Ductular Reaction Research Articles

Pathophysiological Roles of Ductular Reaction in Liver Inflammation and Hepatic Fibrogenesis

Pathophysiological Roles of Ductular Reaction in Liver Inflammation and Hepatic Fibrogenesis

Gypenosides ameliorate ductular reaction and liver fibrosis via inhibition of hedgehog signaling.

Backgroud and aims: Ductular reaction (DR) is a common pathological change and thought to have a key role in the pathogenesis and progression of liver fibrosis. Our previous study reported Gypenosides (GPs) ameliorated liver fibrosis, however, the anti-fibrotic mechanisms of GPs are still unclear. Methods: Liver fibrosis was induced in rats by carbon tetrachloride combining with 2-acerylaminofluorene (CCl4/2-AAF), and Mdr2 knockout (Mdr2 -/-) mice to evaluate the anti-fibrotic role of GPs. In vitro, WB-F344 cells, a hepatic progenitor cells (HPCs) line, with or without Gli1 overexpressing lentiviral vectors, were induced by sodium butyrate (SB) to validate the mechanism of GPs and NPLC0393, the main ingredient of GPs. Results: Both in CCl4/2-AAF-treated rats and Mdr2 -/- mice, GPs obviously reduced the deposition of collagen and hydroxyproline content, inhibited the activation of hepatic stellate cells and inflammatory cell infiltration. Notably, GPs reduced the expressions of Epcam, CK19, CK7, Dhh, Smo, Ptch2, Gli1 and Gli2. Furthermore, CK19+ cells co-expressed Gli1, while the number of CK19+/Gli1+ cells was decreased by GPs. In vitro, GPs and NPLC0393 inhibited the differentiation of WB-F344 cells toward a biliary phenotype. Mechanistically, GPs and NPLC0393 protected against DR by inhibiting hedgehog signaling, which was supported by the results that DR, triggered directly by Gli1 overexpressing lentiviral vector was blocked by administration with GPs or NPLC0393. Conclusion: GPs attenuated DR and liver fibrosis by inhibiting hedgehog signaling, which provided more evidences and a novel mechanism of anti-fibrotic effect of GPs.

Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.

The Protection Conferred by HSD17B13 rs72613567 on Hepatic Fibrosis Is Likely Mediated by Lowering Ballooning and Portal Inflammation

The Protection Conferred by HSD17B13 rs72613567 on Hepatic Fibrosis Is Likely Mediated by Lowering Ballooning and Portal Inflammation

COVID Cholangiopathy Can Occur Despite Mild COVID

Abstract Introduction/Objective Post-COVID-19 cholangiopathy is a novel entity first noted in patients recovering from critical COVID-19 infection. Since its initial description in May 2021, all cases reported to date have been in patients with a history of critical COVID-19, defined as requiring ICU admission, the development of respiratory or circulatory failure requiring intubation or ECMO, or vasopressor support. Here we report three cases of post-COVID-19 cholangiopathy arising in patients who recovered from non-severe COVID-19. Methods/Case Report Six cases of COVID-19-related cholangiopathy were identified by retrospective review, three of which involved patients who verifiably did not develop critical COVID-19. Histology slides for each case were reviewed and all showed features of secondary sclerosing cholangitis. Patient 1 is a 41yo female who developed COVID-19 after liver transplant (LT). Despite administration of monoclonal antibodies, she required re-transplantation 6 weeks later. Explant histology showed bile infarcts, severe hepatocytic and canalicular cholestasis, ductular reaction, organizing portal vein thrombi, and necrotic bile ducts accompanied by bile lakes. Patient 2 is a 47yo male with alcoholic cirrhosis who was diagnosed with COVID-19 at the time of LT workup, and underwent LT 90 days later. In addition to alcohol-related cirrhosis, explant histology showed dilated bile ducts with periductal fibrosis, as well as severe ductular reaction with proliferating ductules containing thick, inspissated bile. Patient 3 is a 54yo male with history of LT for PSC who developed mild COVID-19 five years after LT. Allograft function subsequently worsened and biopsy 6 months later showed bile duct damage and loss of ~35% of bile ducts; repeat biopsy 14 months after his COVID diagnosis showed periportal fibrosis with edema, ductular reaction, marked hepatocellular and canalicular cholestasis, and ductopenia with loss of 60% bile ducts. Average time between COVID-19 diagnosis and onset of COVID-related cholangiopathy was 3 months (range: 6 weeks-6 months). These patients were also all immunocompromised with two due to prior LT and one being cirrhotic. Results (if a Case Study enter NA) NA. Conclusion Although previously reported only in patients with severe COVID-19, the cases described represent the first evidence that cholangiopathy, manifested by sclerosing cholangitis, can arise even in patients who were not critically ill, although this may require an immunocompromised state to develop.

Yap signalling regulates ductular reactions in mice with CRISPR/Cas9-induced glycogen storage disease type Ia

ABSTRACT Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in the glucose-6-phosphatase (G6Pase, G6pc) enzyme, which catalyses the final step of gluconeogenesis and glycogenolysis. Accumulation of G6pc can lead to an increase in glycogen and development of fatty liver. Ductular reactions refer to the proliferation of cholangiocytes and hepatic progenitors, which worsen fatty liver progress. To date, however, ductular reactions in GSD-Ia remain poorly understood. Here, we studied the development and potential underlying mechanism of ductular reactions in GSD-Ia in mice. We first generated GSD-Ia mice using CRISPR/Cas9 to target the exon 3 region of the G6pc gene. The typical GSD-Ia phenotype in G6pc −/− mice was then analysed using biochemical and histological assays. Ductular reactions in G6pc −/− mice were tested based on the expression of cholangiocytic markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM). Yes-associated protein 1 (Yap) signalling activity was measured using western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Verteporfin was administered to the G6pc −/− mice to inhibit Yap signalling. The CRISPR/Cas9 system efficiently generated G6pc −/− mice, which exhibited typical GSD-Ia characteristics, including retarded growth, hypoglycaemia, and fatty liver disease. In addition, CK19- and EpCAM-positive cells as well as Yap signalling activity were increased in the livers of G6pc −/− mice. However, verteporfin treatment ameliorated ductular reactions and decreased Yap signalling activity. This study not only improves our understanding of GSD-Ia pathophysiology, but also highlights the potential of novel therapeutic approaches for GSD-Ia such as drug targeting of ductular reactions.

RF03 | PMON288 Determining Mechanisms of Farnesoid×Receptor Regulation of Heme Biosynthesis and Ductular Reaction

Abstract Bile acids are increasingly becoming recognized as hormones, and their main endogenous receptor is the farnesoid X receptor (FXR). In order to investigate whether FXR is necessary to maintain proliferation of the bile ducts in response to injury, we challenged male and female wild-type (WT) and Fxr knockout (FxrKO) mice with 0.1% 1,4 dihydro 2,4,6 trimethyl 3,5 pyridinediacarboxylic acid diethyl ester (DDC) in the diet for two weeks. DDC is an inhibitor of ferrochelatase, the final enzyme of heme biosynthesis, resulting in a buildup of bile plugs in the liver causing an inflammatory response called ductular reaction. We found that deletion of Fxr led to a dramatic reduction in the ductular reaction independent of the sex. Then we tested if the inhibition of ferrochelatase was compensated for in FxrKO mice by analyzing the expression of genes in the heme biosynthesis pathway. Although we find a decrease in basal Alad gene expression in FxrKO mice in both sexes, upon DDC treatment, expression does not decrease as much as seen in the WT mice. Intriguingly, we also noted sex-specific changes. Cpox expression was increased in male FxrKO mice, but FxrKO mice showed reduced suppression of Cpox expression when treated with DDC. Conversely, Ppox expression was reduced in male FxrKO mice compared to WT mice, and both male WT and FxrKO mice had a comparable reduction in Ppox expression upon DDC treatment. Female FxrKO mice showed a significant decrease in Fech expression basally that did not change with DDC treatment. These results suggest that FXR may contribute towards regulating expression of genes in the heme biosynthesis pathway. However, these observations do not explain the lack of ductular reaction. Since PGC1α was previously shown to control heme biosynthesis through its downstream targets, FOXO1 and NRF1, we examined their transcript expression in our study to determine if the pathway is playing a role in our observations. We examined this pathway and observed a significant decrease in Foxo1 expression in both sexes of WT and FxrKO mice basally but only a reduction in Nrf1 expression upon DDC treatment. We are currently investigating other potential regulators of heme biosynthesis and ductular reaction, such as Klf1 which regulates erythropoiesis and plays a role in ductular reaction and the nuclear receptors Rev-Erbα/β that use heme as a ligand, to determine how FXR is regulating heme biosynthesis. Presentation: Saturday, June 11, 2022 1:24 p.m. - 1:29 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Astragalus saponins and its main constituents ameliorate ductular reaction and liver fibrosis in a mouse model of DDC-induced cholestatic liver disease

Cholestatic liver disease (CLD) is a chronic liver disease characterized by ductular reaction, inflammation and fibrosis. As there are no effective chemical or biological drugs now, majority of CLD patients eventually require liver transplantation. Astragali radix (AR) is commonly used in the clinical treatment of cholestatic liver disease and its related liver fibrosis in traditional Chinese medicine, however its specific active constituents are not clear. Total astragalus saponins (ASTs) were considered to be the main active components of AR. The aim of this study is to investigate the improvement effects of the total astragalus saponins (ASTs) and its main constituents in cholestatic liver disease. The ASTs from AR was prepared by macroporous resin, the content of saponins was measured at 60.19 ± 1.68%. The ameliorative effects of ASTs (14, 28, 56mg/kg) were evaluated by 3, 5-Diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-induced CLD mouse model. The contents of hydroxyproline (Hyp), the mRNA and protein expression of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in liver tissue were dose-dependently improved after treatment for ASTs. 45 astragalus saponins were identified in ASTs by UHPLC-Q-Exactive Orbitrap HRMS, including astragaloside I, astragaloside II, astragaloside III, astragaloside IV, isoastragaloside I, isoastragaloside II, cycloastragenol, etc. And, it was found that ductular reaction in sodium butyrate-induced WB-F344 cell model were obviously inhibited by these main constituents. Finally, the improvement effects of astragaloside I, astragaloside II, astragaloside IV and cycloastragenol (50mg/kg) were evaluated in DDC-induced CLD mice model. The results showed that astragaloside I and cycloastragenol significantly improved mRNA and protein expression of CK19 and α-SMA in liver tissue. It suggested that astragaloside I and cycloastragenol could alleviate ductular reaction and liver fibrosis. In summary, this study revealed that ASTs could significantly inhibit ductular reaction and liver fibrosis, and astragaloside I and cycloastragenol were the key substances of ASTs for treating cholestatic liver disease.

Single-Cell RNA Sequencing Analysis in Murine Biliary Atresia Model Reveals Subpopulation of Biliary Epithelial Cells with Proinflammatory and Fibrogenic Traits

INTRODUCTION: Understanding the pathogenesis of liver fibrosis is critical to impacting outcomes of infants with biliary atresia (BA). In BA, Prominin-1 (Prom1)-expressing hepatic progenitor cell (HPC) populations give rise to biliary epithelial cells (BECs) with ductular reactions (DR). Loss of Prom1 function significantly decreases DRs and fibrosis. Hypothesis: Prom1-expressing HPCs give rise to BECs manifesting activated fibrogenic pathways in BA. METHODS: Experimental BA was induced in neonatal Balb/c mice with intraperitoneal injection of rhesus rotavirus (RRV) vs saline on postnatal day (p) 3. Livers were collected at p3, 10, and 17. EPCAM+/CD31–/CD45–/TER119– live BECs were isolated by fluorescence-activated cell sorting for single-cell RNA sequencing. RESULTS: A total of 44,877 cells were sequenced (saline: 29,195 cells, n = 11; RRV: 15,682 cells, n = 6), graph-based clustering identified 19 clusters of transcriptionally distinct cell types/states. Four distinct BEC subtypes were identified: stem/progenitor, immature, hepatocyte-like, and mature (Figure). All demonstrated activation of inflammation and fibrogenic pathways with RRV treatment. UMAP clustering revealed a unique population of Prom1-expressing immature BECs in RRV p17 samples. Ingenuity pathway analysis (IPA) of this population showed upregulation of several pathways specific to innate immune response, ie natural killer cell and Toll-like receptor signaling. IPA also showed evidence of NFκB signaling with upregulation of proinflammatory cytokines, IL-1β, IL-6, and TNF, and profibrogenic cytokines, Tgfβ1 and Ccl2, in this population.FigureCONCLUSION: A subset of Prom1-expressing immature BECs upregulate proinflammatory and profibrogenic cytokines that likely contribute to fibrosis in BA. These cells and these signaling pathways represent potential therapeutic targets.

Triptolide increases resistance to bile duct ligation-induced liver injury and fibrosis in mice by inhibiting RELB.

Cholestasis is a common, chronic liver disease that may cause fibrosis and cirrhosis. Tripterygium wilfordii Hook.f (TWHF) is a species in the Euonymus family that is commonly used as a source of medicine and food in Eastern and Southern China. Triptolide (TP) is an epoxy diterpene lactone of TWHF, as well as the main active ingredient in TWHF. Here, we used a mouse model of common bile duct ligation (BDL) cholestasis, along with cultured human intrahepatic biliary epithelial cells, to explore whether TP can relieve cholestasis. Compared with the control treatment, TP at a dose of 70 or 140 μg/kg reduced the serum levels of the liver enzymes alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in mice; hematoxylin and eosin staining also showed that TP reduced necrosis in tissues. Both in vitro and in vivo analyses revealed that TP inhibited cholangiocyte proliferation by reducing the expression of RelB. Immunohistochemical staining of CK19 and Ki67, as well as measurement of Ck19 mRNA levels in hepatic tissue, revealed that TP inhibited the BDL-induced ductular reaction. Masson 3 and Sirius Red staining for hepatic hydroxyproline showed that TP alleviated BDL-induced hepatic fibrosis. Additionally, TP substantially inhibited BDL-induced hepatic inflammation. In summary, TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation.

S3170 Syphilitic Hepatitis With Cholestatic Liver Injury

Introduction: The incidence of syphilis in the United States has increased, predominantly among men who have sex with men. Very few patients with early syphilis meet the criteria for syphilitic hepatitis, but many are HIV+ and engage in high-risk sexual behavior. Case Description/Methods: A 30-year-old man who identified as a MSM presented with yellowing of his eyes and hyperbilirubinemia, preceded by one month with a rash and photosensitivity and redness of his right eye. He denied any penile lesions, history of liver disease or alcohol use. A physical examination revealed multiple hyperpigmented macular lesions over his torso, arms, palms, and the soles of his feet and scleral icterus. A slit lamp examination indicated anterior uveitis in both eyes and chorioretinitis/placoid retinitis in the right eye. CBC and iron studies showed mild anemia of chronic disease (Hb 11.7 g/dL). BMP was within normal limits. LFTs were as mentioned in Table 1. Blood tests showed positive HBsAb and T. pallidum antibody with RPR 1:128. Serum markers for viral hepatitis and HIV were negative. CSF from a lumbar puncture had an elevated WBC count (9/mm3). No biliary obstruction or masses were noted during on US and MRCP. Liver biopsy showed bland perivenular cholestasis (zone 3) (Fig. 1A) without necroinflammatory changes, mild to minimal portal inflammation (Fig. 1B), bile duct damage, and ductular reaction. The patient was treated with penicillin G for neurosyphilis, prednisolone and cyclopentolate for ocular syphilis, and ursodiol for cholestasis. Total bilirubin levels decreased to 1.7 mg/dL after 8 weeks of follow-up, and liver enzymes normalized. Discussion: This patient met the criteria for syphilitic hepatitis (elevated liver enzymes, serologic evidence of syphilis, exclusion of other etiology, and improvement following appropriate therapy). He also had bilateral anterior uveitis and placoid retinitis of the right eye, suggesting ocular syphilis. Serum chemistries, testing for T. pallidum and other serologies (including positive anti-smooth muscle antibody) supported hepatic involvement, and CSF studies were consistent with neurosyphilis. Liver biopsy showed a bland cholestatic pattern without evidence of other causes of liver disease. The biopsy results are compatible with syphilitic hepatitis, though histologic features can vary and direct staining for spirochetes has limited sensitivity. These findings, together with his rapid improvement with penicillin therapy, confirmed a diagnosis of syphilitic hepatitis.Figure 1.: (A) Liver specimen (x40 magnification, hematoxylin and eosin stain) showing bland perivenular cholestasis (zone 3 with rare apoptotic hepatocytes. No necroinflammatory activity is present. (B) Liver specimen (x40 magnification, hematoxylin and eosin stain) showing periportal inflammation and bile duct damage with mild bile ductular reaction. Table 1. - Liver function test results AST 41 IU/L ↑ (ULN < 38 IU/L) ALT 53 IU/L ↑ (ULN < 42 IU/L) ALP 210 IU/L ↑↑ (ULN < 129 IU/L) Total bilirubin 11.6 mg/dL ↑↑ (ULN < 1.3 mg/dL) Direct bilirubin 8.8 mg/dL ↑↑ (ULN < 0.4 mg/dL) GGT 53 (ULN < 61 IU/L) Albumin 3.8 g/dL INR 1.0 AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase; INR, international normalized ratio; UNL, upper limits of normal.

S2933 BRIC Type 3? A Case of Benign Recurrent Intrahepatic Cholestasis Unlinked to ATP8B1 and ABCB11

Introduction: Benign Recurrent Intrahepatic Cholestasis (BRIC) is a genetic disorder with two common subtypes: BRIC I (mutated ATP8B1 gene), and BRIC II (mutated ABCB11 gene). Diagnosis requires high degree of suspicion and is confirmed by genetic testing. There are rare cases where patients lack both of the typical mutations. We present a patient with clinical, lab and histological evidence of BRIC without the usual mutations. Case Description/Methods: A 17-year-old male with no medical history presented with yellow eyes, pruritus, and acholic stools for six weeks, without triggering illness or stress. He denied fatigue, fevers, rash, dizziness, nausea, vomiting, diarrhea, constipation, arthralgias, melena, hematochezia, or hematemesis. He had no tattoos, blood transfusions, or allergies. He denied medications, alcohol, smoking, and drug use. On exam, he was icteric, with normal abdominal exam, and Alkaline Phosphatase 370 U/L, ALT 96 U/L, AST 66 U/L, and conjugated bilirubin of 6.7 mg/dL. Serological tests were normal, including antibodies for hepatitis A/B/C, CMV, EBV, HIV; alpha-1 antitrypsin; ceruloplasmin; iron, transferrin, C28Y/H63D mutations; TSH; antinuclear, anti-smooth muscle, anti-liver kidney microsomal, anti-mitochondrial antibodies; serum tissue transglutaminase; urine chlamydia and gonorrhea. Liver ultrasound had no biliary dilation. Genetic testing revealed negative ATP8B1 and ABCB11. Nonfocal liver biopsy showed bland cholestasis with coarse granular material in dilated bile canaliculi, compatible with a disorder of intrahepatic cholestasis, with morphologically similarities to biopsies seen in BRIC. He received ursodiol and symptoms improved. He stopped ursodiol after discharge and presented six months later with similar lab abnormalities. Repeat serological workup was normal. He received ursodiol and symptoms resolved again (Figure). Discussion: Diagnostic criteria for BRIC include episodes of jaundice separated by a symptom-free interval of at least six months, lab and histological evidence of non-inflammatory cholestasis, non-dilated biliary ducts, and no other explanatory causes. Our patient satisfied these diagnostic criteria. Some evidence indicates the presence of additional disease loci for low γ-GT BRIC, explaining why some patients diagnosed with BRIC on clinical and histopathologic evidence lack usual mutations in ATP8B1 or ABCB11. This disorder is informally termed “BRIC 3”; it is unclear which genes are implicated in this form and further research is needed.Figure 1.: Histopathologic appearance of the liver biopsy. Bland centrilobular cholestasis with coarse granular biliary material within dilated bile canaliculi, without any significant inflammation, bile duct damage, or ductular reaction.

Porto-sinusoidal vascular disorder.

It is well established that portal hypertension can occur in the absence of cirrhosis, as reported in patients with immune disorders, infections and thrombophilia. However, similar histological abnormalities primarily affecting the hepatic sinusoidal and (peri)portal vasculature have also been observed in patients without portal hypertension. Thus, the term porto-sinusoidal vascular disorder (PSVD) has recently been introduced to describe a group of vascular diseases of the liver featuring lesions encompassing the portal venules and sinusoids, irrespective of the presence/absence of portal hypertension. Liver biopsy is fundamental for PSVD diagnosis. Specific histology findings include nodular regenerative hyperplasia, obliterative portal venopathy/portal vein stenosis and incomplete septal fibrosis/cirrhosis. Since other conditions including alcohol-related and non-alcoholic fatty liver disease, or viral hepatitis, or the presence of portal vein thrombosis may occur in patients with PSVD, their relative contribution to liver damage should be carefully assessed. In addition to histology and clinical diagnostic criteria, imaging and non-invasive tests such as liver and spleen stiffness measurements could aid in the diagnostic workup. The introduction of PSVD as a novel clinical entity will facilitate collaborative studies and investigations into the underlying molecular pathomechanisms encompassed by this term.

S3007 Glucosamine-Induced Hepatitis - A Case Report

Introduction: Drug-induced liver injury (DILI) is an adverse reaction to drugs that occurs either as a predictable event when an individual is exposed to toxic doses of some compounds or as an unpredictable event at therapeutic dosing. The diagnosis of DILI relies on the exclusion of other etiologies of liver disease as specific biomarkers are still lacking. We present a case of acute hepatocellular hepatitis in the setting of recent glucosamine use. Case Description/Methods: 57-year-old female without significant medical history presents with markedly elevated transaminases noted on routine annual physical. The patient denies any past history of liver disease or abnormal liver enzymes. Baseline weight is around 114 Ibs, stable for years. She exercises regularly by spinning and walking. Pt drinks alcohol socially, average 1 glass of wine daily for 10 years, which she has withheld since onset of illness. Pt denies any history of blood transfusion, IVDU, tattoo, new medications or antibiotics. She started taking glucosamine for joint pain on 4/12. Patient had LFTs drawn on 4/22 as a part of annual evaluation by PCP which were elevated. She was having her LFTs checked daily as an outpatient with progressive rise in her AST and ALT. AST/ALT peaked at 2322 and 3335 around 4/28 accompanied by ALP elevation at 364 with TB 3.4. INR remained within normal limit and mental status has been intact. Workup included normal TSH, normal IgA/IgM/IgG, neg HAV/HBV/HCV serologies, normal Tylenol level, negative ANA/ASMA/Anti LKM and normal ceruloplasmin. RUQUS: contracted gallbladder without gallstones. MRCP: no biliary obstruction. Liver biopsy: pan lobular mixed inflammatory infiltrate with patchy bile duct damage suggestive of DILI. She was recommended to discontinue glucosamine. LFTs slowly down trended during hospitalization without any intervention and she was d/c home on 5/4. On follow up, LFTs continued to improve with the most recent LFTs on 6/15: AST/ALT 43/62, ALP and TB normal. (Figure) Discussion: Glucosamine is used as a nutritional supplement to decrease the pain and cartilage loss in osteoarthritis. Recently, a few case reports of clinically apparent liver injury attributed to glucosamine have been published. The time to onset is usually 1 to 4 weeks and the pattern of injury is typically hepatocellular or mixed. Most patients were reported to recover within 4 to 8 weeks. We want to demonstrate and raise awareness of glucosamine as a potential cause of DILI and to consider it in the differentials for acute hepatitis.Figure 1.: Panlobular mixed inflammatory infiltrate, mild ductular reaction, patchy bile duct damage, hepatocanalicular cholestasis, prominent kupffer cell aggregates and acidophil bodies.

S3125 Overlapping Hepatotoxicity and Colitis Associated With Immune Checkpoint Inhibitors

Introduction: Immunotherapy with immune checkpoint inhibitors (ICIs) has become the standard of care for many solid-organ and hematologic malignancies. Gastrointestinal toxicities are common side effects that typically occur 6 weeks after starting ICI therapy. Overlapping hepatotoxicity and colitis associated with ICIs is rare and may be overlooked, resulting in high mortality. Case Description/Methods: A 73-year-old man with metastatic squamous cell carcinoma (SCC) of the head and neck presented with intermittent, diffuse abdominal discomfort, bloating and painless non-bloody diarrhea (up to 6 episodes daily) triggered by meals. Symptom onset was 6 weeks after starting pembrolizumab for head and neck SCC. Pembrolizumab therapy was held, and supportive therapy with proton pump inhibitors, antacids and antidiarrheals provided partial symptom relief. Laboratory work up showed elevated AST 132 U/L, ALT 220 U/L, ALP 851 U/L and normal total bilirubin. ANA was elevated with titer 1:80, with normal total IgG (699.5 mg/dL) and negative serologies for anti-smooth muscle antibody, CMV, EBV, and HSV. Abdominal CT with contrast showed a dilated common bile duct (CBD) of 11 mm and a new curvilinear filling defect in the distal CBD, without choledocholithiasis. MRCP revealed mildly dilated intrahepatic biliary ducts, CBD dilation (13 mm), and an ovoid 8mm soft tissue nodule in the periampullary duodenum. Upper endoscopy was nondiagnostic. At this time, the patient’s symptoms improved, but there was high suspicion of grade-2 ICI-associated hepatitis (ICIH). Percutaneous liver biopsy showed interlobular bile duct injury with intraepithelial lymphocytes and neutrophils and a neutrophilic ductular reaction consistent with ICIH. Serial lab work demonstrated improvement of liver enzymes, with AST 77 U/L, ALT 69 U/L and ALP 667 U/L at 60 days after initial labs. Diarrhea resumed, up to 6 episodes per day, consistent with the Common Terminology Criteria for Adverse Events diagnosis of ICI-related colitis (ICIC). Initial supportive therapy failed to improve diarrhea; thus, a prednisone taper was started, resolving the colitis. He declined the option to resume immunotherapy and transitioned to comfort care (Figure 1). Discussion: Overlapping ICIH and ICIC is rare and may have discordance between symptomatology and lab findings. Early recognition of overlapping ICIH and ICIC, supportive treatment, permanent cessation of the inciting ICI and switching it with alternative immunosuppressive therapy may improve patient outcomes and quality of life.Figure 1.: Panel A - CT chest/abdomen with contrast (coronal view) showing common bile duct dilation of 1.2 cm (yellow calipers) status post cholecystectomy. Panel B. Upper endoscopy showing prominent ampulla with free-flowing bile. Panel C. Histology of percutaneous, ultrasound-guided liver biopsy with H&E staining (10×) shows preserved lobular hepatic architecture with mixed inflammatory infiltrate including lymphocytes, histiocytes, and scattered eosinophils expanding the portal tracts. Table 1. - Immune Checkpoint Inhibitor Associated Hepatitis Grades, adapted from the American Society of Clinical Oncology, 2021 Grade Criteria 1 Asymptomatic (AST or ALT > ULN to 3.0 X ULN and/or total bilirubin > ULN to 1.5 X ULN) 2 Asymptomatic (AST or ALT > 3.0 to ≤ 5 X ULN and/or total bilirubin > 1.5 to ≤ 3 X ULN) 3 AST or ALT 5-20 X ULN and/or total bilirubin 3-10 X ULN, OR symptomatic liver dysfunction; fibrosis by biopsy; compensated cirrhosis; and reactivation of chronic hepatitis 4 AST or ALT > 20 X ULN and/or total bilirubin > 10 X ULN OR decompensated liver function (e.g., ascites, coagulopathy, encephalopathy, and coma) Abbreviations: AST, aspartate transferase; ALT, alanine transaminase; ULN, upper limit of normal.

S3137 Post-COVID-19 Cholestatic Hepatitis

Introduction: Coronavirus Disease 2019 (COVID-19) caused by SARS-COV-2 has led to a global crisis of unprecedented reach and proportion. In addition to the pulmonary consequences, the incidence of liver injury is increasingly being reported. Understanding the hepatotropism of SARS-COV-2 is of paramount importance as it can lead to a severe COVID-19 course. This case details a unique presentation of cholestatic hepatitis likely a sequela of a severe SARS-COV-2 infection in our patient. Case Description/Methods: A 49-year-old lady with a history of seizure disorder, migraines, hypertension, and recent six-week ICU admission for COVID illness requiring mechanical ventilation (ECMO), presented to our gastroenterology clinic for a post-discharge follow-up. During admission, she consistently had an abnormal elevation of liver enzymes (Peak ALP 2000, AST, ALT 300, Bilirubin 2.5). MRCP was normal and a liver biopsy showed marked intralobular bile duct injury showing severe cytologic atypia, periductal edema, periductal and intraductal neutrophilic, and lymphocytic inflammation and marked ductular reaction, focal mild hepatocellular cholestasis, Kupffer cell hyperplasia. She had no prior history of chronic liver disease and LFT was normal at baseline. Post discharge LFT showed alkaline phosphatase of 1514 with total bilirubin 1.4. Infective (Hepatitis A, B, C panel), Inflammatory/Autoimmune (ANA, ASMA, AMA, P-ANC, Anti-SLA/LP, Anti ds-DNA, ALKM-1, and ALKM-3) workup was negative. Repeat imaging with USG and MRI was unremarkable. She received a course of prednisone after discharge with no improvement in LFT, it was tapered off. She is currently asymptomatic 6 months after acute illness but continues to have persistently elevated liver enzymes with a gradual downtrend with the latest being ALP 490, AST 86, ALT 95, and T. Bilirubin 0.8. Discussion: We describe a unique case of post-COVID-19 cholestatic hepatitis with persistently elevated liver biochemistry with unclear etiology despite extensive workup. It could be a confluence of Secondary Cholangitis in Critically Ill Patients (SSC-CIP) and direct hepatic injury from COVID-19. Further data is needed to understand the patterns of liver injury due to COVID and the development of possible treatment for this newly described condition associated with the virus.

S2984 Drug-Induced Liver Failure Masquerading as Late Onset Wilson Disease: A Case Report

Introduction: The use of herbal and dietary supplements has increased occurrences of drug-induced liver failure. Acute liver failure (ALF) can rapidly progress to multiorgan failure with about 50% of patients requiring a liver transplant. About 10% of all ALF cases have been due to drug injury while only about 2% have been due to Wilson disease (WD). This article provides a detailed case review of ALF due to the use of alkaline water that was originally thought to be due to WD. Case Description/Methods: A 35-year-old male with no known past medical history is brought into the emergency department (ED) with altered mental status for the past 2 to 3 weeks. The patient was found to be in acute renal failure and ALF. Computed tomography (CT) of the head was unremarkable and a CT of the abdomen showed hepatomegaly and hepatic steatosis without evidence of cirrhosis. Abdominal ultrasound was negative for portal vein thrombus, and laboratory findings were significant for reduced ceruloplasmin. Due to the absence of other causes for his ALF, the patient's presentation was thought to be due to WD. An ophthalmologist conducted a slit-lamp exam, which was negative for Kayser-Fleischer rings. A liver biopsy showed focal liver cholestasis, bile ductular reaction, and mixed portal inflammation suggestive of drug-induced liver injury. Examination of the liver core biopsies displayed acute and chronic periportal inflammatory infiltrate with eosinophils. The patient’s acute encephalopathy completely resolved after six days, and the patient subsequently reported drinking an alkaline water daily 10-days before the onset of symptoms. After ten days, the patient was transferred to a transplant center, however, with improving liver function and resolution of symptoms, the patient was discharged home without the need for further treatment. (Figure) (Table). Discussion: Due to increased consumption of herbal and dietary supplements in the United States, the rates of drug-induced liver failure have increased. Many cases of drug-induced liver failure due to these sources are underreported and when they are discovered have been linked to increased rates of liver transplant. This case not only highlights drug-induced acute liver failure and its complicated presentation, but also brings into consideration alkaline water as a potential source for ALF. Cases such as this demonstrate that a thorough history and further understanding of herbal and dietary supplements cannot be overlooked when attempting to elucidate a source in instances of ALF.Figure 1.: Computed tomography of the head and abdomen. (a) Axial view of head. No abnormalities in the basal ganglia. (b) Coronal view of abdomen. Table 1. - Laboratory test results of the patient a day before admission, on the day of admission, five days after admission, ten days after admission, and two days after transfer to an outside facility Variable (normal range) Two days before admission Day of admission Five days after admission Ten days after admission Two days after transfer White Blood Cell k/mm3 (3.10-10.20) 11.13 26.27 14.75 12.20 13.17 Hemoglobin g/dL (13.1-16.8) 14.9 16.3 11.0 9.2 8.9 Platelet k/mm3 (119-332) 314 354 182 163 274 Lactic Acid mmol/L (0.50-1.90) > 11.70 1.08 1.39 BUN mg/dL (5-26) 9 18 102 30 38 Creatinine mg/dL (0.55-1.30) 0.81 2.11 11.11 1.97 2.0 Bilirubin, Total mg/dL (0.0-1.2) 0.5 2.1 5.0 1.7 1.3 Bilirubin, Direct mg/dL (< = 0.5) 1.6 2.9 1.1 AST U/L (8-34) 40 11,867 193 90 65 ALT U/L (10-49) < 7 1,001 171 394 289 Alk-Phos U/L (46-116) 89 152 135 165 126 INR (0.80-1.2) 5.37 1.19 0.95 1.0 Prothrombin time (9.3-12.4 seconds) 53 12.6 10.2 11.8 Ceruloplasmin mg/dL (16.0-31.0) 8.7 13.5 24.6 Copper ug/dL (72-166) 52 82 76 Urine Copper Level ug/L 2787 Urine Copper mcg/24hrs (9-71) 40 Alk-Phos (IU/L) to total bilirubin (mg/dL) ratio 72.38 AST, aspartate transaminase; ALT, alanine transaminase; Alk-Phos, alkaline phosphatase; INR, International Normalization Ratio; aPTT, activated partial thromboplastin

S3114 Painless Jaundice With a Significantly Elevated CA 19-9: It Is Not Always Pancreaticobiliary Cancer

Introduction: Carbohydrate antigen 19-9 (CA 19-9) is often very high in pancreaticobiliary cancers but can be substantially elevated in benign diseases too. We present a case of painless jaundice with a significantly elevated CA 19-9 from vanishing bile duct syndrome (VBDS). Case Description/Methods: 65-year-old white male with history of obesity, hypertension, hyperlipidemia, prior cholecystectomy and no family history of pancreatic cancer presented with two weeks of painless jaundice and 10 pounds unintentional weight loss. Physical exam noted scleral icterus and jaundice with a T.Bili of 23 mg/dL (Table 1). CT scan showed intrahepatic bile duct dilation and a 10 mm common bile duct without a discrete mass. CA 19-9 was elevated to 1,480 U/mL, concerning for malignancy. ERCP found a low-grade main bile duct stricture which was stented. Bile duct brushings and fine needle aspiration of the pancreas were negative for malignancy. Biliary dilation was resolved on follow-up MRCP but his bilirubin remained high. Infectious and autoimmune workup was unremarkable. Repeat cholangiography and brushings confirmed no obstruction or malignancy (Fig. 1a). An infiltrative versus drug induced liver injury was suspected. EUS-guided liver biopsies revealed cholestasis with dilated bile canaliculi, hepatocytes with ballooning degeneration and a paucity of bile ductules. Cytokeratin-7 immunostaining demonstrated >50% ductopenia, confirming VBDS (Fig. 1b and c). Follow up at hepatology clinic determined triamterene-HCTZ, atorvastatin or omeprazole stopped during hospitalization to be the likely culprit. His CA 19-9, LFTs and symptoms improved over the following weeks (Table 1). Discussion: VBDS is an acquired cholestatic liver disease that may mimic the presentation of pancreatic cancer. CA 19-9 can be substantially elevated through T-cell mediated destruction of small bile ducts causing impaired excretion, inflammatory production, and decreased clearance. Severity of liver damage depends on the duration of injury and degree of bile duct loss at the time of diagnosis. Prompt diagnosis is important to prevent cirrhotic progression, improve outcomes, and avoid unnecessary treatments. Recognizing its association with an elevated CA 19-9 may help with this, but is scarcely reported in the literature. CA 19-9 is not typically checked in cases of VBDS but is likely commonly elevated. Providers should be aware of this association when faced with a patient with painless jaundice and a significantly elevated CA 19-9 but no obvious malignancy. Table 1. - LFT Trends During the Course of Presentation Normal Range Hospital Admission * 2 weeks 3 weeks ** 4 weeks *** 7 weeks 13 weeks Total bilirubin 0.2-1.3 mg/dL 23 22.7 22 19.7 4.7 1.3 Direct bilirubin 0.0-0.2 mg/dL >13 n/a 14.9 14.6 4.2 0.6 AST 0-45 U/L 107 134 172 200 121 27 ALT 0-50 U/L 190 130 163 182 120 35 ALP 40-150 U/L 267 179 187 168 132 109 CA 19-9 0-35 U/mL 1480 n/a n/a 85 49 n/a AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; CA19-9, carbohydrate antigen 19-9; n/a, not available. * ANA, anti-AMA, anti-SMA, anti-mitochondrial, anti-TTG, anti-LKM 1/2 IgG, and EBV IgG antibodies were negative. Serum IgA, A1AT, ceruloplasmin, TSH, iron levels, hepatitis panel, serum protein electrophoresis and acetaminophen levels were normal. Acetaminophen, multivitamin, iron, triamterene- HCTZ, omeprazole and atorvastatin were stopped on discharge. ** Three weeks post hospitalization. Repeat EUS/ERCP showed biliary decompression. VBDS diagnosed on liver biopsies. *** Seen in liver clinic. Started ursodiol 2000mg daily. Figure 1.: (A) ERCP with cholangiogram showing a low-grade distal biliary stricture without significant upstream dilation. (B) Liver biopsy with H&E stain, 500 μm magnification showing small portal tracts with no bile ducts (red circle); note marked cholestasis (arrows) in dilated canaliculi and lack of ductular reaction. Also visible at this magnification is the ballooning degeneration of hepatocytes. (C) Liver biopsy with cytokeratin 7 immunostain, 500 μm magnification. Small portal tracts with no terminal/small bile ducts. Early signs of ductular reaction seen by hepatocytes taking up CK7 stain (arrows).

Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response.

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1-2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.

ZO-2/Tjp2 suppresses Yap and Wwtr1/Taz-mediated hepatocyte to cholangiocyte transdifferentiation in the mouse liver

TJP2/ZO-2-inactivating mutations in humans cause progressive cholestatic liver disease. Liver-specific deletion of Tjp2 in the mouse (Tjp2 cKO mice) leads to mild progressive cholestasis without an overt degradation of the bile-blood barrier (BBB). These mice are more susceptible to cholic acid (CA) induced liver injury. Interestingly, while initially also more susceptible, Tjp2 cKO mice develop tolerance to a DDC-supplemented diet. The DDC diet induces an exacerbated ductular reaction in Tjp2 cKO mice, which arises from the transdifferentiation of hepatocytes to cholangiocytes. Consequently, this transdifferentiation is only observed if Tjp2 is inactivated in hepatocytes, but not if deleted in cholangiocytes. The DDC-diet-induced hepatocyte transdifferentiation in Tjp2 cKO mice requires Yap and Wwtr1/Taz, whose protein expression is upregulated in hepatocytes lacking Tjp2, but is independent of Notch2. Although inactivating Tjp2 is sufficient for the upregulation of Yap and Wwtr1/Taz protein, efficient transdifferentiation requires the DDC-diet insult. Thus, Tjp2 negatively regulates Yap/Taz-mediated transdifferentiation of hepatocytes to cholangiocytes in response to DDC-diet-induced liver injury. Furthermore, transdifferentiation is regulated at multiple levels and the type of injury inflicted on the Tjp2 deficient liver plays an important role in the resulting pathophysiology.