Objective: To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis.

Methods: The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family.

Results: Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including SLC25A13 gene variation in 3 patients who were diagnosed with citrin deficiency, JAG1 gene variation in 3 patients who were diagnosed with Alagille syndrome, ABCB11 gene variation in 3 patients who were diagnosed with progressive familial intrahepatic cholestasis type 2, HSD3B7 gene variation in 1 patient who was diagnosed with congenital bile acid synthesis defect type 1, AKR1D1 gene variation in 1 patient who was diagnosed with congenital bile acid synthesis defect type 1, NPC1 gene variation in 1 patient who was diagnosed with Niemann-Pick disease, and CFTR gene variation in 1 patient who was diagnosed with cystic fibrosis.

Conclusions: The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.

目的: 分析婴儿肝内胆汁淤积症患儿的临床资料及基因变异特点。

方法: 收集2017年6月至2019年6月于首都儿科研究所附属儿童医院消化内科就诊的疑似遗传代谢性婴儿肝内胆汁淤积症患儿的临床资料，采用目标基因捕获结合高通量测序技术进行基因检测，并进行Sanger验证。

结果: 共纳入40例患儿，13例（32%）患儿发现致病基因，包括3例SLC25A13基因突变导致的希特林蛋白缺乏症，3例JAG1基因突变导致的Alagille综合征，3例ABCB11基因突变导致的进行性家族性肝内胆汁淤积症2型，1例HSD3B7基因突变导致的先天性胆汁酸合成障碍1型，1例AKR1D1基因突变导致的先天性胆汁酸合成障碍2型，1例NPC1基因突变导致的尼曼匹克病，1例CFTR基因突变导致的囊性纤维化。

结论: 婴儿肝内胆汁淤积症病因繁多，高通量测序技术对临床疑似遗传代谢病的肝内胆汁淤积症患儿的诊断有重要价值。

Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3alpha-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.

Objective To observe the effects of Dahuang Lingxian Capsule (DLC) for regulating and controlling expression of hepatocyte transporters and bile metabolism spectrum in gallstone mice u- sing Western blot and gas chromatography-mass spectrometer ( GC-MS) , and to explore its possible mechanism. Methods Fifty male C57BL/6 mice were randomly divided into five groups, i.e., the normal control group (N), the model group (M) , the ursodeoxycholic acid (UDCA) control group (U) , the drug control group (Y) ,the DLC treatment group (D) , 10 in each group. Mice in group N and group Y were fed with common forage. Forage with high fat, high calorie, high cholesterol was fed to mice in group M, U, and D to induce cholecystolithiasis. Meanwhile, UDCA solution (130 mg kg⁻¹ - d⁻¹) was administered to mice in group U by gastrogavage. DLC decoction (13 g - kg⁻¹ - d1) was administered to mice in group Y and D by gastrogavage. Equal volume of normal saline was administered to mice in group N and M by gastrogavage. After 8-week intervention, gallstone formation rate was observed. Changes of ATP binding cassette subfamily B member 11 (Abcb1l ) and ATP binding cassette subfamily C member 2 (Abcc2) were observed using Western blot. Metabonomic features were detected by GC-MS. Results Improper operation occurred during modeling. One mouse died in group U since its esophagus was pricked by lavage needle. The gallstone formation rate was 100. 00% , higher than that in group N and Y (0.00%, 0.00%; x² =20. 00,P <0. 01). Compared with group M, the gallstone formation rate was reduced in group U and D (44. 44%, x² =7. 54,P = 0. 011 ; 30. 00%, X² = 10. 77, P 0. 003). Cholesterol characteristic absorption peak could be seen at 2 939, 1 446, 1 382, and 1 056 cm - after infrared spec- trum analysis. There was statistical difference in Abcbl1 and Abcc2 among the 5 groups (F =41. 89, P < 0. 01; F=90. 01 , P <0. 01). Compared with group N, expressions of Abcb1 1 and Abcc2 transporters sig- nificantly decreased in group M (P <0. 01). Compared with group M, expressions of Abcb11 and Abcc2 transporters significantly increased in group U, Y, D (P <0. 01). Compared with other groups, concentrations of endogenous metabolites such as alanine, citric acid, lysine, methionine, phenylalanine, tyrosine, cholesterol, low-density lipoprotein (LDL), glycerine, malic acid, acetone increased; concentrations of lactic acid, glutamine, amine glycine, choline, and taurine decreased (P <0. 05, P <0. 01). Con- clusion DLC could stabilize expression of Abcb1 1 and Abcc2, change or improve pathological secretion of bile and its metabolites, thereby achieving the effect of gallstone prevention and treatment.

Bile acids are key components of bile required for human health. In humans and mice, conditions of reduced bile flow, cholestasis, induce bile acid detoxification by producing tetrahydroxylated bile acids (THBA), more hydrophilic and less cytotoxic than the usual bile acids, which are typically di- or tri- hydroxylated. Mice deficient in the Bile Salt Export Pump (Bsep, or Abcb11), the primary bile acid transporter in liver cells, produce high levels of THBA, and avoid the severe liver damage typically seen in humans with BSEP deficiencies. THBA can suppress bile acid-induced liver damage in Mdr2 deficient mice, caused by their lack of phospholipids in bile exposing their biliary tracts to unbound bile acids. Here we review THBA-related works in both animals and humans, and discuss their potential relevance and applications as a class of functional bile acids.

Keywords: BSEP; Bile salt; Cholestasis; Cytochrome P450; MDR; Mouse genetics.

Background and 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/Cas9 genome editing was employed to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice.

Results: In a one-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 MRP9 that belongs to the ATP-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 juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared to WT mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge.

Conclusion: 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.

Keywords: Progressive familial intrahepatic cholestasis; bile duct paucity; mouse; zebrafish.

Objectives: Cholestasis is caused by a wide variety of etiologies, often genetic in origin. Broad overlap in clinical presentations, particularly in newborns, renders prioritizing diagnostic investigations challenging. In this setting, a timely, comprehensive assessment using a multigene panel by a clinical diagnostic laboratory would likely prove useful. We summarize initial findings from a testing program designed to discover genetic causes of cholestasis.

Methods: A neonatal/adult sequencing panel containing 66 genes (originally 57; 9 added March 2017) relevant to cholestasis was used. A broad range of eligible patients were enrolled with current/past history of cholestasis without an identified cause, or unexplained chronic liver disease. DNA sequencing utilized a custom-designed capture library, and variants were classified and reported as benign, likely benign, variant of unknown significance (VOUS), likely pathogenic (LP), or pathogenic (P), according to the clinical interpretation workflow at EGL Genetics.

Results: A total of 2433 samples were submitted between February 2016 and December 2017; 2171 results were reported. Median turnaround time was 21 days. Results from the 2171 subjects (57% <1 year old) included 583 P variants, 79 LP variants, and 3117 VOUS; 166 P/LP variants and 415 VOUS were novel. The panel's overall diagnostic yield was 12% (n=265/2171) representing 32 genes. The top 5 genetic diagnoses for the group, in order: JAG1 + NOTCH2 (Alagille Syndrome), ABCB11, SERPINA1, ABCB4, and POLG.

Conclusions: These findings support the utility of comprehensive rapid multigene testing in diagnosing cholestasis and highlight the evolving understanding of genetic variants contributing to the pathogenesis of cholestasis.

An infographic is available for this article at:http://links.lww.com/MPG/C250.

Benign recurrent intrahepatic cholestasis is a rare disorder characterized by recurrent episodes of cholestatic jaundice without liver damage. A mutation in the ABCB11 gene encoding bile salt export pump protein causes the disease. A 16-year-old boy with severe jaundice is presented here. His laboratory tests were consistent with intrahepatic cholestasis despite having normal gamma-glutamyl transpeptidase levels. Acute and chronic liver diseases with viral, metabolic, and autoimmune etiology were excluded. Magnetic resonance imaging revealed normal intra- and extrahepatic bile ducts. A liver biopsy showed cholestasis in the centrilobular and intermediate zones and sinusoidal dilatation. Genetic testing revealed a homozygous c.3083_3084delCAinsTG (Ala1028Val) mutation in the ABCB11 gene. The patient was treated with ursodeoxycholic acid 20 mg/kg/day and cholestyramine 4 g twice daily, and total bilirubin decreased to normal ranges after two months of therapy. This mutation (c.3083_3084delCAinsTG) in the ABCB11 gene is the first reported in a patient with benign recurrent intrahepatic cholestasis type 2.

Keywords: ABCB11; bile salt export pump; child; cholestatic jaundice; mutation; pruritus.

ABCB11 deficiency, formerly benign recurrent intrahepatic cholestasis (BRIC) is a very rare hereditary disorder characterized by the recurrent and intermittent episodes of cholestasis, jaundice, and pruritus. We report the case of a 12-year-old boy presenting with recurrent episodes of jaundice and severe pruritis since childhood. An extensive workup was done to rule out all the possible etiologies. Liver biopsy was done and histopathology was consistent with intrahepatic cholestasis. Immunohistochemistry, enzyme studies, and genetic testing confirmed the diagnosis. The patient was treated with Ursodeoxycholicacid and is on regular follow-up. We report this case due to the rarity of the disease in South India and to highlight the importance of genetic testing, which is the gold standard for diagnosis as well as for the classification of the disease. These patients should be under regular follow-up as those with fibrosis progression are at a risk for cholangiocarcinoma and hepatocellular carcinoma.

Keywords: ABCB11; Benign Recurrent Intrahepatic Cholestasis; cholestasis; cirrhosis; jaundice.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from 7 donors were maintained as near-confluent collagen-matrigel sandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36 and 43 days. Key hepatocyte genes were evaluated including the differentiation markers albumin (ALB), transferrin (TR) and transthyretin (TTR); the hepatocyte-specific asialoglycoprotein receptor (ASGR1); cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, SLCO2B1; efflux transporter isoforms ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCG2; as well as the nonspecific housekeeping gene hypoxanthine ribosyl transferase (HPRT1). The well-established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 ( <50% decrease in expression), ABCC4 ( >400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. Significance Statement We report that while human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied towards the evaluation of drug properties requiring an extended period of treatment and evaluation.

Keywords: Cell differentiation; Cytochrome P450 (CYP); Uptake transporters (OATP, OAT, OCT, PEPT, MCT, NTCP, ASBT, etc.); efflux transporters (P-gp, BCRP, MRP, MATE, BSEP, etc); hepatocytes.

Introduction: High-fat diet (HFD)-induced obesity impairs clearance of cholesterol through the Reverse Cholesterol Transport (RCT) pathway, with downregulation in hepatic expression of cholesterol and bile acid transporters, ABCG5/8 and ABCB11, and reduced high-density lipoprotein (HDL) cholesterol efflux capacity (CEC). Within the current study we hypothesized that development of hepatosteatosis, secondary to adipose-tissue dysfunction, contributes to obesity-impaired RCT and such effects could be mitigated using the anti-inflammatory drug sodium salicylate (NaS).

Materials and methods: C57BL/6j mice, fed HFD±NaS or low-fat diet (LFD) for 24 weeks, underwent glucose and insulin tolerance testing and ³H-cholesterol movement from macrophage-to-feces was assessed in vivo. HDL-CEC was determined ex vivo. Cytokine secretion from adipose-derived stromal vascular fraction (SVF) cells was measured ex vivo. Liver and HDL proteins were determined by mass spectrometry and analysed using Ingenuity Pathway Analysis.

Results: NaS delayed HFD-induced weight-gain, abrogated priming of pro-IL-1β in SVFs, attenuated insulin resistance and prevented ectopic fat accumulation in the liver. Prevention of hepatosteatosis coincided with increased expression of PPAR-alpha/beta-oxidation proteins with NaS and reduced expression of LXR/RXR-induced proteins including apolipoproteins; these latter effects were mirrored within the HDL proteome in circulation. Despite remarkable protection against steatosis, HFD-induced hypercholesterolemia and repression of the liver-to-bile cholesterol transporter ABCG5/8 were not rescued with NaS.

Discussion/conclusions: The cardiometabolic health benefits of NaS are likely attributable to reprogramming of hepatic metabolic pathways to increase fatty acid utilization in the setting of nutritional overabundance. Reduced hepatic cholesterol levels, coupled with reduced LXR/RXR-induced proteins, may underlie lack of rescue of ABCG5/8 expression with NaS. This remarkable protection against HFD-induced hepatosteatosis did not translate to improvements in cholesterol homeostasis.

Keywords: HDL proteomics; Metabolic Inflammation; Reverse Cholesterol transport; Sodium Salicylate; hepatosteatosis; liver proteomics.

Introduction: Heterozygous defects in genes implicated in Progressive Familial Intrahepatic Cholestasis have been described in milder forms of cholestatic diseases. Our aim is to describe clinical, laboratory and imaging characteristics as well as treatment and outcome of a cohort of pediatric patients with heterozygous mutations in ATP8B1, ABCB11 or ABCB4.

Patients and methods: We present a retrospective descriptive study including pediatric patients with at least one heterozygosis defect in ATP8B1, ABCB11 or ABCB4 diagnosed after a cholestatic episode. Clinical, diagnostic and outcome data were collected including gene analysis (panel of PFIC NextGeneDx®).

Results: 7 patients showed a heterozygous mutation: 3 patients in ABCB4, 1 in ABCB11, 2 in ABCB4 and ABCB11 and 1 in ATP8B1. The median onset age was 5.5 years with a median time of follow-up of 6 years. The initial presentation was pruritus followed by asymptomatic hypertransaminasemia and persistent cholestasis. Two patients had family history of gallbladder stones and mild hepatitis. All showed elevated transaminases and bile acids, high gamma glutamyl-transferase (GGT) in 3 and conjugated bilirubin in 2 patients. Liver biopsy showed inflammatory infiltrate or mild fibrosis with normal immunohistochemistry. All patients were treated with ursodeoxycholic acid, two patients requiring the addition of resincholestyramine. During follow-up, 3 patients suffered limited relapses of pruritus. No disease progression was observed.

Conclusion: Heterozygous mutations in genes coding proteins of the hepatocellular transport system can cause cholestatic diseases with great phenotypic variability. The presence of repeated episodes of hypertransaminasemia or cholestasis after a trigger should force us to rule out the presence of these heterozygous mutations in genes involved in CIFP.

Keywords: ABCB11; ABCB4; ATP8B1; BSEP; Colestasis intrahepática progresiva familiar; FIC1; MDR3; Progressive Familial Intrahepatic Cholestasis.

Biliary atresia (BA) is the most serious type of obstructive cholangiopathy that occurs in infants. BA can be the cause of death in children under 2 years if untreated early. However, the etiology of the disease is not known. BA is considered to be the result of the destruction of the bile duct system including the accumulation of bile acids. The bile salt export pump, a transporter protein encoded by the ABCB11 gene, plays the main role in the exportation and accumulation of bile acids. The p.Val444Ala variant in this gene is known to be associated with many cholestatic diseases. However, to date no study have been performed to evaluate the association of this variant with susceptibility to the risk of BA. In this study, we aimed to identify the frequency of p.Val444Ala variant and the risk of BA in Vietnamese patients.The polymerase chain reaction (PCR)- restriction fragment length polymorphism method was used to determine the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene in 266 Vietnamese patients with BA and 150 healthy people. The gene segment containing the variant was amplified by PCR with specific primers, after that the PCR products were cut by HaeIII restriction enzyme and analyzed on agarose gel to determine the genotypes. The frequency of alleles was assessed statistically to determine the association between these alleles and the risk of disease in patients.In our study, the frequency of alleles c.1331T>C (p.Val444Ala, rs2287622) in the ABCB11 gene was investigated the first time in the patients with BA. The results showed that CC and TC genotypes were significantly different between BA patients and healthy people (P < .01), and the C allele was associated with an increased risk of BA (odds ratio = 2.47; 95% confidence interval: 1.84-3.32; P < .01). The initial results of clinical, biochemical, and genetic analysis in our study suggested that the p.Val444Ala variant in the ABCB11 gene may be a susceptibility factor for the disease in Vietnamese patients with BA. These results provided new insights into the role of this ABCB11 variant in the pathogenesis of BA.

The aim of the present study was to investigate the effects and the underlying mechanisms of Yinchenhao Decoction (YCHD), a traditional Chinese medicine formulation, on C57BL/6 mice with lithogenic diet (LD)-induced cholelithiasis. The condition of cholelithiasis was evaluated using a six-level criteria. Levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) in the serum and liver tissue were measured using enzyme colorimetry. Concentrations of TC, phospholipids (PL) and total bile acids (TBA) in the bile were measured to calculate the cholesterol saturation index. Liver histopathology was microscopically observed and mRNA expression levels of ABCG5, ABCG8, SRBI, ABCB4, ABCB11 and NPC1L1 involved in cholesterol metabolism were measured using reverse transcription-quantitative PCR. The results showed that feeding mice the LD induced cholelithiasis, along with abnormal serum biochemical indices and imbalances in biliary cholesterol homeostasis. Increased ALT and ALP levels in the serum and ALT, ALP, TC and LDL-C levels in the serum and liver indicated the existence of hepatocyte injury, which were consistent with the pathological changes. YCHD treatment ameliorated the serum and hepatic biochemical abnormalities and adjusted the biliary imbalance. In addition, elevated expression of ATP-binding cassette subfamily G member 5/8, scavenger receptor class B type I and Niemann-Pick C1 Like 1 in the liver and small intestine were observed at the onset of cholelithiasis but were reversed by YCHD. Taken together, results from the present study suggest that YCHD ameliorated LD-induced cholelithiasis mice, which may be caused by improvements in biliary cholesterol supersaturation and regulation of cholesterol metabolism.

Keywords: Yinchenhao decoction; biliary cholesterol supersaturation; cholelithiasis; cholesterol metabolism.

Purpose: Irinotecan (IR) displays significant PK/PD variability. This study evaluated functional hepatic imaging (HNI) and extensive pharmacogenomics (PGs) to explore associations with IR PK and PD (toxicity and response).

Methods: Eligible patients (pts) suitable for Irinotecan-based therapy. At baseline: (i) PGs: blood analyzed by the Affymetrix-DMET™-Plus-Array (1936 variants: 1931 single nucleotide polymorphisms [SNPs] and 5 copy number variants in 225 genes, including 47 phase I, 80 phase II enzymes, and membrane transporters) and Sanger sequencing (variants in HNF1A, Topo-1, XRCC1, PARP1, TDP, CDC45L, NKFB1, and MTHFR), (ii) HNI: pts given IV 250 MBq-^99mTc-IDA, data derived for hepatic extraction/excretion parameters (CL_HNI, T_1/2-HNI, 1hRET, HEF, T_d1/2). In cycle 1, blood was taken for IR analysis and PK parameters were derived by non-compartmental methods. Associations were evaluated between HNI and PGs, with IR PK, toxicity, objective response rate (ORR) and progression-free survival (PFS).

Results: N = 31 pts. The two most significant associations between PK and PD with gene variants or HNI parameters (P < 0.05) included: (1) PK: SN38-Metabolic Ratio with CL_HNI, 1hRET, (2) Grade 3+ diarrhea with SLC22A2 (rs 316019), GSTM5 (rs 1296954), (3) Grade 3+ neutropenia with CL_HNI, 1hRET, SLC22A2 (rs 316019), CYP4F2 (rs2074900) (4) ORR with ALDH2 (rs 886205), MTHFR (rs 1801133). (5) PFS with T_1/2-HNI, XDH (rs 207440), and ABCB11 (rs 4148777).

Conclusions: Exploratory associations were observed between Irinotecan PK/PD with hepatic functional imaging and extensive pharmacogenomics. Further work is required to confirm and validate these findings in a larger cohort of patients.

Australian new zealand clinical trials registry (anzctr) number: ACTRN12610000897066, Date registered: 21/10/2010.

Keywords: DMET; Hepatic functional imaging; Irinotecan; Pharmacodynamics; Pharmacokinetics.

Background: Infants who are born in The Netherlands receive oral vitamin K to prevent bleeding due to a vitamin K deficiency. However the incidence of such bleedings are higher compared to other European countries. Therefore, the Dutch Health Council advised in 2017 to change this guideline from oral to intramuscular administration.

Case description: A 2 months old girl presented with a fatal intracranial hemorrhage. A day before she developed a hematoma on her foot and orbit. Despite daily oral vitamin K, blood results revealed a severe vitamin K deficiency-related bleeding. Postmortem liver biopsy and genetic studies showed cholestasis as the most likely cause of malabsorption of fat soluble vitamins due to a heterozygous pathogenic variant in the ABCB11 gene, which could possibly be transient.

Conclusion: Our case illustrates the importance of revising the national guideline for vitamin K prophylaxis to intramuscular administration, according to the recommendation of the Dutch Health Council.

Background & aim: Persistent hepatocellular secretory failure (PHSF) is a rare condition of severe cholestasis caused by drugs, toxins, infection, or temporary biliary obstruction. Real-world data on rifampicin in cholestasis, particularly among patients with deep jaundice, are scarce. We aimed to expand the knowledge on the efficacy and safety of rifampicin treatment in PHSF patients.

Methods: Sixteen patients with PHSF who had received rifampicin treatment (150-300 mg/d) at our institution from September 2016 to July 2020 were included. Treatment efficacy was assessed by 20% improvement in serum total bilirubin (TBIL) concentration at 4 weeks. Follow-up was continued until the concentration of TBIL returned to normal.

Results: Among the 16 enrolled patients, 12 had predisposing factors (drugs, infection or transient biliary obstruction). ATP8B1 or ABCB11 mutations were detected in the other 4 patients without trigger events. UGT1A1 mutations were found in 7/10 patients. Before rifampicin treatment, the median TBIL level was 352 μmol/L (range 171-591 μmol/L). TBIL > 20% improvement was observed in 14 patients at 4 weeks. TBIL levels of 14 patients eventually returned to normal after 6-12 weeks of rifampicin treatment. The remaining 2 patients who did not respond to rifampicin finally recovered after nasobiliary drainage. Except for one patient with transient drug-induced hepatitis, no other serious adverse events were observed.

Conclusions: Rifampicin could be a promising option for most PHSF patients. Most PHSF patients have UGT1A1 deficiency, which may be the target of rifampicin.

Keywords: ABCB11; ATP8B1; UGT1A1; cholestasis; rifampicin.

Arazyme and extracts of soy leaves (ESLs) are used as ingredients for functional foods; however, their combined administration has not been studied. This study assessed the combined effect of Arazyme and ESLs in high-fat-diet (HFD)-induced obese C57BL/6J mice fed 2 mg/kg Arazyme, 50 mg/kg ESLs, or a combination of 2 mg/kg Arazyme and 50 mg/kg ESLs by oral gavage for 13 weeks. Individually, Arazyme and ESLs had no effect on the HFD-induced phenotypes. The combination of Arazyme and ESLs significantly suppressed body weight gain, improved glucose and insulin tolerance, and suppressed hepatic steatosis by reducing lipid synthesis and enhancing lipid utilization gene expression. Furthermore, the combination significantly reduced HFD-induced plasma bile acid reabsorption by suppressing bile acid transporter expression, including the ATP biding cassette subfamily B member 11 (<i><em>Abcb11</em></i>), solute carrier family 10 member 1 (<i>Slc10a1</i>), <i>Slc10a</i><i>2</i>, <i>Slc51a</i>, and <i>Slc51b</i> in the liver and gut. Moreover, the combination of Arazyme and ESLs significantly prevented HFD-induced islet compensation in the pancreas. These results suggest that the incorporation of Arazyme combined with ESLs reduces HFD-induced body weight, hyperglycemia, and hepatic steatosis by regulating liver-gut bile acid circulation in HFD-fed mice. This combination can markedly reduce treatment doses and enhance their therapeutic effects, thereby reducing therapeutic healthcare costs.

Keywords: Arazyme; diet therapy; hepatic steatosis; hyperglycemia; obesity; soy leaf.

Background: We have recently reported a mouse model of PNAC in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid transporter Abcb11, bilirubin exporter Abcc2, and sterol transporters Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice.

Methods: (1) Recombinant TNFα was administered to mice as well as in hepatocyte cell culture. (2) Tnfr1/2^-/- or WT 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). (3) WT/DSS-PN mice were also infused with infliximab at 10mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum AST, ALT, total bile acids and bilirubin.

Results: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed mRNA transcription of bile (Abcb11[BSEP], Abcc2[MRP2]) and sterol transporters (Abcg5, Abcg8) and their regulators Nr1h3(FXR) and Nr1h4(LXR). DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3 and Nr1h4. In contrast, PNAC development was prevented, and mRNA expression normalized, in both Tnfr1/2^-/- /DSS-PN mice and DSS-PN mice treated with infliximab.

Conclusions: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2 and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation. This article is protected by copyright. All rights reserved.

Keywords: Cholestasis; Infliximab; Intestinal failure associated liver disease; Tumor Necrosis Factor alpha; hepatic macrophages.

ABCB11 encodes bile salt export pump (BSEP), a key regulator in maintaining bile acid (BA) homeostasis. Although inherited ABCB11 mutations have previously been linked to primary liver cancer, whether ABCB11 deficiency leads to liver cancer remains unknown. Here, we analyzed ABCB11 mRNA expression levels in liver tumor specimens (29 with hepatocellular carcinoma (HCC), 1 with intrahepatic cholangiocarcinoma (ICC) and 1 with mixed HCC/ICC) with adjacent normal specimens and published human data sets. Liver tissues obtained from Abcb11-deficient (Abcb11^-/- ) mice and wild-type mice at different ages were compared by histologic, RNA-sequencing, and BA analyses. ABCB11 was significantly downregulated in human liver tumors compared with normal controls. Abcb11^-/- mice demonstrated progressive intrahepatic cholestasis and liver fibrosis, and spontaneously developed HCC and ICC over 12 months of age. Abcb11 deficiency increased BAs in the liver and serum in mice, most of which are farnesoid X receptor (FXR) antagonists/non-agonists. Accordingly, the hepatic expression and transcriptional activity of FXR were downregulated in Abcb11^-/- mouse livers. Administration of the FXR agonist obeticholic acid reduced liver injury and tumor incidence in Abcb11^-/- mice. In conclusion, ABCB11 is aberrantly downregulated and plays a vital role in liver carcinogenesis. The cholestatic liver injury and liver tumors developed in Abcb11^-/- mice are associated with increased FXR antagonist BAs and thereby decreased activation of FXR. FXR activation might be a therapeutic strategy in ABCB11 deficiency diseases. This article is protected by copyright. All rights reserved.

Keywords: ABCB11/BSEP; bile acids; farnesoid X receptor; hepatocellular carcinoma; intrahepatic cholangiocarcinoma.

Background and aims: Parenteral nutrition (PN) associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1β derived from activated hepatic macrophages to suppress hepatocyte FXR signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model.

Methods: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sodium sulfate for 4 days) followed by central venous catheterization and 14 day-infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, biliary and sterol transporter expression were determined.

Results: GW4064 (30mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis, reversed the suppressed mRNA expression of Nr1h4/FXR, Abcb11/BSEP, Abcc2, Abcb4, and Abcg5/8, and normalized serum bile acids. Chromatin-immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll1b, Ccr2, Itgam, Ly6C), and hepatic macrophage cytokine transcription in response to LPS in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1β exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15 and Ostα) were not different among groups, supporting a liver specific effect of GW4064 in this model CONCLUSION: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling resulting in increased expression of canalicular bile, sterol and phospholipid transporters, and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

Keywords: GW4064; IL-1β; Intestinal failure associated liver disease; Liver X receptor; bile acids.

Background: ABCB4-gene mutations are responsible for several cholestatic diseases with a heterogeneous clinical spectrum.

Aims: To analyse phenotype/genotype relationships in ABCB4-mutations.

Methods: Retrospective characterization of adult patients with ABCB4-variations diagnosed between 2015 and 2020. Genotype-phenotype correlations were analysed and compared with previously reported data.

Results: Twenty patients from 12 families were included. Thirteen patients presented recurrent elevated liver tests, eight fulfilled Low-Phospholipid-Associated-Cholelithiasis syndrome criteria, five had Intrahepatic Cholestasis of Pregnancy and three patients developed Drug-Induced-Liver-Injury. ABCB4 screening identified eight different mutations. Five patients were homozygotes to the variant c.504T > C. Ten patients had one mutation in heterozygote-state and five patients had two mutations in compound-heterozygosity. Portal fibrosis occurred in two patients. One of these patients presented progressive fibrosis and progression of cholestasis despite ursodeoxycholic-acid treatment, this patient also harbours a ABCB11 polymorphism.

Conclusion: Although, phenotype-genotype relationships have not been clearly defined, an early diagnosis of ABCB4-variants may have an important role in management decisions and patient outcomes. To our knowledge, we describe a not previously reported deletion (c.1181delT) in ABCB4. The c.504T > C polymorphism, although a silent mutation at the protein level, seems to be associated to different cholestatic diseases. The role of other genes variants, namely ABCB11, as co-factor for progression, needs to be clarified.

Keywords: ABCB4; Idiopathic chronic cholestasis; Intrahepatic cholestasis of pregnancy; Low phospholipid-associated cholelithiasis syndrome; MDR3 protein.

We report a newborn who presented with multiple limb and facial anomalies, endocrine disorders, and progressively worsening low-GGT cholestasis. A liver biopsy revealed hepatocellular cholestasis with giant cell transformation. Immunohistochemical staining revealed complete absence of BSEP protein compared to control liver. A large 2q24-32.2 deletion leading to loss of 78 OMIM genes. Multiple structural anomalies, epilepsy and endocrine anomalies have been described with hemizygous loss of these genes. This deletion also resulted in complete heterozygous deletion of ABCB11, which encodes the bile salt export pump (BSEP). Genetic analysis did not reveal any pathogenic variants, deletions, or duplications in the other ABCB11 allele. A heterozygous variant in NR1H4, which causes the autosomal recessive progressive familial intrahepatic cholestasis type 5, was also detected. The possible explanations for the PFIC type 2 phenotype in heterozygous loss of ABCB11 include genetic modifiers or di-genic disease with a compound ABCB11 deletion and an NR1H4 missense variant; or undetected pathogenic variants in the other ABCB11 or NR1H4 alleles.

Keywords: congenital heart disease; congenital hypothyroidism; dysmorphology; farnesoid X-activated receptor; metabolic liver; neonatal cholestasis.