMS: Focal zone 1 steatosis, although rare in adults with nonalcoholic fatty liver disease (NAFLD), does occur in children with NAFLD. We investigated whether focal zone 1 steatosis and focal zone 3 steatosis are distinct subphenotypes of pediatric NAFLD. We aimed to determine associations between the zonality of steatosis and demographic, clinical, and histologic features in children with NAFLD. METHODS: We performed a cross-sectional study of baseline data from 813 children (age <18 years; mean age, 12.8 ± 2.7 years). The subjects had biopsy-proven NAFLD and were enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network. Liver histology was reviewed using the Nonalcoholic Steatohepatitis Clinical Research Network scoring system. RESULTS: Zone 1 steatosis was present in 18% of children with NAFLD (n = 146) and zone 3 steatosis was present in 32% (n = 244). Children with zone 1 steatosis were significantly younger (10 vs 14 years; P < .001) and a significantly higher proportion had any fibrosis (81% vs 51%; P < .001) or advanced fibrosis (13% vs 5%; P < .001) compared with children with zone 3 steatosis. In contrast, children with zone 3 steatosis were significantly more likely to have steatohepatitis (30% vs 6% in children with zone 1 steatosis; P < .001). CONCLUSIONS: Children with zone 1 or zone 3 distribution of steatosis have an important subphenotype of pediatric NAFLD. Children with zone 1 steatosis are more likely to have advanced fibrosis and children with zone 3 steatosis are more likely to have steatohepatitis. To achieve a comprehensive understanding of pediatric NAFLD, studies of pathophysiology, natural history, and response to treatment should account for the zonality of steatosis.

Initial assessment of the infant with neonatal cholestasis-Is this biliary atresia?

INTRODUCTION: Optimizing outcome in biliary atresia (BA) requires timely diagnosis. Cholestasis is a presenting feature of BA, as well as other diagnoses (Non-BA). Identification of clinical features of neonatal cholestasis that would expedite decisions to pursue subsequent invasive testing to correctly diagnose or exclude BA would enhance outcomes. The analytical goal was to develop a predictive model for BA using data available at initial presentation. METHODS: Infants at presentation with neonatal cholestasis (direct/conjugated bilirubin >2 mg/dl [34.2 µM]) were enrolled prior to surgical exploration in a prospective observational multi-centered study (PROBE-NCT00061828). Clinical features (physical findings, laboratory results, gallbladder sonography) at enrollment were analyzed. Initially, 19 features were selected as candidate predictors. Two approaches were used to build models for diagnosis prediction: a hierarchical classification and regression decision tree (CART) and a logistic regression model using a stepwise selection strategy. RESULTS: In PROBE April 2004-February 2014, 401 infants met criteria for BA and 259 for Non-BA. Univariate analysis identified 13 features that were significantly different between BA and Non-BA. Using a CART predictive model of BA versus Non-BA (significant factors: gamma-glutamyl transpeptidase, acholic stools, weight), the receiver operating characteristic area under the curve (ROC AUC) was 0.83. Twelve percent of BA infants were misclassified as Non-BA; 17% of Non-BA infants were misclassified as BA. Stepwise logistic regression identified seven factors in a predictive model (ROC AUC 0.89). Using this model, a predicted probability of >0.8 (n = 357) yielded an 81% true positive rate for BA; <0.2 (n = 120) yielded an 11% false negative rate. CONCLUSION: Despite the relatively good accuracy of our optimized prediction models, the high precision required for differentiating BA from Non-BA was not achieved. Accurate identification of BA in infants with neonatal cholestasis requires further evaluation, and BA should not be excluded based only on presenting clinical features.

MYO5B mutations cause cholestasis with normal serum gamma-glutamyl transferase activity in children without microvillous inclusion disease.

Some patients with microvillus inclusion disease due to myosin 5B (MYO5B) mutations may develop cholestasis characterized by a progressive familial intrahepatic cholestasis-like phenotype with normal serum gamma-glutamyl transferase activity. So far MYO5B deficiency has not been reported in patients with such a cholestasis phenotype in the absence of intestinal disease. Using a new-generation sequencing approach, we identified MYO5B mutations in five patients with progressive familial intrahepatic cholestasis-like phenotype with normal serum gamma-glutamyl transferase activity without intestinal disease. CONCLUSION: These data show that MYO5B deficiency may lead to isolated cholestasis and that MYO5B should be considered as an additional progressive familial intrahepatic cholestasis gene. (Hepatology 2017;65:164-173).

Infantile Cirrhosis, Growth Impairment, and Neurodevelopmental Anomalies Associated with Deficiency of PPP1R15B.

OBJECTIVE: To assess the utility of whole-exome sequencing (WES) in a sibling pair with undetermined liver disease and describe the phenotype associated with mutations discovered therein. STUDY DESIGN: Next-generation WES was performed on 2 siblings (S1 and S2) who were born to nonconsanguineous parents of European extraction. Both siblings developed cirrhosis of indeterminate etiology and required liver transplantation; S1 at 7 months and S2 at 22 months. RESULTS: Sequencing of germline DNA identified compound heterozygous mutations in PPP1R15B resulting in increased levels of phosphorylated eukaryotic translation initiation factor 2α. CONCLUSIONS: The first demonstration of PPP1R15B associated with liver disease expands the phenotypic spectrum of PPP1R15B related diseases. Our findings validate the application of WES in the diagnosis of children with undetermined liver disease. Understanding the genetic basis of liver disease may allow the development of targeted therapies for treatment and adequate counseling of families.

In Children With Nonalcoholic Fatty Liver Disease, Cysteamine Bitartrate Delayed Release Improves Liver Enzymes but Does Not Reduce Disease Activity Scores.

BACKGROUND & AIMS: No treatment for nonalcoholic fatty liver disease (NAFLD) has been approved by regulatory agencies. We performed a randomized controlled trial to determine whether 52 weeks of cysteamine bitartrate delayed release (CBDR) reduces the severity of liver disease in children with NAFLD. METHODS: We performed a double-masked trial of 169 children with NAFLD activity scores of 4 or higher at 10 centers. From June 2012 to January 2014, the patients were assigned randomly to receive CBDR or placebo twice daily (300 mg for patients weighing ≤65 kg, 375 mg for patients weighing >65 to 80 kg, and 450 mg for patients weighing >80 kg) for 52 weeks. The primary outcome from the intention-to-treat analysis was improvement in liver histology over 52 weeks, defined as a decrease in the NAFLD activity score of 2 points or more without worsening fibrosis; patients without biopsy specimens from week 52 (17 in the CBDR group and 6 in the placebo group) were considered nonresponders. We calculated the relative risks (RR) of improvement using a stratified Cochran-Mantel-Haenszel analysis. RESULTS: There was no significant difference between groups in the primary outcome (28% of children in the CBDR group vs 22% in the placebo group; RR, 1.3; 95% confidence interval [CI], 0.8-2.1; P = .34). However, children receiving CBDR had significant changes in prespecified secondary outcomes: reduced mean levels of alanine aminotransferase (reduction, 53 ± 88 U/L vs 8 ± 77 U/L in the placebo group; P = .02) and aspartate aminotransferase (reduction, 31 ± 52 vs 4 ± 36 U/L in the placebo group; P = .008), and a larger proportion had reduced lobular inflammation (36% in the CBDR group vs 21% in the placebo group; RR, 1.8; 95% CI, 1.1-2.9; P = .03). In a post hoc analysis of children weighing 65 kg or less, those taking CBDR had a 4-fold better chance of histologic improvement (observed in 50% of children in the CBDR group vs 13% in the placebo group; RR, 4.0; 95% CI, 1.3-12.3; P = .005). CONCLUSIONS: In a randomized trial, we found that 1 year of CBDR did not reduce overall histologic markers of NAFLD compared with placebo in children. Children receiving CBDR, however, had significant reductions in serum aminotransferase levels and lobular inflammation. ClinicalTrials.gov no: NCT01529268.

Biliary atresia and other cholestatic childhood diseases: Advances and future challenges.

Biliary Atresia and other cholestatic childhood diseases are rare conditions affecting the function and/or anatomy along the canalicular-bile duct continuum, characterised by onset of persistent cholestatic jaundice during the neonatal period. Biliary atresia (BA) is the most common among these, but still has an incidence of only 1 in 10-19,000 in Europe and North America. Other diseases such as the genetic conditions, Alagille syndrome (ALGS) and Progressive Familial Intrahepatic Cholestasis (PFIC), are less common. Choledochal malformations are amenable to surgical correction and require a high index of suspicion. The low incidence of such diseases hinder patient-based studies that include large cohorts, while the limited numbers of animal models of disease that recapitulate the spectrum of disease phenotypes hinders both basic research and the development of new treatments. Despite their individual rarity, collectively BA and other cholestatic childhood diseases are the commonest indications for liver transplantation during childhood. Here, we review the recent advances in basic research and clinical progress in these diseases, as well as the research needs. For the various diseases, we formulate current key questions and controversies and identify top priorities to guide future research.

Neonatal acute liver failure.

Neonatal acute liver failure (NALF) is a rare disease about which there is little published data; however, NALF is an extremely important condition as it is distinct from acute liver failure seen in older children and adults. First, unlike acute liver failure in older patients, NALF can be diagnosed in an infant with cirrhosis. This is due to the fetal-neonatal continuum of liver disease, or the principle that neonatal liver failure may be the result of a liver disease that began in utero. Further differences exist in the mechanism of disease, diagnostic principles, and the common etiologies of NALF when compared with pediatric and adult acute liver failure. This review will address many of the distinguishing features of NALF and focus on the most common etiologies of NALF, including gestational alloimmune liver disease (GALD), the most common cause of NALF. Additionally, this review will provide insight into the pathogenesis, diagnosis, and treatment of this rare condition. Liver Transplantation 22 677-685 2016 AASLD.

Total Serum Bilirubin within 3 Months of Hepatoportoenterostomy Predicts Short-Term Outcomes in Biliary Atresia.

OBJECTIVES: To prospectively assess the value of serum total bilirubin (TB) within 3 months of hepatoportoenterostomy (HPE) in infants with biliary atresia as a biomarker predictive of clinical sequelae of liver disease in the first 2 years of life. STUDY DESIGN: Infants with biliary atresia undergoing HPE between June 2004 and January 2011 were enrolled in a prospective, multicenter study. Complications were monitored until 2 years of age or the earliest of liver transplantation (LT), death, or study withdrawal. TB below 2 mg/dL (34.2 µM) at any time in the first 3 months (TB <2.0, all others TB ≥ 2) after HPE was examined as a biomarker, using Kaplan-Meier survival and logistic regression. RESULTS: Fifty percent (68/137) of infants had TB < 2.0 in the first 3 months after HPE. Transplant-free survival at 2 years was significantly higher in the TB < 2.0 group vs TB ≥ 2 (86% vs 20%, P < .0001). Infants with TB ≥ 2 had diminished weight gain (P < .0001), greater probability of developing ascites (OR 6.4, 95% CI 2.9-14.1, P < .0001), hypoalbuminemia (OR 7.6, 95% CI 3.2-17.7, P < .0001), coagulopathy (OR 10.8, 95% CI 3.1-38.2, P = .0002), LT (OR 12.4, 95% CI 5.3-28.7, P < .0001), or LT or death (OR 16.8, 95% CI 7.2-39.2, P < .0001). CONCLUSIONS: Infants whose TB does not fall below 2.0 mg/dL within 3 months of HPE were at high risk for early disease progression, suggesting they should be considered for LT in a timely fashion. Interventions increasing the likelihood of achieving TB <2.0 mg/dL within 3 months of HPE may enhance early outcomes. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00061828 and NCT00294684.

Factors Determining δ-Bilirubin Levels in Infants With Biliary Atresia.

OBJECTIVES: δ-Bilirubin (Bδ) forms when bilirubin conjugates covalently bind to albumin by way of nonenzymatic transesterification in patients with cholestasis. Infants with cholestasis with biliary atresia form Bδ. The aim of the present study was to investigate the factors determining serum Bδ concentrations in infants with biliary atresia. METHODS: Study patients were infants enrolled in a prospective study (PROBE: Clinicaltrials.gov NCT00061828) of biliary atresia. We acquired data of concurrently measured serum bilirubin analytes (total bilirubin [TB], conjugated bilirubin [Bc], and unconjugated bilirubin) and applied graphical methods and linear mixed effects model to study factors contributing to Bδ variability. RESULTS: Bδ level increased with increasing levels of Bc and TB. In addition, the length of time cholestasis persisted partially determined the level of Bδ. An increase of 1 mg/dL in Bc is related to approximately 0.36 mg/dL increase in Bδ (P < 0.0001); every 100 days of cholestasis is associated with an approximately 1.0 mg/dL increase in Bδ (P < 0.0001) given the same level of Bc. Serum albumin levels are not significantly related to Bδ (P = 0.89). CONCLUSIONS: Bδ levels in infants with biliary atresia increase with increasing levels of Bc and longer duration of cholestasis. Understanding the relation among Bδ, Bc, TB, and direct-reacting bilirubin levels can help in interpretation of the clinical extent of cholestasis in infants and children with biliary atresia, assisting in the diagnosis and management of these infants.

Prevalence of groups A and C rotavirus antibodies in infants with biliary atresia and cholestatic controls.

OBJECTIVE: To analyze the prevalence of acute asymptomatic group A and C rotavirus (RV-A and RV-C) infection in neonates with cholestasis. STUDY DESIGN: Participants were infants <180 days of age with cholestasis (serum direct or conjugated bilirubin >20% of total and ≥2 mg/dL) enrolled in the Childhood Liver Disease Research and Education Network during RV season (December-May). Forty infants with biliary atresia (BA), age 62 ± 29 days (range, 4.7-13 weeks) and 38 infants with cholestasis, age 67 ± 44 days (range, 3-15.8 weeks) were enrolled. RESULTS: At enrollment, RV-A IgM positivity rates did not differ between infants with BA (10%) vs those without (18%) (P = .349). RV-C IgM was positive in 0% of infants with BA vs 3% in those without BA (P = .49). RV-A IgG was lower in infants with BA: 51 ± 39 vs 56 ± 44 enzyme-linked immunoassay unit, P = .045 but this difference may lack biological relevance as maternal RV-A IgG titers were similar between groups. Infant RV-A IgM titers at 2-6 months follow-up increased markedly vs at presentation in both infants with BA (50 ± 30 vs 9 ± 9) and those without (43 ± 18 vs 16 ± 20 enzyme-linked immunoassay unit) (P < .0001), without differences between groups. CONCLUSIONS: RV-A infection in the first 6 months of life is common in infants with cholestasis of any cause. RV-A could have different pathogenetic effects by initiating different hepatic immune responses in infants with vs without BA or could lack pathogenetic significance.

Elaboration of tubules with active hedgehog drives parenchymal fibrogenesis in gestational alloimmune liver disease.

Gestational alloimmune liver disease (GALD) produces severe neonatal liver disease that is notable for paucity of hepatocytes, large numbers of parenchymal tubules, and extensive fibrosis. Liver specimens from 19 GALD cases were studied in comparison with 14 infants without liver disease (normal newborn liver; NNL) to better understand the pathophysiology that would produce this characteristic histopathology. GALD liver parenchyma contained large numbers of tubules comprising epithelium expressing KRT7/19, EPCAM, and SOX9, suggesting biliary progenitor status. Quantitative morphometry demonstrated that in GALD, the area density of KRT19+ tubules was 16.4 ± 6.2 versus 2.0 ± 2.6 area% in NNL (P < .0001). Functional hepatocyte mass was markedly reduced in GALD, 16.3 ± 6.2 versus 61.9 ± 11.0 area% of CPS1+ cells in NNL (P < .0001). A strong inverse correlation was established between CPS1+ area density and KRT19+ area density (r(2) = 0.66, P < .0001). Tubules showed active hedgehog signaling as determined by SHH and nuclear GLI2 expression and expressed the profibrogenic cytokine SPP1. SPP1 protein content and SPP1 expression were greater in GALD than NNL (15- and 13-fold respectively; P = .002). GALD liver contained large numbers of activated myofibroblasts and showed greater than 10-fold more fibrosis than NNL. The extent of fibrosis correlated with the area density of KRT19+ tubules (r(2) = 0.387, P = .001). The data support a pathogenic model in which immune injury to fetal hepatocytes provides a stimulus for expansion of parenchymal tubules, which, by way of Hh activation, produce fibrogenic signals leading to vibrant fibrosis.

Bile acid pool dynamics in progressive familial intrahepatic cholestasis with partial external bile diversion.

OBJECTIVES: Partial external bile diversion (PEBD) is an established therapy for low-γ-glutamyl transferase (GGT) progressive familial intrahepatic cholestasis (PFIC). This study sought to determine whether the dynamics of the cholic acid (CA) and chenodeoxycholic acid (CDCA) pools in subjects with low-GGT-PFIC with successful PEBD were equivalent to those achieved with successful liver transplantation (LTX). METHODS: The kinetics of CA and CDCA metabolism were measured by stable isotope dilution in plasma samples in 5 subjects with PEBD, all with intact canalicular bile salt export pump expression and compared with subjects with low-GGT-PFIC with successful LTX. Stomal loss of bile acids was measured in subjects with PEBD. RESULTS: The fractional turnover rate for CA in the PEBD group ranged from 0.5 to 4.2/day (LTX group, range 0.2-0.9/day, P = 0.076) and for CDCA from 0.7 to 4.5/day (LTX group 0.3-0.4/day, P = 0.009). The CA and CDCA pool sizes were equivalent between groups; however, pool composition in PEBD was somewhat more hydrophilic. The CA/CDCA ratio in PEBD ranged from 0.9 to 19.5, whereas in LTX it ranged from 0.5 to 2.6. Synthesis rates computed from isotope dilution correlated well with timed output for both CA (r2 = 0.760, P = 0.024) and CDCA (r2 = 0.690, P = 0.021). CONCLUSIONS: PEBD results in bile acid fractional turnover rates greater than LTX, pool sizes equivalent to LTX, and pool composition that is at least as hydrophilic as produced by LTX.

Total serum bilirubin predicts fat-soluble vitamin deficiency better than serum bile acids in infants with biliary atresia.

OBJECTIVE: Fat-soluble vitamin (FSV) deficiency is a well-recognized consequence of cholestatic liver disease and reduced intestinal intraluminal bile acid. We hypothesized that serum bile acid (SBA) would predict biochemical FSV deficiency better than serum total bilirubin (TB) level in infants with biliary atresia. METHODS: Infants enrolled in the Trial of Corticosteroid Therapy in Infants with Biliary Atresia after hepatoportoenterostomy were the subjects of this investigation. Infants received standardized FSV supplementation and monitoring of TB, SBA, and vitamin levels at 1, 3, and 6 months. A logistic regression model was used with the binary indicator variable insufficient/sufficient as the outcome variable. Linear and nonparametric correlations were made between specific vitamin measurement levels and either TB or SBA. RESULTS: The degree of correlation for any particular vitamin at a specific time point was higher with TB than with SBA (higher for TB in 31 circumstances vs 3 circumstances for SBA). Receiver operating characteristic curve shows that TB performed better than SBA (area under the curve 0.998 vs 0.821). Including both TB and SBA did not perform better than TB alone (area under the curve 0.998). CONCLUSIONS: We found that TB was a better predictor of FSV deficiency than SBA in infants with biliary atresia. The role of SBA as a surrogate marker of FSV deficiency in other cholestatic liver diseases, such as progressive familial intrahepatic cholestasis, α-1-antitrypsin deficiency, and Alagille syndrome in which the pathophysiology is dominated by intrahepatic cholestasis, warrants further study.

Use of corticosteroids after hepatoportoenterostomy for bile drainage in infants with biliary atresia: the START randomized clinical trial.

IMPORTANCE: Biliary atresia is the most common cause of end-stage liver disease in children. Controversy exists as to whether use of steroids after hepatoportoenterostomy improves clinical outcome. OBJECTIVE: To determine whether the addition of high-dose corticosteroids after hepatoportoenterostomy is superior to surgery alone in improving biliary drainage and survival with the native liver. DESIGN, SETTING, AND PATIENTS: The multicenter, double-blind Steroids in Biliary Atresia Randomized Trial (START) was conducted in 140 infants (mean age, 2.3 months) between September 2005 and February 2011 in the United States; follow-up ended in January 2013. INTERVENTIONS: Participants were randomized to receive intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks) followed by a tapering protocol for 9 weeks (n = 70) or placebo (n = 70) initiated within 72 hours of hepatoportoenterostomy. MAIN OUTCOMES AND MEASURES: The primary end point (powered to detect a 25% absolute treatment difference) was the percentage of participants with a serum total bilirubin level of less than 1.5 mg/dL with his/her native liver at 6 months posthepatoportoenterostomy. Secondary outcomes included survival with native liver at 24 months of age and serious adverse events. RESULTS: The proportion of participants with improved bile drainage was not statistically significantly improved by steroids at 6 months posthepatoportoenterostomy (58.6% [41/70] of steroids group vs 48.6% [34/70] of placebo group; adjusted relative risk, 1.14 [95% CI, 0.83 to 1.57]; P = .43). The adjusted absolute risk difference was 8.7% (95% CI, -10.4% to 27.7%). Transplant-free survival was 58.7% in the steroids group vs 59.4% in the placebo group (adjusted hazard ratio, 1.0 [95% CI, 0.6 to 1.8]; P = .99) at 24 months of age. The percentage of participants with serious adverse events was 81.4% [57/70] of the steroids group and 80.0% [56/70] of the placebo group (P > .99); however, participants receiving steroids had an earlier time of onset of their first serious adverse event by 30 days posthepatoportoenterostomy (37.2% [95% CI, 26.9% to 50.0%] of steroids group vs 19.0% [95% CI, 11.5% to 30.4%] of placebo group; P = .008). CONCLUSIONS AND RELEVANCE: Among infants with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6 months, although a small clinical benefit could not be excluded. Steroid treatment was associated with earlier onset of serious adverse events in children with biliary atresia. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00294684.