Biliary atresia is a severe cholangiopathy of early infancy that destroys extrahepatic bile ducts and disrupts bile flow. With a poorly defined disease pathogenesis, treatment consists of the ...surgical removal of duct remnants followed by hepatoportoenterostomy. Although this approach can improve the short-term outcome, the liver disease progresses to end-stage cirrhosis in most children. Further improvement in outcome will require a greater understanding of the mechanisms of biliary injury and fibrosis. Here, we review progress in the field, which has been fuelled by collaborative studies in larger patient cohorts and the development of cell culture and animal model systems to directly test hypotheses. Advances include the identification of phenotypic subgroups and stages of disease based on clinical, pathological and molecular features. Stronger evidence exists for viruses, toxins and gene sequence variations in the aetiology of biliary atresia, triggering a proinflammatory response that injures the duct epithelium and produces a rapidly progressive cholangiopathy. The immune response also activates the expression of type 2 cytokines that promote epithelial cell proliferation and extracellular matrix production by nonparenchymal cells. These advances provide insight into phenotype variability and might be relevant to the design of personalized trials to block progression of liver disease.
Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low‐phospholipid “toxic” bile causing progressive cholestasis. We hypothesize that ...pharmacological inhibition of the ileal, apical sodium‐dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2–/– mice. Thirty‐day‐old, female mdr2–/– mice were fed high‐fat chow containing 0.006% SC‐435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry.
Compared with untreated mdr2–/– mice, SC‐435 treatment for 14 days increased fecal BA excretion by 8‐fold, lowered total BA concentration in liver by 65%, reduced total BA and individual hydrophobic BA concentrations in serum by >98%, and decreased plasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levels by 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved, and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenic gene expression. Biliary BA concentrations significantly decreased and phospholipids remained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratio in treated mice corrected toward a ratio of 0.28 found in wild‐type mice, indicating decreased bile toxicity. Hepatic RNA sequencing studies revealed up‐regulation of putative anti‐inflammatory and antifibrogenic genes, including Ppara and Igf1, and down‐regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated in leukocyte recruitment. Flow cytometric analysis revealed significant reduction of frequencies of hepatic CD11b+F4/80+ Kupffer cells and CD11b+Gr1+ neutrophils, accompanied by expansion of anti‐inflammatory Ly6C– monocytes in treated mdr2–/– mice. Conclusion: Inhibition of ASBT reduces BA pool size and retention of hydrophobic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic transcriptome, attenuates recruitment of leukocytes, and abrogates progression of murine sclerosing cholangitis. (Hepatology 2016;63:512–523)
Purpose
To assess longitudinal changes in quantitative MRI metrics in pediatric and young adult patients with autoimmune liver disease (AILD).
Methods
This prospective, IRB-approved study included 20 ...children and young adults (median age = 15 years) with primary sclerosing cholangitis (PSC)/autoimmune sclerosing cholangitis (ASC) and 19 (median age = 17 years) with autoimmune hepatitis (AIH). At a field strength of 1.5-T, T2*-corrected T1 mapping (cT1), 3D fast spin-echo MRCP, and 2D gradient recalled echo MR elastography (MRE) were performed at baseline, one year, and two years. cT1 and quantitative MRCP were processed using LiverMultiScan and MRCP + , respectively (Perspectum Ltd, Oxford, UK). Linear mixed models were used to assess longitudinal changes in quantitative MRI metrics. Spearman rank-order correlation was used to assess relationships between changes in quantitative MRI metrics.
Results
Changes in quantitative MRI metrics greater than established repeatability coefficients were measured in six (cT1) and five (MRE) patients with PSC/ASC as well as in six patients (cT1 and MRE) with AIH, although linear mixed models identified no significant changes for the subgroups as a whole. For PSC/ASC, there were positive correlations between change in liver stiffness and changes in bile duct strictures (
ρ
= 0.68;
p
= 0.005) and bile duct dilations (
ρ
= 0.70;
p
= 0.004) between baseline and Year 2.
Conclusion
On average, there were no significant changes in quantitative MRI metrics over a two-year period in children and young adults with AILD. However, worsening cholangiopathy was associated with increasing liver stiffness by MRE in patients with PSC/ASC.
The etiology of acute liver failure (ALF) remains elusive in almost half of affected children. We hypothesized that inherited mitochondrial and fatty acid oxidation disorders were occult etiological ...factors in patients with idiopathic ALF and impaired energy metabolism.
Twelve patients with elevated blood molar lactate/pyruvate ratio and indeterminate etiology were selected from a retrospective cohort of 74 subjects with ALF because their fixed and frozen liver samples were available for histological, ultrastructural, molecular and biochemical analysis.
A customized next-generation sequencing panel for 26 genes associated with mitochondrial and fatty acid oxidation defects revealed mutations and sequence variants in five subjects. Variants involved the genes ACAD9, POLG, POLG2, DGUOK, and RRM2B; the latter not previously reported in subjects with ALF. The explanted livers of the patients with heterozygous, truncating insertion mutations in RRM2B showed patchy micro- and macrovesicular steatosis, decreased mitochondrial DNA (mtDNA) content <30% of controls, and reduced respiratory chain complex activity; both patients had good post-transplant outcome. One infant with severe lactic acidosis was found to carry two heterozygous variants in ACAD9, which was associated with isolated complex I deficiency and diffuse hypergranular hepatocytes. The two subjects with heterozygous variants of unknown clinical significance in POLG and DGUOK developed ALF following drug exposure. Their hepatocytes displayed abnormal mitochondria by electron microscopy.
Targeted next generation sequencing and correlation with histological, ultrastructural and functional studies on liver tissue in children with elevated lactate/pyruvate ratio expand the spectrum of genes associated with pediatric ALF.
The purpose of this study was to assess relationships between liver-corrected T1 (cT1) values (adjusted for T2* effect, MRI system manufacturer, and field strength) and histologic inflammation and ...fibrosis in 35 participants (15 women and girls, 20 boys and men; median age, 16.0 years) with autoimmune liver disease. At multivariable analysis, inflammation score (β = 15.5) and sex (β = 56.0 female) were independent predictors of cT1, and fibrosis score (β = 32.3) and age (β = 5.5) were independent predictors of cT1 IQR. Liver T1 may have relevance for assessing liver inflammatory activity and fibrosis stage.
In the multidrug resistance protein 2 (Mdr2)‐/‐ mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms ...implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2‐/‐ mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14‐30 of life. Antibody‐mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro‐fibrogenic cytokine. Depletion of intrahepatic Tregs with anti‐CD25 antibody between days 7‐30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti‐interleukin 2 immune complexes (IL‐2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL‐2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro‐inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2‐/‐ mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg‐associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL‐2c, suggesting that Treg‐directed low‐dose Il‐2 treatment may be considered as therapy for SC.
Bile salt export pump (BSEP) adenosine triphosphate–binding cassette B11 (ABCB11) is a liver‐specific ABC transporter that mediates canalicular bile salt excretion from hepatocytes. Human mutations ...in ABCB11 cause progressive familial intrahepatic cholestasis type 2. Although over 150 ABCB11 variants have been reported, our understanding of their biological consequences is limited by the lack of an experimental model that recapitulates the patient phenotypes. We applied CRISPR/Cas9‐based genome editing technology to knock out abcb11b, the ortholog of human ABCB11, in zebrafish and found that these mutants died prematurely. Histological and ultrastructural analyses showed that abcb11b mutant zebrafish exhibited hepatocyte injury similar to that seen in patients with progressive familial intrahepatic cholestasis type 2. Hepatocytes of mutant zebrafish failed to excrete the fluorescently tagged bile acid that is a substrate of human BSEP. Multidrug resistance protein 1, which is thought to play a compensatory role in Abcb11 knockout mice, was mislocalized to the hepatocyte cytoplasm in abcb11b mutant zebrafish and in a patient lacking BSEP protein due to nonsense mutations in ABCB11. We discovered that BSEP deficiency induced autophagy in both human and zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage, and extended the life span of abcb11b mutant zebrafish, correlating with the recovery of canalicular multidrug resistance protein 1 localization. Conclusions: Collectively, these data suggest a model that rapamycin rescues BSEP‐deficient phenotypes by prompting alternative transporters to excrete bile salts; multidrug resistance protein 1 is a candidate for such an alternative transporter. (Hepatology 2018;67:1531‐1545).
CD8 T‐lymphocytes are effector cells of cholangiocyte injury in human and in rhesus rotavirus (RRV)‐induced experimental biliary atresia (BA). Here we hypothesize that neonatal deficiency in ...CD25+CD4+ regulatory T cells (Tregs) leads to aberrant activation of hepatic T‐lymphocytes in BA. We found that adoptive transfer of total CD4 cells, but not of CD25‐depleted CD4 cells, prior to RRV inoculation reduced expansion of CD8 cells, plasma bilirubin levels, ductal inflammation, and bile duct epithelial injury at 7 days postinfection (dpi) compared with age‐matched infected controls without adoptive transfer. Searching for mechanisms, we found that in vitro production of interferon‐gamma (IFN‐γ) by naïve CD8 cells upon polyclonal stimulation was enhanced in coculture with hepatic dendritic cells (DCs) from RRV‐infected, but not with DCs from noninfected mice, which was correlated with an increased proportion of CD11b+ myeloid (m)DCs and up‐regulation of the costimulatory molecule CD86 on RRV‐primed DCs. Furthermore, DC‐dependent T‐lymphocyte activation was blocked by anti‐CD86 antibody in dose‐dependent fashion. Importantly, expression of CD86 on mDCs was down‐regulated by Tregs in vitro, and adoptive transfer of Treg‐containing CD4 cells decreased expression of CD86 on hepatic mDCs at 7 dpi. On the contrary, in mice resistant to experimental BA, CD25+ cell depletion aggravated bile duct injury at 12 dpi after RRV inoculation, as plasma bilirubin levels were elevated by >20‐fold compared with nondepleted infected controls. Increased susceptibility to hepatobiliary injury in Treg‐depleted mice was linked to hepatic CD8 expansion and enhanced stimulatory capacity of hepatic DCs. Conclusion: Activation of hepatic T‐lymphocytes driving biliary obstruction in BA is regulated by mDCs by way of CD86‐dependent costimulation and is susceptible to inhibition by Tregs. (HEPATOLOGY 2012;56:219–227)
Purpose
Autoimmune liver diseases (AILD), including primary sclerosing cholangitis (PSC), autoimmune sclerosing cholangitis (ASC), and autoimmune hepatitis (AIH), have overlapping clinical features ...but distinct management strategies and outcomes. The purpose of this study was to assess the diagnostic performance of quantitative magnetic resonance cholangiopancreatography (MRCP) parameters for distinguishing PSC/ASC from AIH in children and young adults.
Materials and methods
This IRB-approved, cross-sectional study included participants from an institutional AILD registry that underwent baseline serum liver biochemistry testing and 3D fast spin-echo MRCP. The biliary tree was extracted and modeled from MRCP images using novel proprietary software (MRCP+ ™; Perspectum Diagnostics; Oxford, United Kingdom), and quantitative parameters were generated (e.g., biliary tree volume; number and length of bile ducts, strictures, and dilations; bile duct median/maximum diameters). Mann–Whitney
U
tests were performed to compare laboratory values and MRCP metrics between patient cohorts (clinical diagnosis of PSC/ASC versus AIH). Receiver operating characteristic (ROC) curves and multivariable logistic regression were used to assess diagnostic performance of serum biochemistry values and MRCP parameters for discriminating PSC/ASC from AIH.
Results
Thirty percent (14/47) of MRCP exams failed post-processing due to motion artifact. The remaining 33 patients included 20 males and 13 females, with a mean age of 15.1 ± 3.9 years. Eighteen patients were assigned the clinical diagnosis of PSC or ASC and 15 of AIH. All but one quantitative MRCP parameter were significantly different between cohorts (
p
< 0.05) and predictive of diagnosis (ROC
p
< 0.05), including numbers of bile duct strictures (area under curve AUC = 0.86,
p
< 0.0001) and dilations (AUC = 0.87,
p
< 0.0001) and total length of dilated ducts (AUC = 0.89,
p
< 0.0001). Laboratory values were not significantly different between cohorts (
p
> 0.05). The best multivariable model for distinguishing PSC/ASC from AIH included total length of dilated ducts (odds ratio OR, 1.08; 95% CI 1.02–1.14) and maximum left hepatic duct diameter (OR, 1.21; 95% CI 0.57–2.56) AUC = 0.92.
Conclusion
Quantitative MRCP parameters provide good discrimination of PSC/ASC from AIH.
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