Diagnostic accuracy of serum matrix metalloproteinase-7 as a biomarker of biliary atresia in a large North American cohort.

BACKGROUND AIMS: High levels of serum matrix metalloproteinase-7 (MMP-7) have been linked to biliary atresia (BA), with wide variation in concentration cutoffs. We investigated accuracy of serum MMP-7 as a diagnostic biomarker in a large North American cohort. APPROACH RESULTS: MMP-7 was measured in serum samples of 399 cholestatic infants in the Prospective Database of Infants with Cholestasis study of the Childhood Liver Disease Research Network, 201 infants with BA and 198 with non-BA cholestasis (age median: 64 and 59 days, p=0.94). MMP-7 was assayed on antibody-bead fluorescence (single-plex) and time resolved-fluorescence energy transfer (TR-FRET) assays. Discriminative performance of MMP-7 was compared with other clinical markers. On the single-plex assay, MMP-7 generated an area under receiver operating curve (AUROC) of 0.90 (confidence interval [CI] 0.87-0.94). At cutoff 52.8 ng/mL, it produced sensitivity=94.03%, specificity=77.78%, positive predictive value=64.46%, and negative predictive value=96.82% for BA. AUROC for gamma-glutamyl transferase (GGT)=0.81 (CI 0.77-0.86), stool color=0.68 (CI 0.63-0.73), and pathology=0.84 (CI 0.76-0.91). Logistic regression models of MMP-7 with other clinical variables individually or combined showed an increase for MMP-7+GGT AUROC to 0.91 (CI 0.88-0.95). Serum concentrations produced by TR-FRET differed from single-plex, with optimal cutoff of 18.2 ng/mL. Results were consistent within each assay technology and generated similar AUROCs. CONCLUSIONS: Serum MMP-7 has high discriminative properties to differentiate BA from other forms of neonatal cholestasis. MMP-7 cutoff values vary according to assay technology. Using MMP-7 in evaluation of cholestatic infants may simplify diagnostic algorithms and shorten time to hepatoportoenterostomy.

Protein biomarkers GDF15 and FGF21 to differentiate mitochondrial hepatopathies from other pediatric liver diseases.

BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.

Neonatal cholestasis in children with Alpha-1-AT deficiency is a risk for earlier severe liver disease with male predominance.

BACKGROUND: Our objective was to better understand the natural history and disease modifiers of Alpha-1-antitrypsin deficiency (AATD), a common genetic liver disease causing hepatitis and cirrhosis in adults and children. The clinical course is highly variable. Some infants present with neonatal cholestasis, which can resolve spontaneously or progress to cirrhosis; others are well in infancy, only to develop portal hypertension later in childhood. METHODS: The Childhood Liver Disease Research Network has been enrolling AATD participants into longitudinal, observational studies at North American tertiary centers since 2004. We examined the clinical courses of 2 subgroups of participants from the several hundred enrolled; first, those presenting with neonatal cholestasis captured by a unique study, enrolled because of neonatal cholestasis but before specific diagnosis, then followed longitudinally (n=46); second, separately, all participants who progressed to liver transplant (n=119). RESULTS: We found male predominance for neonatal cholestasis in AATD (65% male, p=0.04), an association of neonatal gamma-glutamyl transpeptidase elevation to more severe disease, and a higher rate of neonatal cholestasis progression to portal hypertension than previously reported (41%) occurring at median age of 5 months. Participants with and without preceding neonatal cholestasis were at risk of progression to transplant. Participants who progressed to liver transplant following neonatal cholestasis were significantly younger at transplant than those without neonatal cholestasis (4.1 vs. 7.8 years, p=0.04, overall range 0.3-17 years). Neonatal cholestasis had a negative impact on growth parameters. Coagulopathy and varices were common before transplant, but gastrointestinal bleeding was not. CONCLUSIONS: Patients with AATD and neonatal cholestasis are at risk of early progression to severe liver disease, but the risk of severe disease extends throughout childhood. Careful attention to nutrition and growth is needed.

LRH-1 agonist DLPC through STAT6 promotes macrophage polarization and prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition-associated cholestasis (PNAC) is an important complication in patients with intestinal failure with reduced LRH-1 expression. Here, we hypothesized that LRH-1 activation by its agonist, dilauroylphosphatidylcholine (DLPC), would trigger signal transducer and activator of transcription 6 (STAT6) signaling and hepatic macrophage polarization that would mediate hepatic protection in PNAC. APPROACH AND RESULTS: PNAC mouse model (oral DSSx4d followed by PNx14d; DSS-PN) was treated with LRH-1 agonist DLPC (30 mg/kg/day) intravenously. DLPC treatment prevented liver injury and cholestasis while inducing hepatic mRNA expression of Nr5a2 (nuclear receptor subfamily 5 group A member 2), Abcb11 (ATP binding cassette subfamily B member 11), Abcg5 (ATP-binding cassette [ABC] transporters subfamily G member 5), Abcg8 (ATP-binding cassette [ABC] transporters subfamily G member 8), nuclear receptor subfamily 0, and ATP-binding cassette subfamily C member 2 ( Abcc2) mRNA, all of which were reduced in PNAC mice. To determine the mechanism of the DLPC effect, we performed RNA-sequencing analysis of the liver from Chow, DSS-PN, and DSS-PN/DLPC mice, which revealed DLPC upregulation of the anti-inflammatory STAT6 pathway. In intrahepatic mononuclear cells or bone-marrow derived macrophages (BMDM) from PNAC mice, DLPC treatment prevented upregulation of pro-inflammatory (M1) genes, suppressed activation of NFκB and induced phosphorylation of STAT6 and its target genes, indicating M2 macrophage polarization. In vitro, incubation of DLPC with cultured macrophages showed that the increased Il-1b and Tnf induced by exposure to lipopolysaccharides or phytosterols was reduced significantly, which was associated with increased STAT6 binding to promoters of its target genes. Suppression of STAT6 expression by siRNA in THP-1 cells exposed to lipopolysaccharides, phytosterols, or both resulted in enhanced elevation of IL-1B mRNA expression. Furthermore, the protective effect of DLPC in THP-1 cells was abrogated by STAT6 siRNA. CONCLUSIONS: These results indicate that activation of LRH-1 by DLPC may protect from PNAC liver injury through STAT6-mediated macrophage polarization.

Sarcopenia is associated with osteopenia and impaired quality of life in children with genetic intrahepatic cholestatic liver disease.

BACKGROUND: Sarcopenia occurs in pediatric chronic liver disease, although the prevalence and contributing factors in genetic intrahepatic cholestasis are not well-described. The objective of this study was to measure muscle mass in school-aged children with genetic intrahepatic cholestasis and assess relationships between sarcopenia, clinical variables, and outcomes. METHODS: Estimated skeletal muscle mass (eSMM) was calculated on dual-energy x-ray absorptiometry obtained in a Childhood Liver Disease Research Network study of children with bile acid synthesis disorders(BASD) alpha-1 antitrypsin deficiency (a1ATd), chronic intrahepatic cholestasis (CIC), and Alagille syndrome (ALGS). Relationships between eSMM, liver disease, and transplant-free survival were assessed. RESULTS: eSMM was calculated in 127 participants (5-18 y): 12 BASD, 41 a1ATd, 33 CIC, and 41 ALGS. eSMM z-score was lower in CIC (-1.6 ± 1.3) and ALGS (-2.1 ± 1.0) than BASD (-0.1 ± 1.1) and a1ATd (-0.5 ± 0.8, p < 0.001). Sarcopenia (defined as eSMM z-score ≤- 2) was present in 33.3% of CIC and 41.5% of ALGS participants. eSMM correlated with bone mineral density in the 4 disease groups (r=0.52-0.55, p < 0.001-0.07), but not serum bile acids, bilirubin, aspartate aminotransferase/platelet ratio index, or clinically evident portal hypertension. Of the 2 patients who died (1 with sarcopenia) and 18 who underwent liver transplant (LT, 4 with sarcopenia), eSMM z-score did not predict transplant-free survival. eSMM z-score correlated with the Physical Pediatric Quality of Life Inventory score (r=0.38-0.53, p = 0.007-0.04) in CIC and a1ATd. CONCLUSION: Severe sarcopenia occurs in some children with ALGS and CIC. The lack of correlation between eSMM and biochemical cholestasis suggests mechanisms beyond cholestasis contribute to sarcopenia. While sarcopenia did not predict transplant-free survival, LT and death were infrequent events. Future studies may define mechanisms of sarcopenia in genetic intrahepatic cholestasis.

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

BACKGROUND: We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. METHODS: WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1ß (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. RESULTS: In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1ß or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. CONCLUSIONS: Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1ß and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.

Interim results from an ongoing, open-label, single-arm trial of odevixibat in progressive familial intrahepatic cholestasis.

Background & Aims: PEDFIC 2, an ongoing, open-label, 72-week study, evaluates odevixibat, an ileal bile acid transporter inhibitor, in patients with progressive familial intrahepatic cholestasis. Methods: PEDFIC 2 enrolled and dosed 69 patients across two cohorts; all received odevixibat 120 µg/kg per day. Cohort 1 comprised children from PEDFIC 1, and cohort 2 comprised new patients (any age). We report data through 15 July 2020, with Week 24 of PEDFIC 2 the main time point analysed. This represents up to 48 weeks of cumulative exposure for patients treated with odevixibat from the 24-week PEDFIC 1 study (cohort 1A) and up to 24 weeks of treatment for those who initiated odevixibat in PEDFIC 2 (patients who received placebo in PEDFIC 1 [cohort 1B] or cohort 2 patients). Primary endpoints for this prespecified interim analysis were change from baseline to Weeks 22-24 in serum bile acids (sBAs) and proportion of positive pruritus assessments (≥1-point drop from PEDFIC 2 baseline in pruritus on a 0-4 scale or score ≤1) over the 24-week period. Safety monitoring included evaluating treatment-emergent adverse events (TEAEs). Results: In cohort 1A, mean change from PEDFIC 1 baseline to Weeks 22-24 of PEDFIC 2 in sBAs was -201 µmol/L (p <0.0001). For cohort 1B and cohort 2, mean changes from odevixibat initiation to weeks 22-24 in sBAs were -144 and -104 µmol/L, respectively. The proportion of positive pruritus assessments in the first 24-week period of PEDFIC 2 was 33%, 56%, and 62% in cohorts 1A, 1B, and 2, respectively. Most TEAEs were mild or moderate. No drug-related serious TEAEs occurred. Conclusions: Odevixibat in patients with progressive familial intrahepatic cholestasis was generally well tolerated and associated with sustained reductions in sBAs and pruritus. Clinical Trials Registration: This study is registered at ClinicalTrials.gov (NCT03659916). Impact and Implications: Disrupted bile flow is a hallmark feature of patients with progressive familial intrahepatic cholestasis and can result in build-up of bile constituents in the liver with spill over into the bloodstream; other effects that patients can experience include extremely itchy skin, and because not enough bile reaches the gut, patients can have problems digesting food, which may lead to poor growth. Odevixibat is an orally administered medication that shunts bile acids away from the liver. The current study, called PEDFIC 2, suggested that odevixibat can improve the problematic signs and symptoms of progressive familial intrahepatic cholestasis and was generally safe for patients.

Cytomegalovirus in biliary atresia is associated with increased pretransplant death, but not decreased native liver survival.

BACKGROUND: Biliary atresia (BA) is likely caused by a common phenotypic response to various triggers; one proposed trigger, cytomegalovirus (CMV), may lead to worse outcomes. The aim of this study was to determine the severity of disease and pretransplant outcomes of infants with BA, who have evidence of CMV (CMV+) at diagnosis compared with CMV-negative (CMV-) infants. METHODS: The study used data and biospecimens from the Childhood Liver Disease Research Network PROBE study of cholestatic infants. Plasma obtained at the time of hepatic portoenterostomy (HPE) of 249 infants with BA was tested for CMV by DNA-PCR and CMV-IgM. Comparisons between CMV+ and CMV- infants were made using Wilcoxon rank sum, Student t test, chi-square, or Fisher exact test. Native liver survival (NLS) outcomes were analyzed using Kaplan-Meier and Cox regression adjusting for age at HPE; pretransplant patient survival outcomes were analyzed using a competing risk model and adjusting for age at HPE. RESULTS: CMV+ infants (n = 29, 12%) underwent HPE later (67.8±13.6 d vs. 55.1±18.5 d, p = 0.0005) and had higher baseline alkaline phosphatase and aminotransferases. There was no difference between groups in jaundice clearance or NLS. The subdistribution HR of pretransplant death for CMV+ infants adjusted for age at HPE was 3.8 (p = 0.034). CONCLUSIONS: CMV infection at the time of HPE in infants with BA is not associated with worse NLS despite the association with worse liver injury, older age at HPE, and increased risk of pretransplant death adjusted for age at HPE. Continued evaluation of the consequences of CMV infection and the effects of antiviral treatment should be explored.

Predictors of 6-year event-free survival in Alagille syndrome patients treated with maralixibat, an ileal bile acid transporter inhibitor.

BACKGROUND AND AIMS: Refractory pruritus and other complications of cholestasis are indications for liver transplantation (LT) in patients with Alagille syndrome (ALGS). We evaluated predictors of event-free survival and transplant-free survival in patients with ALGS treated with maralixibat (MRX), an ileal bile acid transporter inhibitor. APPROACH AND RESULTS: We assessed patients with ALGS from 3 clinical trials of MRX with up to 6 years of follow-up. Event-free survival was defined as the absence of LT, surgical biliary diversion, hepatic decompensation, or death; transplant-free survival was the absence of LT or death. Forty-three potential predictors were evaluated, including age, pruritus (ItchRO[Obs] 0-4 scale), biochemistries, platelets, and serum bile acids. Harrell's concordance statistic assessed goodness-of-fit, and then, Cox proportional hazard models confirmed the statistical significance of the predictors identified. A further analysis was performed to identify cutoffs using a grid search. Seventy-six individuals met the criteria of receiving MRX for ≥48 weeks with laboratory values available at week 48 (W48). The median duration of MRX was 4.7 years (IQR: 1.6-5.8); 16 had events (10 LT, 3 decompensation, 2 death, and 1 surgical biliary diversion). The 6-year event-free survival improved with a clinically meaningful >1-point ItchRO(Obs) reduction from baseline to W48 (88% vs. 57%; p = 0.005), W48 bilirubin < 6.5 mg/dL (90% vs. 43%; p < 0.0001), and W48 serum bile acid < 200 µmol/L (85% vs. 49%; p = 0.001). These parameters were also predictive of 6-year transplant-free survival. CONCLUSIONS: Improvement in pruritus by 48 weeks, and lower W48 bilirubin and serum bile acid levels were associated with fewer events. These data may help identify potential markers of disease progression for ALGS patients treated with MRX.

Pharmacologic inhibition of HNF4α prevents parenteral nutrition associated cholestasis in mice.

Prolonged parenteral nutrition (PN) can lead to PN associated cholestasis (PNAC). Intestinally derived lipopolysaccharides and infused PN phytosterols lead to activation of NFκB, a key factor in PNAC. Our objective was to determine if inhibition of HNF4α could interfere with NFκB to alleviate murine PNAC. We showed that HNF4α antagonist BI6015 (20 mg/kg/day) in DSS-PN (oral DSS x4d followed by Total PN x14d) mice prevented the increased AST, ALT, bilirubin and bile acids and reversed mRNA suppression of hepatocyte Abcg5/8, Abcb11, FXR, SHP and MRP2 that were present during PNAC. Further, NFκB phosphorylation in hepatocytes and its binding to LRH-1 and BSEP promoters in liver, which are upregulated in DSS-PN mice, were inhibited by BI6015 treatment. BI6015 also prevented the upregulation in liver macrophages of Adgre1 (F4/80) and Itgam (CD11B) that occurs in DSS-PN mice, with concomitant induction of anti-inflammatory genes (Klf2, Klf4, Clec7a1, Retnla). In conclusion, HNF4α antagonism attenuates PNAC by suppressing NFκB activation and signaling while inducing hepatocyte FXR and LRH-1 and their downstream bile and sterol transporters. These data identify HNF4α antagonism as a potential therapeutic target for prevention and treatment of PNAC.

Clinical spectrum and genetic causes of mitochondrial hepatopathy phenotype in children.

BACKGROUND: Alterations in both mitochondrial DNA (mtDNA) and nuclear DNA genes affect mitochondria function, causing a range of liver-based conditions termed mitochondrial hepatopathies (MH), which are subcategorized as mtDNA depletion, RNA translation, mtDNA deletion, and enzymatic disorders. We aim to enhance the understanding of pathogenesis and natural history of MH. METHODS: We analyzed data from patients with MH phenotypes to identify genetic causes, characterize the spectrum of clinical presentation, and determine outcomes. RESULTS: Three enrollment phenotypes, that is, acute liver failure (ALF, n = 37), chronic liver disease (Chronic, n = 40), and post-liver transplant (n = 9), were analyzed. Patients with ALF were younger [median 0.8 y (range, 0.0, 9.4) vs 3.4 y (0.2, 18.6), p < 0.001] with fewer neurodevelopmental delays (40.0% vs 81.3%, p < 0.001) versus Chronic. Comprehensive testing was performed more often in Chronic than ALF (90.0% vs 43.2%); however, etiology was identified more often in ALF (81.3% vs 61.1%) with mtDNA depletion being most common (ALF: 77% vs Chronic: 41%). Of the sequenced cohort (n = 60), 63% had an identified mitochondrial disorder. Cluster analysis identified a subset without an underlying genetic etiology, despite comprehensive testing. Liver transplant-free survival was 40% at 2 years (ALF vs Chronic, 16% vs 65%, p < 0.001). Eighteen (21%) underwent transplantation. With 33 patient-years of follow-up after the transplant, 3 deaths were reported. CONCLUSIONS: Differences between ALF and Chronic MH phenotypes included age at diagnosis, systemic involvement, transplant-free survival, and genetic etiology, underscoring the need for ultra-rapid sequencing in the appropriate clinical setting. Cluster analysis revealed a group meeting enrollment criteria but without an identified genetic or enzymatic diagnosis, highlighting the need to identify other etiologies.

Stat3 role in the protective effect of FXR Agonist in parenteral nutrition-associated cholestasis.

BACKGROUND AND AIMS: Parenteral nutrition (PN) in patients with intestinal failure can lead to cholestasis (PNAC). In a PNAC mouse model, farnesoid X receptor (FXR) agonist (GW4064) treatment alleviated IL-1ß-dependent cholestatic liver injury. The objective of this study was to determine whether this hepatic protection of FXR activation is mediated through IL-6-STAT3 signaling. APPROACH AND RESULTS: Hepatic apoptotic pathways [Fas-associated protein with death domain (Fas) mRNA, caspase 8 protein, and cleaved caspase 3] and IL-6-STAT3 signaling, and expression of its downstream effectors Socs1/3 were all upregulated in the mouse PNAC model (dextran sulfate sodium enterally × 4 d followed by total PN for 14 d). Il1r-/- mice were protected from PNAC in conjunction with suppression of the FAS pathway. GW4064 treatment in the PNAC mouse increased hepatic FXR binding to the Stat3 promoter, further increased STAT3 phosphorylation and upregulated Socs1 and Socs3 mRNA, and prevented cholestasis. In HepG2 cells and primary mouse hepatocytes, IL-1ß induced IL-6 mRNA and protein, which were suppressed by GW4064. In IL-1ß or phytosterols treated HepG2 and Huh7 cells, siRNA knockdown of STAT3 significantly reduced GW4064-upregulated transcription of hepatoprotective nuclear receptor subfamily 0, group B, member 2 (NR0B2) and ABCG8. CONCLUSIONS: STAT3 signaling mediated in part the protective effects of GW4064 in the PNAC mouse, and in HepG2 cells and hepatocytes exposed to either IL-1ß or phytosterols, 2 factors critical in PNAC pathogenesis. These data demonstrate that FXR agonists may mediate hepatoprotective effects in cholestasis by inducing STAT3 signaling.

Thioredoxin reductase 1 regulates hepatic inflammation and macrophage activation during acute cholestatic liver injury.

BACKGROUND AND AIMS: Cholestatic liver diseases, including primary sclerosing cholangitis, are characterized by periportal inflammation with progression to hepatic fibrosis and ultimately cirrhosis. We recently reported that the thioredoxin antioxidant response is dysregulated during primary sclerosing cholangitis. The objective of this study was to examine the impact of genetic and pharmacological targeting of thioredoxin reductase 1 (TrxR1) on hepatic inflammation and liver injury during acute cholestatic injury. APPROACH AND RESULTS: Primary mouse hepatocytes and intrahepatic macrophages were isolated from 3-day bile duct ligated (BDL) mice and controls. Using wildtype and mice with a liver-specific deletion of TrxR1 (TrxR1LKO), we analyzed the effect of inhibition or ablation of TrxR1 signaling on liver injury and inflammation. Immunohistochemical analysis of livers from BDL mice and human cholestatic patients revealed increased TrxR1 staining in periportal macrophages and hepatocytes surrounding fibrosis. qPCR analysis of primary hepatocytes and intrahepatic macrophages revealed increased TrxR1 mRNA expression following BDL. Compared with sham controls, BDL mice exhibited increased inflammation, necrosis, and increased mRNA expression of pro-inflammatory cytokines, fibrogenesis, the NLRP3 inflammatory complex, and increased activation of NFkB, all of which were ameliorated in TrxR1LKO mice. Importantly, following BDL, TrxR1LKO induced periportal hepatocyte expression of Nrf2-dependent antioxidant proteins and increased mRNA expression of basolateral bile acid transporters with reduced expression of bile acid synthesis genes. In the acute BDL model, the TrxR1 inhibitor auranofin (10 mg/kg/1 d preincubation, 3 d BDL) ameliorated BDL-dependent increases in Nlrp3, GsdmD, Il1ß, and TNFα mRNA expression despite increasing serum alanine aminotransferase, aspartate aminotransferase, bile acids, and bilirubin. CONCLUSIONS: These data implicate TrxR1-signaling as an important regulator of inflammation and bile acid homeostasis in cholestatic liver injury.

Serum biomarkers correlated with liver stiffness assessed in a multicenter study of pediatric cholestatic liver disease.

BACKGROUND AND AIMS: Detailed investigation of the biological pathways leading to hepatic fibrosis and identification of liver fibrosis biomarkers may facilitate early interventions for pediatric cholestasis. APPROACH AND RESULTS: A targeted enzyme-linked immunosorbent assay-based panel of nine biomarkers (lysyl oxidase, tissue inhibitor matrix metalloproteinase (MMP) 1, connective tissue growth factor [CTGF], IL-8, endoglin, periostin, Mac-2-binding protein, MMP-3, and MMP-7) was examined in children with biliary atresia (BA; n = 187), alpha-1 antitrypsin deficiency (A1AT; n = 78), and Alagille syndrome (ALGS; n = 65) and correlated with liver stiffness (LSM) and biochemical measures of liver disease. Median age and LSM were 9 years and 9.5 kPa. After adjusting for covariates, there were positive correlations among LSM and endoglin ( p = 0.04) and IL-8 ( p < 0.001) and MMP-7 ( p < 0.001) in participants with BA. The best prediction model for LSM in BA using clinical and lab measurements had an R2 = 0.437; adding IL-8 and MMP-7 improved R2 to 0.523 and 0.526 (both p < 0.0001). In participants with A1AT, CTGF and LSM were negatively correlated ( p = 0.004); adding CTGF to an LSM prediction model improved R2 from 0.524 to 0.577 ( p = 0.0033). Biomarkers did not correlate with LSM in ALGS. A significant number of biomarker/lab correlations were found in participants with BA but not those with A1AT or ALGS. CONCLUSIONS: Endoglin, IL-8, and MMP-7 significantly correlate with increased LSM in children with BA, whereas CTGF inversely correlates with LSM in participants with A1AT; these biomarkers appear to enhance prediction of LSM beyond clinical tests. Future disease-specific investigations of change in these biomarkers over time and as predictors of clinical outcomes will be important.

Serum bile acids as a prognostic biomarker in biliary atresia following Kasai portoenterostomy.

BACKGROUND AND AIMS: In biliary atresia, serum bilirubin is commonly used to predict outcomes after Kasai portoenterostomy (KP). Infants with persistently high levels invariably need liver transplant, but those achieving normalized levels have a less certain disease course. We hypothesized that serum bile acid levels could help predict outcomes in the latter group. APPROACH AND RESULTS: Participants with biliary atresia from the Childhood Liver Disease Research Network were included if they had normalized bilirubin levels 6 months after KP and stored serum samples from the 6-month post-KP clinic visit ( n  = 137). Bile acids were measured from the stored serum samples and used to divide participants into ≤40 µmol/L ( n  = 43) or >40 µmol/L ( n  = 94) groups. At 2 years of age, the ≤40 µmol/L compared with >40 µmol/L group had significantly lower total bilirubin, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, bile acids, and spleen size, as well as significantly higher albumin and platelet counts. Furthermore, during 734 person-years of follow-up, those in the ≤40 µmol/L group were significantly less likely to develop splenomegaly, ascites, gastrointestinal bleeding, or clinically evident portal hypertension. The ≤40 µmol/L group had a 10-year cumulative incidence of liver transplant/death of 8.5% (95% CI: 1.1%-26.1%), compared with 42.9% (95% CI: 28.6%-56.4%) for the >40 µmol/L group ( p  = 0.001). CONCLUSIONS: Serum bile acid levels may be a useful prognostic biomarker for infants achieving normalized bilirubin levels after KP.

Real-World Evidence of Neutralizing Monoclonal Antibodies for Preventing Hospitalization and Mortality in COVID-19 Outpatients.

BACKGROUND: Neutralizing monoclonal antibodies (mAbs) were authorized for the treatment of COVID-19 outpatients based on clinical trials completed early in the pandemic, which were underpowered for mortality and subgroup analyses. Real-world data studies are promising for further assessing rapidly deployed therapeutics. RESEARCH QUESTION: Did mAb treatment prevent progression to severe disease and death across pandemic phases and based on risk factors, including prior vaccination status? STUDY DESIGN AND METHODS: This observational cohort study included nonhospitalized adult patients with SARS-CoV-2 infection from November 2020 to October 2021 using electronic health records from a statewide health system plus state-level vaccine and mortality data. Using propensity matching, we selected approximately 2.5 patients not receiving mAbs for each patient who received mAb treatment under emergency use authorization. The primary outcome was 28-day hospitalization; secondary outcomes included mortality and hospitalization severity. RESULTS: Of 36,077 patients with SARS-CoV-2 infection, 2,675 receiving mAbs were matched to 6,677 patients not receiving mAbs. Compared with mAb-untreated patients, mAb-treated patients had lower all-cause hospitalization (4.0% vs 7.7%; adjusted OR, 0.48; 95% CI, 0.38-0.60) and all-cause mortality (0.1% vs 0.9%; adjusted OR, 0.11; 95% CI, 0.03-0.29) to day 28; differences persisted to day 90. Among hospitalized patients, mAb-treated patients had shorter hospital length of stay (5.8 vs 8.5 days) and lower risk of mechanical ventilation (4.6% vs 16.6%). Results were similar for preventing hospitalizations during the Delta variant phase (adjusted OR, 0.35; 95% CI, 0.25-0.50) and across subgroups. Number-needed-to-treat (NNT) to prevent hospitalization was lower for subgroups with higher baseline risk of hospitalization; for example, multiple comorbidities (NNT = 17) and not fully vaccinated (NNT = 24) vs no comorbidities (NNT = 88) and fully vaccinated (NNT = 81). INTERPRETATION: Real-world data revealed a strong association between receipt of mAbs and reduced hospitalization and deaths among COVID-19 outpatients across pandemic phases. Real-world data studies should be used to guide practice and policy decisions, including allocation of scarce resources.

The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease.

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction.

The discriminatory ability of FibroScan liver stiffness measurement, controlled attenuation parameter, and FibroScan-aspartate aminotransferase to predict severity of liver disease in children.

Vibration controlled transient elastography (FibroScan) is used to predict the severity of liver fibrosis and steatosis. In pediatrics, few studies have been performed directly comparing liver histologic features with FibroScan liver stiffness measurements (LSMs) and controlled attenuation parameters (CAPs). The FibroScan-aspartate aminotransferase (FAST) score, which predicts liver disease severity in adult nonalcoholic fatty liver disease (NAFLD), has not been analyzed in children. The aims of this study were to determine if LSM and CAP correlated with liver histologic fibrosis stage and steatosis grade, respectively, and to determine the predictive capacity of FAST in pediatric NAFLD. Research participants (n = 216) included those with FibroScan within 90 days of a liver biopsy. The ability of LSM, CAP, and FAST to predict severity of liver disease was analyzed by Spearman correlation, linear regression, and receiver operating characteristic and C statistic. Significant correlations were identified between LSM and Ishak fibrosis stages, with the strongest correlation occurring in the non-NAFLD group (Spearman r = 0.47, p < 0.0001). LSM adequately predicted Ishak stages F0-2 versus F3-F6 (area under the receiver operating characteristic curve [AUROC], 0.73 for all; 0.77 for non-NAFLD). CAP strongly predicted histologic steatosis grade (r = 0.84; p < 0.0001; AUROC, 0.98). FAST had acceptable discriminatory ability for significant liver disease (AUROC, 0.75). A FAST cutoff ≥0.67 had a sensitivity of 89% but a specificity of only 62% at determining significant liver disease. This study encompasses one of the largest pediatric cohorts describing the accuracy of FibroScan LSM and CAP to predict liver histologic fibrosis stage and steatosis grade, respectively. In order to determine specific LSM, CAP, and FAST cut-off values for fibrosis stages, steatosis grades, and significant liver disease, respectively, a much larger cohort is necessary and will likely entail the need for multicentered studies.