Publications by authors named "Alexander Miethke"

47 Publications

Sarcopenia is highly prevalent in children with autoimmune liver diseases and is linked to visceral fat and parent-perceived general health.

Liver Int 2021 Nov 24. Epub 2021 Nov 24.

Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Background: Patients with autoimmune hepatitis (AIH) and primary or autoimmune sclerosing cholangitis are at nutritional risk; their body composition and has not been extensively studied. We aimed to describe their body composition and identify clinical links.

Methods: Using magnetic resonance imaging (MRI), two reviewers segmented total psoas muscle area (tPMSA), visceral fat area (VFA) and subcutaneous fat area (mm ) and measured visceral and subcutaneous thickness (mm). Clinical, laboratory and quality of life (QoL; using PedsQL) data were collected. Sarcopenia was defined as tPMSA ≤5th percentile. Analysis of variance, Wilcoxon rank test and multivariable modelling were performed. A paediatric cohort with non-alcoholic fatty liver disease (NAFLD) was used as a comparator following propensity score matching.

Results: Fifty-eight patients with autoimmune liver disease (AILD) (33 [57%] with AIH) were included: median age 16 years (interquartile range [IQR]: 13-18), 33 (57%) male. Median time from diagnosis to MRI was 15 months (IQR: 2-39 months). Two patients (3%) had a BMIz indicative of mild malnutrition. tPMSA was measurable in 52 subjects (90%). Of those, 25 (48%) had sarcopenia. Sarcopenic patients had a lower blood urea nitrogen compared to non-sarcopenic (median [IQR]: 9.5 [8.0, 12.0] vs 11 [10, 14] mg/dL; P = .023). There was no difference in corticosteroid use between groups. The VFA of sarcopenic patients was higher (3156 [2064, 7492]) vs 2084 [688, 3092]) mm ; P = .005). Patient-reported QoL negatively associated with VFA and general health negatively associated with VFA. Compared with NAFLD, the odds ratio for sarcopenia with AILD was 14.5 (95% confidence interval: 2.3-90.7).

Conclusion: In autoimmune liver diseases, sarcopenia is highly prevalent, associated with increased visceral fat and QoL.
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http://dx.doi.org/10.1111/liv.15108DOI Listing
November 2021

Isolation and Culturing Primary Chaolangiocytes from Mouse Liver.

Bio Protoc 2021 Oct 20;11(20):e4192. Epub 2021 Oct 20.

Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Cholangiocytes are epithelial cells lining the intrahepatic and extrahepatic bile ducts. Cholangiocytes perform key physiological functions in the liver. Bile synthesized by hepatocytes is secreted into bile canaliculi, further stored in the gallbladder, and finally discharged into the duodenum. Due to liver injury, biliary epithelial proliferate in response to endogenous or exogenous signals leading to cholangiopathies, inflammation, fibrosis, and cholangiocarcinoma. Cholangiocytes exhibit anatomical and functional heterogeneity, and understanding such diversified functions will potentially help in finding effective therapies for various cholestatic liver diseases. To perform such functional studies, effective cholangiocyte isolation and culture procedures are needed. This protocol will aid in easy isolation and expansion of cholangiocytes from the liver.
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http://dx.doi.org/10.21769/BioProtoc.4192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8554810PMC
October 2021

Assessment of agreement between manual and automated processing of liver MR elastography for shear stiffness estimation in children and young adults with autoimmune liver disease.

Abdom Radiol (NY) 2021 08 2;46(8):3927-3934. Epub 2021 Apr 2.

Department of Radiology, Imaging Research Center (IRC), Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.

Purpose: To compare automated versus standard of care manual processing of 2D gradient recalled echo (GRE) liver MR Elastography (MRE) in children and young adults.

Materials And Methods: 2D GRE liver MRE data from research liver MRI examinations performed as part of an autoimmune liver disease registry between March 2017 and March 2020 were analyzed retrospectively. All liver MRE data were acquired at 1.5 T with 60 Hz mechanical vibration frequency. For manual processing, two independent readers (R1, R2) traced regions of interest on scanner generated shear stiffness maps. Automated processing was performed using MREplus+ (Resoundant Inc.) using 90% (A90) and 95% (A95) confidence masks. Agreement was evaluated using intra-class correlation coefficients (ICC) and Bland-Altman analyses. Classification performance was evaluated using receiver operating characteristic curve (ROC) analyses.

Results: In 65 patients with mean age of 15.5 ± 3.8 years (range 8-23 years; 35 males) median liver shear stiffness was 2.99 kPa (mean 3.55 ± 1.69 kPa). Inter-reader agreement for manual processing was very strong (ICC = 0.99, mean bias = 0.01 kPa [95% limits of agreement (LoA): - 0.41 to 0.44 kPa]). Correlation between manual and A95 automated processing was very strong (R1: ICC = 0.988, mean bias = 0.13 kPa [95% LoA: - 0.40 to 0.68 kPa]; R2: ICC = 0.987, mean bias = 0.13 kPa [95% LoA: - 0.44 to 0.69 kPa]). Automated measurements were perfectly replicable (ICC = 1.0; mean bias = 0 kPa).

Conclusion: Liver shear stiffness values obtained using automated processing showed excellent agreement with manual processing. Automated processing of liver MRE was perfectly replicable.
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http://dx.doi.org/10.1007/s00261-021-03073-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292210PMC
August 2021

Deleterious Variants in ABCC12 are Detected in Idiopathic Chronic Cholestasis and Cause Intrahepatic Bile Duct Loss in Model Organisms.

Gastroenterology 2021 07 23;161(1):287-300.e16. Epub 2021 Mar 23.

Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio. Electronic address:

Background & Aims: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models.

Method: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice.

Results: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge.

Conclusions: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.
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http://dx.doi.org/10.1053/j.gastro.2021.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238842PMC
July 2021

Serum Matrix Metalloproteinase 7 Is a Diagnostic Biomarker of Biliary Injury and Fibrosis in Pediatric Autoimmune Liver Disease.

Hepatol Commun 2020 Nov 24;4(11):1680-1693. Epub 2020 Sep 24.

Division of Gastroenterology, Hepatology and Nutrition Cincinnati Children's Hospital Medical Center Cincinnati OH USA.

In autoimmune liver disease (AILD), including autoimmune hepatitis (AIH), primary sclerosing cholangitis (PSC), and overlap syndrome of AIH and PSC (ASC), the presence of biliary injury portends a worse prognosis. We studied serum matrix metalloproteinase 7 (sMMP7) as a biomarker for pediatric sclerosing cholangitis (SC). We prospectively enrolled 54 children (median age, 16 years) with AILD (AIH, n = 26; ASC, n = 16; and PSC, n = 12) at our center. The sMMP7 concentrations were higher in patients with SC compared to those without cholangiopathy ( < 0.001). An sMMP7 concentration >23.7 ng/mL had a sensitivity and specificity of 79% and 96%, respectively, and outperformed alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) in segregating patients with SC. Serum concentrations correlated with liver gene expression levels for ( = 0.70;  < 0.001). Using immunofluorescence, MMP7 was localized primarily to the cholangiocytes of patients with SC. In 46 subjects with liver biopsy available for blinded review, elevation in sMMP7 concentrations segregated with the presence of lymphocytic and neutrophilic cholangitis and periductal fibrosis and correlated with Ishak, Ludwig, and Nakanuma scoring systems. Liver stiffness measured by magnetic resonance elastography also correlated with sMMP7 concentrations ( = 0.56;  < 0.01). Using magnetic resonance cholangiopancreatography plus (MRCP+), sMMP7 in 34 patients correlated with the number of biliary dilatations ( = 0.54;  < 0.01) and strictures ( = 0.56;  < 0.01). MMP7 as a marker of biliary injury was validated in an independent cohort of children with ulcerative colitis. Higher sMMP7 concentrations also correlated with a history of SC-related complication. : MMP7 is a promising biomarker for pediatric SC that diagnostically outperforms ALP and GGT. sMMP7 may directly reflect biliary injury and fibrosis, the main drivers of disease progression in SC.
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http://dx.doi.org/10.1002/hep4.1589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603534PMC
November 2020

Oral Vancomycin, Ursodeoxycholic Acid, or No Therapy for Pediatric Primary Sclerosing Cholangitis: A Matched Analysis.

Hepatology 2021 03;73(3):1061-1073

Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Background And Aims: Many children with primary sclerosing cholangitis (PSC) receive oral vancomycin therapy (OVT) or ursodeoxycholic acid (UDCA). There is a paucity of data on whether these medications improve outcomes.

Approach And Results: We analyzed retrospective data from the Pediatric PSC Consortium. Children treated with OVT were matched 1:1:1 to those treated with UDCA or managed with observation (no treatment) based on the closest propensity score, ensuring similar baseline characteristics. Two hundred sixty-four patients (88 each with OVT, UDCA, or observation) had matching propensity scores and were similar in demographics, phenotype, immunosuppression, baseline biochemistry, and hepatic fibrosis. After 1 year in an intention-to-treat analysis, all outcome metrics were similar regardless of treatment group. In OVT, UDCA, and untreated groups, respectively: Gamma-glutamyltransferase normalized in 53%, 49%, and 52% (P = not significant [NS]), liver fibrosis stage was improved in 20%, 13%, and 18% and worsened in 11%, 29%, and 18% (P = NS), and the 5-year probability of liver transplant listing was 21%, 10%, and 12% (P = NS). Favorable outcome was associated with having a mild phenotype of PSC and minimal hepatic fibrosis.

Conclusions: We presented the largest-ever description of outcomes on OVT in PSC and compared them to carefully matched patients on UDCA or no therapy. Neither OVT nor UDCA showed improvement in outcomes compared to a strategy of observation. Patients progressed to end-stage liver disease at similar rates. Spontaneous normalization of biochemistry is common in children receiving no therapy, particularly in the majority of children with a mild phenotype and an early stage of disease. Placebo-controlled treatment trials are needed to identify effective treatments for pediatric PSC.
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http://dx.doi.org/10.1002/hep.31560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8557636PMC
March 2021

Natural Course of Pediatric Portal Hypertension.

Hepatol Commun 2020 Sep 16;4(9):1346-1352. Epub 2020 Jul 16.

Division of Pediatric General and Thoracic Surgery Cincinnati Children's Hospital Medical Center Cincinnati OH.

The etiology of portal hypertension (pHTN) in children differs from that of adults and may require different management strategies. We set out to review the etiology, management, and natural history of pHTN at a pediatric liver center. From 2008 to 2018, 151 children and adolescents with pHTN were identified at a free-standing children's hospital. Patients were stratified by etiology of pHTN (intrahepatic disease [IH], defined as cholestatic disease and fibrotic or hepatocellular disease; extrahepatic disease [EH], defined as hepatic vein obstruction and prehepatic pHTN). Patients with EH were more likely to undergo an esophagoduodenscopy for a suspected gastrointestinal bleed (77% vs. 41%;  < 0.01). Surgical interventions differed based on etiology ( < 0.01), with IH more likely resulting in a transplant only (65%) and EH more likely to result in a shunt only (43%); 30% of patients with IH and 47% of patients with EH did not undergo an intervention for pHTN. Kaplan-Meier analysis revealed a significant increase in mortality in the group that received no intervention compared to shunt, transplant, or both and lower mortality in patients with prehepatic pHTN compared to other etiologies ( < 0.01 each). Multivariate analysis revealed increased odds of mortality in patients with refractory ascites (odds ratio [OR], 4.34; 95% confidence interval [CI], 1.00, 18.88;  = 0.05) and growth failure (OR, 13.49; 95% CI, 3.07, 58.99;  < 0.01). In this single institution study, patients with prehepatic pHTN had better survival and those who received no intervention had higher mortality than those who received an intervention. Early referral to specialized centers with experience managing these complex disease processes may allow for improved risk stratification and early intervention to improve outcomes.
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http://dx.doi.org/10.1002/hep4.1560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471417PMC
September 2020

Unraveling the Relationship Between Itching, Scratch Scales, and Biomarkers in Children With Alagille Syndrome.

Hepatol Commun 2020 Jul 26;4(7):1012-1018. Epub 2020 May 26.

Baylor College of Medicine Texas Children's Hospital Houston TX.

Pruritus is a debilitating symptom for patients with Alagille syndrome (ALGS). In a previously reported trial of maralixibat, an investigational antipruritic agent, itching was assessed using a digital diary based on twice-daily caregiver observation of itching severity (Itch Reported Outcome, ItchRO[Observer]). The goal of this study was to characterize pruritus in participants with ALGS at baseline in this trial, as assessed by the ItchRO instrument and the physician-observed clinician scratch scale (CSS), relative to biomarkers putatively associated with pruritus and health-related quality of life assessment. Thirty-seven participants with ALGS (median age of 6 years; range 1-17 years) were enrolled. No association was identified between CSS and ItchRO(Obs) ( = 0.22,  = 0.2). Neither CSS nor ItchRO were associated with serum bile acids ( = -0.08,  = 0.6 for both) or autotaxin ( = 0.22,  = 0.2;  = 0.28,  = 0.12). There was no significant association between Pediatric Quality of Life Inventory total parent scores and CSS or ItchRO ( = -0.23,  = 0.2;  = -0.16,  = 0.36). There was a significant association between ItchRO and Multidimensional Fatigue Scale and Family Impact Module total scores (Pearson correlation coefficient -0.575,  = 0.0005; 0.504,  = 0.002). In exploratory analysis, selected questions relating to fatigue and sleep disturbance (n = 12) from Pediatric Quality of Life Inventory, Multidimensional Fatigue Scale, and Family Impact Module were correlated with pruritus scores; positive associations were identified. Itching scores did not correlate with each other, nor with putative serum biomarkers of pruritus, and further, did not correlate with quality of life. Hypothesis-generating analyses implicate sleep disturbance and fatigue as key associations with caregiver observations of itching. This is highly relevant to the selection of surrogate endpoints for clinical trials of pruritus therapies.
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http://dx.doi.org/10.1002/hep4.1522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327199PMC
July 2020

The Sclerosing Cholangitis Outcomes in Pediatrics (SCOPE) Index: A Prognostic Tool for Children.

Hepatology 2021 03 19;73(3):1074-1087. Epub 2020 Dec 19.

Boston Children's Hospital and Harvard Medical School, Boston, MA.

Background And Aims: Disease progression in children with primary sclerosing cholangitis (PSC) is variable. Prognostic and risk-stratification tools exist for adult-onset PSC, but not for children. We aimed to create a tool that accounts for the biochemical and phenotypic features and early disease stage of pediatric PSC.

Approach And Results: We used retrospective data from the Pediatric PSC Consortium. The training cohort contained 1,012 patients from 40 centers. We generated a multivariate risk index (Sclerosing Cholangitis Outcomes in Pediatrics [SCOPE] index) that contained total bilirubin, albumin, platelet count, gamma glutamyltransferase, and cholangiography to predict a primary outcome of liver transplantation or death (TD) and a broader secondary outcome that included portal hypertensive, biliary, and cancer complications termed hepatobiliary complications (HBCs). The model stratified patients as low, medium, or high risk based on progression to TD at rates of <1%, 3%, and 9% annually and to HBCs at rates of 2%, 6%, and 13% annually, respectively (P < 0.001). C-statistics to discriminate outcomes at 1 and 5 years were 0.95 and 0.82 for TD and 0.80 and 0.76 for HBCs, respectively. Baseline hepatic fibrosis stage was worse with increasing risk score, with extensive fibrosis in 8% of the lowest versus 100% with the highest risk index (P < 0.001). The model was validated in 240 children from 11 additional centers and performed well.

Conclusions: The SCOPE index is a pediatric-specific prognostic tool for PSC. It uses routinely obtained, objective data to predict a complicated clinical course. It correlates strongly with biopsy-proven liver fibrosis. SCOPE can be used with families for shared decision making on clinical care based on a patient's individual risk, and to account for variable disease progression when designing future clinical trials.
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http://dx.doi.org/10.1002/hep.31393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8557635PMC
March 2021

Association between liver diffusion-weighted imaging apparent diffusion coefficient values and other measures of liver disease in pediatric autoimmune liver disease patients.

Abdom Radiol (NY) 2021 01;46(1):197-204

Center for Autoimmune Liver Disease (CALD), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Background: Multiple quantitative magnetic resonance imaging (MRI) methods have been described to noninvasively detect and characterize liver fibrosis, including diffusion-weighted imaging (DWI).

Purpose: To evaluate associations between liver MRI DWI apparent diffusion coefficient (ADC) values and clinical factors and other quantitative liver MRI metrics in pediatric patients with autoimmune liver disease (AILD).

Materials And Methods: Fifty-seven research liver MRI examinations performed from January 2017 to August 2018 for pediatric AILD registry participants were evaluated. Liver DWI ADC values, liver and spleen stiffness (kPa), and iron-corrected T1 (cT1; Perspectum Diagnostics) were measured at four anatomic levels. Participant age, sex, and laboratory data (alanine aminotransferase [ALT], total bilirubin, alkaline phosphatase, gamma-glutamyl transferase [GGT]) were recorded. Spearman's rank-order correlation (rho) and multiple linear regression were used to evaluate the associations between liver ADC values and predictor variables.

Results: Mean (SD) participant age was 14.8 (4.0) years, 45.6% (26/57) were girls. Mean liver DWI ADC value was 1.34 (0.14 × 10) mm/s. Liver ADC values showed weak to moderate correlations with liver stiffness (r = - 0.42, p = 0.001), spleen stiffness (r = - 0.34; p = 0.015), whole-liver mean cT1 (r = - 0.39; p = 0.007), ALT (r = - 0.50; p = 0.0001), and GGT (r = - 0.48; p = 0.0004). Multiple linear regression showed liver stiffness (p = 0.0009) and sex (p = 0.023) to be independent predictors of liver ADC values.

Conclusion: Liver DWI ADC values are significantly associated with liver and spleen stiffnesses, liver cT1, ALT, GGT, and participant sex, with liver stiffness and sex remaining significant at multivariable regression. Liver ADC ultimately may play a role in multi-parametric prediction of chronic liver disease/fibrosis severity.
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http://dx.doi.org/10.1007/s00261-020-02595-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530174PMC
January 2021

Comparison of liver T1 relaxation times without and with iron correction in pediatric autoimmune liver disease.

Pediatr Radiol 2020 06 14;50(7):935-942. Epub 2020 May 14.

Department of Radiology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH, 45229, USA.

Background: Magnetic resonance imaging (MRI) T1 relaxometry (mapping) has been reported as a quantitative biomarker of liver injury due to inflammation and fibrosis.

Objective: To assess the relationship between liver MRI T1 relaxometry measurements obtained using a modified Look-Locker inversion recovery (MOLLI) pulse sequence without and with iron (T2*) correction (cT1) in pediatric autoimmune liver disease.

Materials And Methods: This cross-sectional study was institutional review board-approved, with informed consent obtained. MRI was acquired at 1.5 T in patients participating in an autoimmune liver disease registry. T1 relaxometry was performed using a MOLLI sequence with a 5(3)3-s acquisition strategy. A multi-echo gradient echo sequence was used to measure liver T2*. Non-iron-corrected native T1 (ms), calculated as the mean of four slices through the mid-liver, was measured using T1 parametric maps generated off-line. A proprietary T2* correction (Perspectum Diagnostics, Oxford, UK), blinded to native T1 values, calculated cT1 values. The relationship between native T1 and cT1 measurements was assessed using Spearman rank correlation and Bland-Altman analyses.

Results: Forty-eight patients with a mean (standard deviation [SD]) age of 15.2 (4.1) years were included. Mean (SD) liver native T1 was 651.2 (123.9) ms and mean (SD) cT1 was 919.5 (86.8) ms, with excellent positive correlation between values (r=0.91 [95% confidence interval (CI): 0.85-0.95]; P<0.0001). Mean bias between native T1 and cT1 measurements was 268.3 ms (95% limits of agreement: 131.9-404.7 ms).

Conclusion: There is excellent positive correlation between liver native T1 and cT1 measurements in pediatric patients with autoimmune liver disease. This relationship brings into question the need to perform T1 iron correction in this patient population. T1 and cT1 measurements are not interchangeable, however, due to considerable systematic bias with cT1 values being considerably higher.
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http://dx.doi.org/10.1007/s00247-020-04663-8DOI Listing
June 2020

Colorectal Dysplasia and Cancer in Pediatric-Onset Ulcerative Colitis Associated With Primary Sclerosing Cholangitis.

Clin Gastroenterol Hepatol 2021 05 29;19(5):1067-1070.e2. Epub 2020 Apr 29.

Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut.

Inflammatory bowel disease (IBD), especially when associated with primary sclerosing cholangitis (PSC), is a risk factor for developing colorectal cancer (CRC). We aimed to determine the incidence of CRC in a large cohort of pediatric-onset PSC-ulcerative colitis (UC) patients.
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http://dx.doi.org/10.1016/j.cgh.2020.04.055DOI Listing
May 2021

Relationship between magnetic resonance imaging spleen T1 relaxation and other radiologic and clinical biomarkers of liver fibrosis in children and young adults with autoimmune liver disease.

Abdom Radiol (NY) 2020 11;45(11):3709-3715

Department of Radiology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 5031, Cincinnati, OH, 45229-3026, USA.

Background: Little is known about the relationships between MRI splenic T1 relaxation measurements and other radiologic and clinical markers of chronic liver disease, including the presence of radiologic portal hypertension.

Objective: To evaluate the relationships between MRI splenic T1 relaxation and other radiologic and clinical biomarkers of liver fibrosis, including the presence of radiologic portal hypertension, in children and young adults with autoimmune liver diseases (AILD).

Materials And Methods: Research MRI examinations performed at 1.5 T from 63 AILD registry participants were identified. Spleen T1 and iron-corrected T1 (cT1) relaxation measurements, liver cT1, liver/spleen stiffness, splenic length percentile for age, and presence of radiologic portal hypertension were recorded, along with demographic and laboratory data. The Mann-Whitney U test was used to compare continuous data between groups; Spearman correlation was used to evaluate associations. Areas-under-the-receiver operating characteristic curve (AuROC) was used to assess diagnostic performance.

Results: Mean age was 15.2 ± 4.1 years. Mean splenic T1 and cT1 values for the study population were 1158.0 ± 70.9 ms and 1436.0 ± 68.9 ms, respectively. Splenic T1 and cT1 values positively correlated with APRI and FIBROSIS-4 scores, splenic length percentile, liver cT1 values, and liver and spleen stiffnesses (p-values < 0.05). There was no significant relationship between splenic T1/cT1 and age (p-values > 0.05). Splenic T1 and cT1 values were higher in participants with vs. without radiologic portal hypertension (n = 18) (1213.4 ± 69.6 vs. 1135.4 ± 58.5 ms; p = 0.0001, and 1488.2 ± 64.8 vs. 1415.1 ± 59.1 ms; p = 0.0002). Splenic T1 and cT1 both demonstrated an AuROC of 0.81 for discriminating patients without and with portal hypertension (p-values < 0.0001).

Conclusion: Splenic T1 relaxation is associated with other radiologic and clinical biomarkers of liver fibrosis, including radiologic portal hypertension, in children and young adults with AILD.
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http://dx.doi.org/10.1007/s00261-020-02536-0DOI Listing
November 2020

Four Biomarkers Linked to Activation of Cluster of Differentiation 8-Positive Lymphocytes Predict Clinical Outcomes in Pediatric Acute Liver Failure.

Hepatology 2021 01;73(1):233-246

Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine and Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Background And Aims: Immune dysregulation contributes to the pathogenesis of pediatric acute liver failure (PALF). Our aim was to identify immune activation markers (IAMs) in PALF that are associated with a distinct clinical phenotype and outcome.

Approach And Results: Among 47 PALF study participants, 12 IAMs collected ≤6 days after enrollment were measured by flow cytometry and IMMULITE assay on blood natural killer and cluster of differentiation 8-positive (CD8 ) lymphocytes and subjected to unsupervised hierarchical analyses. A derivation cohort using 4 of 12 IAMs which were available in all participants (percent perforin-positive and percent granzyme-positive CD8 cells, absolute number of CD8 cells, soluble interleukin-2 receptor level) were sufficient to define high (n = 10), medium (n = 15), and low IAM (n = 22) cohorts. High IAM was more frequent among those with indeterminate etiology than those with defined diagnoses (80% versus 20%, P < 0.001). High IAM was associated with higher peak serum total bilirubin levels than low IAM (median peak 21.7 versus 4.8 mg/dL, P < 0.001) and peak coma grades. The 21-day outcomes differed between groups, with liver transplantation more frequent in high IAM participants (62.5%) than those with medium (28.2%) or low IAM (4.8%) (P = 0.002); no deaths were reported. In an independent validation cohort (n = 71) enrolled in a prior study, segregation of IAM groups by etiology, initial biochemistries, and short-term outcomes was similar, although not statistically significant. High serum aminotransferases, total bilirubin levels, and leukopenia at study entry predicted a high immune activation profile.

Conclusion: Four circulating T-lymphocyte activation markers identify a subgroup of PALF participants with evidence of immune activation associated with a distinct clinical phenotype and liver transplantation; these biomarkers may identify PALF participants eligible for future clinical trials of early targeted immunosuppression.
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http://dx.doi.org/10.1002/hep.31271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530172PMC
January 2021

Lipopolysaccharide Reverses Hepatic Stellate Cell Activation Through Modulation of cMyb, Small Mothers Against Decapentaplegic, and CCAAT/Enhancer-Binding Protein C/EBP Transcription Factors.

Hepatology 2020 11 22;72(5):1800-1818. Epub 2020 Oct 22.

Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

Background And Aims: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFβR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs.

Approach And Results: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFβR, transforming growth factor beta receptor 1 (TGFβR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFβR, αSMA, TGFβR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ.

Conclusions: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.
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http://dx.doi.org/10.1002/hep.31188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009050PMC
November 2020

Evaluation of a change in cytomegalovirus prevention strategy following pediatric solid organ transplantation.

Transpl Infect Dis 2020 Apr 30;22(2):e13232. Epub 2019 Dec 30.

Department of Pediatrics, Cincinnati Children's Medical Center, Cincinnati, Ohio.

Background: An optimal cytomegalovirus (CMV) prevention strategy following solid organ transplantation (SOT) remains uncertain. This study reports on the rates of CMV events following a change in a local prevention guideline involving increased surveillance, earlier transition to oral valganciclovir, and decreased CMV-immunoglobulin use.

Methods: A retrospective cohort study utilizing historical controls evaluated the rates of CMV invasive disease pre- and post-intervention among pediatric heart, liver, and kidney recipients. Outcomes were recorded for the 4 years pre- and post-intervention, 9/2009-10/2017. Logistic regression was used to estimate the risk of a CMV event.

Results: There was no difference in the rates of CMV invasive disease between the two study groups (P = 1). An increase in the detection of CMV events occurred (P = .04), predominantly asymptomatic CMV infection. This increase was independently associated with increased surveillance testing among high-risk heart and liver recipients, aOR 1.08 (1.06-1.12). Surprisingly, 28.9% of CMV events occurred during antiviral prophylaxis.

Conclusions: Modification of the local CMV prevention guideline did not result in an increase in CMV invasive disease. CMV events occurred while on prophylaxis, highlighting a potential difference from adult solid organ transplant (SOT) and emphasizing the potential need for monitoring on prophylaxis in the pediatric population.
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http://dx.doi.org/10.1111/tid.13232DOI Listing
April 2020

Differentiating pediatric autoimmune liver diseases by quantitative magnetic resonance cholangiopancreatography.

Abdom Radiol (NY) 2020 01;45(1):168-176

Department of Radiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

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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http://dx.doi.org/10.1007/s00261-019-02184-zDOI Listing
January 2020

Ursodeoxycholic Acid Therapy in Pediatric Primary Sclerosing Cholangitis: Predictors of Gamma Glutamyltransferase Normalization and Favorable Clinical Course.

J Pediatr 2019 06 14;209:92-96.e1. Epub 2019 Mar 14.

Department of Pediatrics, University of Utah, Salt Lake City, UT.

Objective: To investigate patient factors predictive of gamma glutamyltransferase (GGT) normalization following ursodeoxycholic acid (UDCA) therapy in children with primary sclerosing cholangitis.

Study Design: We retrospectively reviewed patient records at 46 centers. We included patients with a baseline serum GGT level ≥50 IU/L at diagnosis of primary sclerosing cholangitis who initiated UDCA therapy within 1 month and continued therapy for at least 1 year. We defined "normalization" as a GGT level <50 IU/L without experiencing portal hypertensive or dominant stricture events, liver transplantation, or death during the first year.

Results: We identified 263 patients, median age 12.1 years at diagnosis, treated with UDCA at a median dose of 15 mg/kg/d. Normalization occurred in 46%. Patients with normalization had a lower prevalence of Crohn's disease, lower total bilirubin level, lower aspartate aminotransferase to platelet ratio index, greater platelet count, and greater serum albumin level at diagnosis. The 5-year survival with native liver was 99% in those patients who achieved normalization vs 77% in those who did not.

Conclusions: Less than one-half of the patients treated with UDCA have a complete GGT normalization in the first year after diagnosis, but this subset of patients has a favorable 5-year outcome. Normalization is less likely in patients with a Crohn's disease phenotype or a laboratory profile suggestive of more advanced hepatobiliary fibrosis. Patients who do not achieve normalization could reasonably stop UDCA, as they are likely not receiving clinical benefit. Alternative treatments with improved efficacy are needed, particularly for patients with already-advanced disease.
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http://dx.doi.org/10.1016/j.jpeds.2019.01.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535363PMC
June 2019

Diagnostic performance of quantitative magnetic resonance imaging biomarkers for predicting portal hypertension in children and young adults with autoimmune liver disease.

Pediatr Radiol 2019 03 3;49(3):332-341. Epub 2019 Jan 3.

Center for Autoimmune Liver Disease (CALD), Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Background: Primary sclerosing cholangitis, autoimmune hepatitis and autoimmune sclerosing cholangitis are forms of chronic, progressive autoimmune liver disease (AILD) that can affect the pediatric population.

Objective: To determine whether quantitative MRI- and laboratory-based biomarkers are associated with conventional imaging findings of portal hypertension (radiologic portal hypertension) in children and young adults with AILD.

Materials And Methods: Forty-four patients with AILD enrolled in an institutional registry underwent a research abdominal MRI examination at 1.5 tesla (T). Five quantitative MRI techniques were performed: liver MR elastography, spleen MR elastography, liver iron-corrected T1 mapping, liver T2 mapping, and liver diffusion-weighted imaging (DWI, quantified as apparent diffusion coefficients). Two anatomical sequences were used to document splenomegaly, varices and ascites. We calculated aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores - laboratory-based biomarkers of liver fibrosis. We used receiver operating characteristic (ROC) curve analyses to establish the diagnostic performance of quantitative MRI and laboratory biomarkers for indicating the presence of radiologic portal hypertension.

Results: Twenty-three (52%) patients were male; mean age was 15.2±4.0 years. Thirteen (30%) patients had radiologic portal hypertension. Liver and spleen stiffness demonstrated the greatest diagnostic performance for indicating the presence of portal hypertension (area-under-the-ROC-curve [AUROC]=0.98 and 0.96, respectively). The APRI and FIB-4 scores also demonstrated good diagnostic performance (AUROC=0.87 and 0.88, respectively).

Conclusion: MRI-derived measures of liver and spleen stiffness as well as laboratory-based APRI and FIB-4 scores are highly associated with imaging findings of portal hypertension in children and young adults with AILD and thus might be useful for predicting portal hypertension impending onset and directing personalized patient management.
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http://dx.doi.org/10.1007/s00247-018-4319-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530173PMC
March 2019

Placebo-Controlled Randomized Trial of an Intestinal Bile Salt Transport Inhibitor for Pruritus in Alagille Syndrome.

Hepatol Commun 2018 Oct 24;2(10):1184-1198. Epub 2018 Sep 24.

Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics University of Colorado School of Medicine Aurora CO.

Medically refractory, severe, cholestasis-induced pruritus in Alagille syndrome may be improved by surgical interruption of the enterohepatic circulation. This multicenter trial (NCT02057692) tested the hypothesis that the intestinal bile acid transport inhibitor maralixibat would similarly reduce pruritus in Alagille syndrome. Thirty-seven children with Alagille syndrome were randomly assigned to double-blinded administration of placebo, 70, 140, or 280 µg/kg/day of maralixibat for 13 weeks. Pruritus was assessed by caregiver (itch-reported outcome instrument [ItchRO]) and clinician report (range, 0-4 [severe]). Liver chemistries and serum bile acids were measured. The primary outcome was the change from baseline to week 13 in ItchRO relative to placebo. In the first analysis of the primary efficacy endpoint, the mean adjusted difference between participants receiving 140 or 280 µg/kg/day and placebo was -0.47 (95% confidence interval [CI], -1.14, 0.20; 0.16). Statistically significant decreases were observed with doses of 70 and 140 µg/kg/day (mean adjusted difference, -0.89; 95% CI, -1.70, -0.08; 0.032; and mean adjusted difference, -0.91; 95% CI, -1.62, -0.19; 0.014) but not 280 µg/kg/day (mean adjusted difference, -0.04; 95% CI, -0.94, 0.86; 0.44) or all doses combined (mean adjusted difference, -0.61; 95% CI, -1.24, 0.20; 0.055). A 1-point reduction in pruritus was more common in maralixibat-treated versus placebo-treated participants (caregiver ItchRO, 65% versus 25%; 0.06; clinician score, 76% versus 25%; 0.01). There were no significant changes in liver chemistries or bile acids relative to placebo. Adverse and serious adverse events were similar between maralixibat and placebo. Although the prespecified primary analyses of ItchRO were not all statistically significant, the data suggest that maralixibat is safe and may reduce pruritus in Alagille syndrome.
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http://dx.doi.org/10.1002/hep4.1244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167076PMC
October 2018

Putting it all together: established and emerging MRI techniques for detecting and measuring liver fibrosis.

Pediatr Radiol 2018 08 4;48(9):1256-1272. Epub 2018 Aug 4.

Department of Radiology, MLC 5031, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH, 45229, USA.

Chronic injury to the liver leads to inflammation and hepatocyte necrosis, which when untreated can lead to myofibroblast activation and fibrogenesis with deposition of fibrous tissue. Over time, liver fibrosis can accumulate and lead to cirrhosis and end-stage liver disease with associated portal hypertension and liver failure. Detection and accurate measurement of the severity of liver fibrosis are important for assessing disease severity and progression, directing patient management, and establishing prognosis. Liver biopsy, generally considered the clinical standard of reference for detecting and measuring liver fibrosis, is invasive and has limitations, including sampling error, relatively high cost, and possible complications. For these reasons, liver biopsy is suboptimal for fibrosis screening, longitudinal monitoring, and assessing therapeutic efficacy. A variety of established and emerging qualitative and quantitative noninvasive MRI methods for detecting and staging liver fibrosis might ultimately serve these purposes. In this article, we review multiple MRI methods for detecting and measuring liver fibrosis and discuss the diagnostic performance and specific strengths and limitations of the various techniques.
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http://dx.doi.org/10.1007/s00247-018-4083-2DOI Listing
August 2018

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

Hepatology 2018 11 30;68(5):1905-1921. Epub 2018 Sep 30.

Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

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.
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http://dx.doi.org/10.1002/hep.30061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203671PMC
November 2018

Zebrafish abcb11b mutant reveals strategies to restore bile excretion impaired by bile salt export pump deficiency.

Hepatology 2018 04 23;67(4):1531-1545. Epub 2018 Feb 23.

Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

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).
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http://dx.doi.org/10.1002/hep.29632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480337PMC
April 2018

Hepatic MDR3 expression impacts lipid homeostasis and susceptibility to inflammatory bile duct obstruction in neonates.

Pediatr Res 2017 07 3;82(1):122-132. Epub 2017 May 3.

Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, Ohio.

BackgroundHeterozygous mutations in the gene ABCB4, encoding the phospholipid floppase MDR3 (Mdr2 in mice), are associated with various chronic liver diseases. Here we hypothesize that reduced ABCB4 expression predisposes to extrahepatic biliary atresia (EHBA).MethodsLivers from neonatal wild-type (wt) and heterozygous Mdr2-deficient mice were subjected to mass spectrometry-based lipidomics and RNA sequencing studies. Following postnatal infection with rhesus rotavirus (RRV), liver immune responses and EHBA phenotype were assessed. Hepatic microarray data from 40 infants with EHBA were mined for expression levels of ABCB4.ResultsPhosphatidylcholine (PC) and phosphatidylethanolamine (PE) were increased, whereas the PC/PE ratio was decreased in neonatal Mdr2 mice compared with wt mice. Following RRV challenge, hepatic expression of IFNγ and infiltration with CD8+ and NK+ lymphocytes were increased in Mdr2 mice. Plasma total bilirubin levels and prevalence of complete ductal obstruction were higher in these mice. In infants with EHBA, hepatic gene expression of ABCB4 was downregulated in those with an inflammatory compared with a fibrosing molecular phenotype.ConclusionDecreased expression of ABCB4 causes dysregulation in (phospho)lipid homeostasis, and predisposes to aberrant pro-inflammatory lymphocyte responses and an aggravated phenotype of EHBA in neonatal mice. Downregulated ABCB4 is associated with an inflammatory transcriptome signature in infants with EHBA.
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http://dx.doi.org/10.1038/pr.2017.78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509537PMC
July 2017

Analysis of surgical interruption of the enterohepatic circulation as a treatment for pediatric cholestasis.

Hepatology 2017 05 22;65(5):1645-1654. Epub 2017 Mar 22.

Children's Hospital Los Angeles, Los Angeles, CA.

To evaluate the efficacy of nontransplant surgery for pediatric cholestasis, 58 clinically diagnosed children, including 20 with Alagille syndrome (ALGS), 16 with familial intrahepatic cholestasis-1 (FIC1), 18 with bile salt export pump (BSEP) disease, and 4 others with low γ-glutamyl transpeptidase disease (levels <100 U/L), were identified across 14 Childhood Liver Disease Research Network (ChiLDReN) centers. Data were collected retrospectively from individuals who collectively had 39 partial external biliary diversions (PEBDs), 11 ileal exclusions (IEs), and seven gallbladder-to-colon (GBC) diversions. Serum total bilirubin decreased after PEBD in FIC1 (8.1 ± 4.0 vs. 2.9 ± 4.1 mg/dL, preoperatively vs. 12-24 months postoperatively, respectively; P = 0.02), but not in ALGS or BSEP. Total serum cholesterol decreased after PEBD in ALGS patients (695 ± 465 vs. 457 ± 319 mg/dL, preoperatively vs. 12-24 months postoperatively, respectively; P = 0.0001). Alanine aminotransferase levels increased in ALGS after PEBD (182 ± 70 vs. 260 ± 73 IU/L, preoperatively vs. 24 months; P = 0.03), but not in FIC1 or BSEP. ALGS, FIC1, and BSEP patients experienced less severely scored pruritus after PEBD (ALGS, 100% vs. 9% severe; FIC1, 64% vs. 10%; BSEP, 50% vs. 20%, preoperatively vs. >24 months postoperatively, respectively; P < 0.001). ALGS patients experienced a trend toward greater freedom from xanthomata after PEBD. There was a trend toward decreased pruritus in FIC1 after IE and GBC. Vitamin K supplementation increased in ALGS after PEBD (33% vs. 77%; P = 0.03). Overall, there were 15 major complications after surgery. Twelve patients (3 ALGS, 3 FIC1, and 6 BSEP) subsequently underwent liver transplantation.

Conclusion: This was a multicenter analysis of nontransplant surgical approaches to intrahepatic cholestasis. Approaches vary, are well tolerated, and generally, although not uniformly, result in improvement of pruritus and cholestasis. (Hepatology 2017;65:1645-1654).
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http://dx.doi.org/10.1002/hep.29019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397365PMC
May 2017

The dendritic cell-T helper 17-macrophage axis controls cholangiocyte injury and disease progression in murine and human biliary atresia.

Hepatology 2017 01 10;65(1):174-188. Epub 2016 Nov 10.

Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4 lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6C macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A cells was positively correlated with the degree of CD68 macrophage infiltration at diagnosis. Hepatic CD4 lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation.

Conclusion: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).
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http://dx.doi.org/10.1002/hep.28851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5191928PMC
January 2017

Deep Sequencing Reveals Novel Genetic Variants in Children with Acute Liver Failure and Tissue Evidence of Impaired Energy Metabolism.

PLoS One 2016 2;11(8):e0156738. Epub 2016 Aug 2.

Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, United States of America.

Background & Aims: 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.

Methods: 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.

Results: 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.

Conclusion: 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156738PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970743PMC
August 2017

Two Case Reports of FGF23-Induced Hypophosphatemia in Childhood Biliary Atresia.

Pediatrics 2016 08;138(2)

Divisions of Endocrinology.

Cholestatic liver disease has long been associated with childhood rickets, secondary to impaired absorption of fat-soluble vitamin D. Elevated serum levels of fibroblast growth factor 23 (FGF23), secondary to genetic defects or tumor-induced osteomalacia, causes hypophosphatemic rickets in childhood. We present 2 infants with end-stage liver disease due to biliary atresia (BA) who developed hypophosphatemia with renal phosphate wasting. Serum FGF23 levels were elevated more than 8 times the upper limit of normal, and the older infant showed radiographic evidence of rickets. Both infants required large supplements of phosphate in addition to calcitriol. Following liver transplantation, FGF23 normalized in both patients and phosphate and calcitriol supplementation were discontinued. Immunohistochemistry revealed ectopic overexpression of FGF23 by hepatocytes in the BA liver. These observations highlight a unique cause of hypophosphatemic rickets in childhood and suggest the need for further investigation into the relationship between BA and other cholestatic disorders, and bone metabolism.
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http://dx.doi.org/10.1542/peds.2015-4453DOI Listing
August 2016
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