Vanishing bile duct syndrome following pembrolizumab infusion: case report and review of the literature.

PD-1/PD-L1 inhibitors demonstrate high efficacy in non-small-cell lung cancer and are now routinely used in clinical practice. Severe immune-related adverse events are reported in about 5% of patients, requiring hospitalization and possibly leading to death. We present a rare case of vanishing bile duct syndrome that arose a few days after the first pembrolizumab infusion. Laboratory tests and radiological imaging studies were performed to orient diagnosis and monitor the disease, while the evidence of ductal loss on the histological sample was pathognomonic for vanishing bile duct syndrome. High-dose steroid therapy and immunosuppressors were administered, resulting in scarce efficacy. Prompt recognition and management of similar conditions is crucial to avoid fatal events. Further studies are needed to investigate new drugs for steroid-refractory conditions.

Plain language summary Immunotherapy has demonstrated high efficacy in lung cancer and is commonly used in clinical practice. Despite the good tolerability, severe immune-related adverse events may occur, requiring hospitalization and possibly leading to death. We present a case of vanishing bile duct syndrome (a rare and potentially lethal condition characterized by progressive destruction of small bile ducts) which arose a few days after the first pembrolizumab infusion. Laboratory tests and radiological imaging were performed to orient diagnosis and monitor disease; a histological sample was required for vanishing bile duct syndrome diagnosis. High-dose steroid therapy and immunosuppressors were administered, with scarce efficacy. Prompt recognition and management of similar conditions is crucial to avoid fatal events. Further studies are needed to investigate new drugs for steroid-refractory conditions.

Pantoprazole ameliorates liver fibrosis and suppresses hepatic stellate cell activation in bile duct ligation rats by promoting YAP degradation.

Liver fibrosis is one of the most severe pathologic consequences of chronic liver diseases, and effective therapeutic strategies are urgently needed. Proton pump inhibitors (PPIs) are H+/K+-ATPase inhibitors and currently used to treat acid-related diseases such as gastric ulcers, which have shown other therapeutic effects in addition to inhibiting acid secretion. However, few studies have focused on PPIs from the perspective of inhibiting hepatic fibrosis. In the present study, we investigated the effects of pantoprazole (PPZ), a PPI, against liver fibrosis in a bile duct ligation (BDL) rat model, human hepatic stellate cell (HSC) line LX-2 and mouse primary HSCs (pHSCs), and explored the potential mechanisms underlying the effects of PPZ in vitro and in vivo. In BDL rats, administration of PPZ (150 mg· kg-1· d-1, i.p. for 14 d) significantly attenuated liver histopathological injury, collagen accumulation, and inflammatory responses, and suppressed fibrogenesis-associated gene expression including Col1a1, Acta2, Tgfß1, and Mmp-2. In LX-2 cells and mouse pHSCs, PPZ (100-300 µM) dose-dependently suppressed the levels of fibrogenic markers. We conducted transcriptome analysis and subsequent validation in PPZ-treated LX-2 cells, and revealed that PPZ inhibited the expression of Yes-associated protein (YAP) and its downstream targets such as CTGF, ID1, survivin, CYR61, and GLI2. Using YAP overexpression and silencing, we demonstrated that PPZ downregulated hepatic fibrogenic gene expression via YAP. Furthermore, we showed that PPZ promoted the proteasome-dependent degradation and ubiquitination of YAP, thus inhibiting HSC activation. Additionally, we showed that PPZ destabilized YAP by disrupting the interaction between a deubiquitinating enzyme OTUB2 and YAP, and subsequently blocked the progression of hepatic fibrosis.

Yes-associated protein regulates the hepatoprotective effect of vitamin D receptor activation through promoting adaptive bile duct remodeling in cholestatic mice.

Mounting clinical evidence has revealed that the vitamin D receptor (VDR) is associated with cholestatic liver injury, although the functions of VDR in this condition remain largely unexplored. Here, we investigated the effects of VDR activation on bile duct ligation (BDL) mice, and the underlying mechanisms were further investigated. A low-calcemic VDR agonist, paricalcitol (PAL, 200 ng/kg), was intraperitoneally injected into BDL mice every other day for 5 days or 28 days. Liver histology, liver function indicators, cholangiocyte proliferation, fibrosis scores, and inflammation were evaluated. Mice treated with PAL were rescued from the decreased survival rate induced by BDL and liver damage was reduced. Mechanistically, PAL promoted cholangiocyte proliferation, which was likely conducive to proliferating bile duct maturation and increased branching of bile ducts. PAL treatment also increased the expression of Yes-associated protein (YAP) and its target protein epithelial cell adhesion molecule (EpCam) and decreased the level of inactive cytoplasmic phosphorylated YAP. YAP knockdown abrogated PAL-induced primary bile duct epithelial cell proliferation, confirmed with YAP inhibitor administration. In addition, BDL-induced liver fibrosis and inflammatory cell infiltration were reduced by PAL treatment at both day 5 and day 28 post-BDL. In conclusion, VDR activation mitigates cholestatic liver injury by promoting adaptive bile duct remodeling through cholangiocytic YAP upregulation. Because PAL is an approved clinical drug, it may be useful for treatment of cholestatic liver disease. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

1,2-Dichloropropane induces γ-H2AX expression in human cholangiocytes only in the presence of macrophages.

Recent epidemiological studies reported cases of cholangiocarcinoma in workers exposed to 1,2-dichloropropane (1,2-DCP) in an offset proof printing factory in Japan. The present study investigated the effects of 1,2-DCP on the expression of histone family member X (H2AX) phosphorylated on Ser 139 (γ-H2AX), a marker of DNA double strand break, in human immortalized cholangiocytes MMNK-1 cells. Mono-cultures of MMNK-1 cells and co-cultures of MMNK-1 cells with THP-1 macrophages were exposed to 1,2-DCP at concentrations of 100 and 500 µM for 24 h. Expression of γ-H2AX was visualized by immunofluorescence staining. Exposure to 1,2-DCP had no effect on the expression of γ-H2AX in mono-cultured MMNK-1 cells, but significantly increased the number of nuclear foci stained by γ-H2AX in MMNK-1 cells co-cultured with THP-1 macrophages. Exposure to 1,2-DCP also significantly increased the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in co-cultured MMNK-1 cells. The results suggest that macrophages play a critical role by producing cytokines in 1,2-DCP-induced DNA double strand break in MMNK-1 cells.

Formyl Peptide Receptor 2 Alleviates Hepatic Fibrosis in Liver Cirrhosis by Vascular Remodeling.

Hexapeptide WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met), a ligand of formyl peptide receptor 2, exhibits anti-inflammatory and angiogenic properties in disease models. However, the therapeutic effects of WKYMVm on hepatic fibrosis have not been evaluated to date. Therefore, we investigated whether WKYMVm exerts antifibrotic effects and induces vascular regeneration in a rat model of bile duct ligation (BDL). The antifibrotic and angiogenic effects of WKYMVm on liver regeneration in the BDL rat model were analyzed using biochemical assays, qRT-PCR, western blotting, immunofluorescence, and immunohistochemistry. To determine the effects of WKYMVm on hepatic fibrosis and angiogenesis in vitro, we measured the expression levels of fibrotic factors in hepatic stellate cells (HSCs) and angiogenic factors in human umbilical vein endothelial cells (HUVECs). WKYMVm attenuated the expression of collagen type I (Col I) and α-smooth muscle actin (α-SMA) and significantly increased the levels of angiogenetic factors in the BDL model (p < 0.05). WKYMVm reduced fibrotic marker expression in transforming growth factor (TGF)-ß-induced HSCs and promoted angiogenic activity through tube formation in 5-Fluorouracil (FU)-treated HUVECs (p < 0.05). Also, WKYMVm administration enhanced hepatocyte proliferation in BDL rats (p < 0.05). The WKYMVm alleviates hepatic fibrosis by inhibiting HSC activation and promotes hepatic regeneration via vascular remodeling. These data suggest that the WKYMVm may be a new therapeutic agent for liver fibrosis.

Epigenomic Evaluation of Cholangiocyte Transforming Growth Factor-ß Signaling Identifies a Selective Role for Histone 3 Lysine 9 Acetylation in Biliary Fibrosis.

BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.

Targeting UBC9-mediated protein hyper-SUMOylation in cystic cholangiocytes halts polycystic liver disease in experimental models.

BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. METHODS: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. RESULTS: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdh1-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUMO1-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. CONCLUSIONS: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy. LAY SUMMARY: Protein SUMOylation is a dynamic post-translational event implicated in numerous cellular processes. This study revealed dysregulated protein SUMOylation in polycystic liver disease, which promotes hepatic cystogenesis. Administration of S-adenosylmethionine (SAMe), a natural UBC9-dependent SUMOylation inhibitor, halted polycystic liver disease in experimental models, thus representing a potential therapeutic agent for patients.

Carbon Dots from Paeoniae Radix Alba Carbonisata: Hepatoprotective Effect.

INTRODUCTION: The charcoal processed product of Paeoniae Radix Alba (PRA), PRA Carbonisata (PRAC), has long been used for its hepatoprotective effects. However, the material basis and mechanism of action of PRAC remain unclear. AIM: To explore the hepatoprotective effects of Paeoniae Radix Alba Carbonisata-derived carbon dots (PRAC-CDs). METHODS: PRAC-CDs were characterized using transmission electron microscopy, high-resolution transmission electron microscopy, ultraviolet, fluorescence, Fourier transform infrared and X-ray photoelectron spectroscopy, X-ray diffraction, and high-performance liquid chromatography. The hepatoprotective effect of PRAC-CDs was evaluated and confirmed using the classic carbon tetrachloride acute liver injury model. RESULTS: PRAC-CDs averaged 1.0-2.4 nm in size and exhibited a quantum yield of 5.34% at a maximum excitation wavelength of 320 nm and emission at 411 nm. PRAC-CDs can reduce the ALT and AST levels of mice with carbon tetrachloride-induced acute liver injury and have a mitigating effect on the rise in TBA and TBIL. More interestingly, PRAC-CDs can significantly reduce MDA and increase SOD levels, demonstrating that PRAC-CDs can improve the body's ability to scavenge oxygen free radicals and inhibit free radical-induced liver cell lipid peroxidation, thereby preventing liver cell damage. CONCLUSION: These results demonstrate the remarkable hepatoprotective effects of PRAC-CDs against carbon tetrachloride-induced acute liver injury, which provide new insights into potential biomedical and healthcare applications of CDs.

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

Long-term Proton Pump Inhibitor Administration Caused Physiological and Microbiota Changes in Rats.

Proton pump inhibitors (PPIs) are used for the long-term treatment of gastroesophageal disorders and the non-prescription medicines for acid reflux. However, there is growing concerns about PPI misuse, overuse and abuse. This study aimed to develop an animal model to examine the effects of long-term use of PPI in vivo. Twenty one Wistar rats were given omeprazole orally or intravenously for 30 days, and caerulein as a positive control. After euthanization, the serum and stool were collected to perform MS-based quantitative analysis of metabolites. We carried out 16S-based profiling of fecal microbiota, assessed the expression of bile acid metabolism regulators and examined the immunopathological characteristics of bile ducts. After long-term PPI exposure, the fecal microbial profile was altered and showed similarity to those observed in high-fat diet studies. The concentrations of several metabolites were also changed in various specimens. Surprisingly, morphological changes were observed in the bile duct, including ductal epithelial proliferation, micropapillary growth of biliary epithelium, focal bile duct stricture formation and bile duct obstruction. These are characteristics of precancerous lesions of bile duct. FXR and RXRα expressions were significantly reduced, which were similar to that observed in cholangiocarcinoma in TCGA and Oncomine databases. We established a novel animal model to examine the effects of long-term use of omeprazole. The gut microbes and metabolic change are consequences of long-term PPI exposure. And the results showed the environment in vivo tends to a high-fat diet. More importantly, we observed biliary epithelial hyperplasia, which is an indicator of a high-fat diet.

The Effect of Lecithins Coupled Decorin Nanoliposomes on Treatment of Carbon Tetrachloride-Induced Liver Fibrosis.

This study aimed to investigate the effect of bile duct-targeting lecithins- (PC-) coupled decorin (DCN) (PC-DCN) nanoliposomes against liver fibrosis in vitro and in vivo. We prepared PC-DCN nanoliposomes by using rat astrocytes, HSC-T6, to verify the antifibrosis effect of PC-DCN in vitro. First, we established a rat model of carbon tetrachloride-induced fibrosis. PC-DCN nanoliposomes were then injected into fibrotic rats via the portal vein or bile duct. The EdU assay was performed to analyze cell proliferation. Immunofluorescence staining was used to detect α-smooth muscle actin (α-SMA) expression. Western blot was performed to examine the expression of α-SMA, collagen type I alpha 1 (COL1A1), and transforming growth factor-ß (TGF-ß) protein. The levels of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin (TBIL) were examined by enzyme-linked immunosorbent assay (ELISA) analysis. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were used to determine liver tissue lesions and liver fibrosis. Compared with TGF-ß group, PC-DCN treatment could significantly reduce cell proliferation. Western blot analysis indicated that the expression of α-SMA, COL1A1, and TGF-ß was downregulated after treatment with PC-DCN in vitro and in vivo. Immunofluorescence staining confirmed that α-SMA expression was reduced by PC-DCN. Furthermore, H&E staining and Masson trichrome staining showed that the administration of PC-DCN nanoliposomes via the bile duct could reduce the extent of liver fibrosis. PCR analysis showed that PC-DCN administration could reduce proinflammatory cytokines IL-6, TNF-α, and IL-1ß expression via the bile duct. The administration of PC-DCN nanoliposomes also significantly downregulated liver function indicators ALT, AST, and TBIL. The results of our study indicated that PC-DCN could effectively reduce the extent of liver fibrosis.

Metformin ameliorates bile duct ligation-induced acute hepatic injury via regulation of ER stress.

Cholestasis is a condition in which the bile duct becomes narrowed or clogged by a variety of factors and bile acid is not released smoothly. Bile acid-induced liver injury is facilitated by necrotic cell death, neutrophil infiltration, and inflammation. Metformin, the first-line treatment for type 2 diabetes, is known to reduce not only blood glucose but also inflammatory responses. In this study, we investigated the effects of metformin on liver injury caused by cholestasis with bile acid-induced hepatocyte injury. Static bile acid-induced liver injury is thought to be related to endoplasmic reticulum (ER) stress, inflammatory response, and chemokine expression. Metformin treatment reduced liver injury caused by bile acid, and it suppressed ER stress, inflammation, chemokine expression, and neutrophil infiltration. Similar results were obtained in mouse primary hepatocytes exposed to bile acid. Hepatocytes treated with tauroursodeoxycholic acid, an ER stress inhibitor, showed inhibition of ER stress, as well as reduced levels of inflammation and cell death. These results suggest that metformin may protect against liver injury by suppressing ER stress and inflammation and reducing chemokine expression. [BMB Reports 2020; 53(6): 311-316].

Aldehyde dehydrogenase-2 activation by Alda-1 decreases necrosis and fibrosis after bile duct ligation in mice.

BACKGROUND AND AIM: Liver fibrosis is a leading cause of mortality worldwide. Oxidative stress is a key component in the pathogenesis of liver fibrosis. We investigated the role of aldehyde formation resulting from lipid peroxidation in cholestatic liver injury and fibrosis. METHODS: C57Bl/6J mice underwent bile duct ligation (BDL) or sham operation. One hour after surgery and daily thereafter, animals were given Alda-1 (20 mg/kg, s.c.), an aldehyde dehydrogenase-2 activator, or equivalent volume of vehicle. Blood and livers were collected after 3 and 14 days. RESULTS: Serum alanine aminotransferase (ALT) increased from 39.8 U/L after sham operation to 537 U/L 3 days after BDL, which Alda-1 decreased to 281 U/L. Biliary infarcts with a periportal distribution developed with an area of 7.8% at 14 days after BDL versus 0% area after sham operation. Alda-1 treatment with BDL decreased biliary infarcts to 1.9%. Fibrosis detected by picrosirius red staining increased from 1.6% area in sham to 7.3% after BDL, which decreased to 3.8% with Alda-1. Alda-1 suppression of fibrosis was additionally confirmed by second harmonic generation microscopy. After BDL, collagen-I mRNA increased 12-fold compared to sham, which decreased to 6-fold after Alda-1 treatment. Smooth muscle α-actin expression in the liver, a marker of activated stellate cells, increased from 1% area in sham to 18.7% after BDL, which decreased to 5.3% with Alda-1. CD68-positive macrophages increased from 33.4 cells/field in sham to 134.5 cells/field after BDL, which decreased to 64.9 cells/field with Alda-1. Lastly, 4-hydroxynonenal adduct (4-HNE) immunofluorescence increased from 2.5% area in sham to 14.1% after BDL. Alda-1 treatment decreased 4-HNE to 2.2%. CONCLUSION: Accelerated aldehyde degradation by Alda-1 decreases BDL-induced liver necrosis, inflammation, and fibrosis, implying that aldehydes play an important role in the pathogenesis of cholestatic liver injury and fibrosis.

The application of Compont gel in chronic obstructive jaundice rats model.

PURPOSE: To establish a new rat model, the pathogenesis of which is closer to the clinical occurrence of chronic obstructive jaundice with liver fibrosis. METHODS: 90 SD rats were randomly divided into 3 groups. Group A common bile duct ligation, group B common bile duct injection compont and group C injection saline. The serum of three groups was extracted, and the liver function was detected by ELISA. HE staining, Masson staining and immunohistochemistry were used to detect liver pathology. RESULTS: Group B showed a fluctuant development of jaundice, obstructive degree reached a peak at 2 weeks, and decreased from 3 weeks. HA, LA and PCIII were significantly higher than control group. 3 weeks after surgery, liver tissue fibrosis occurred in group B, and a wide range of fiber spacing was formed at 5 weeks. Immunohistochemistry showed that hepatic stellate cells were more active than the control group. CONCLUSION: Intra-biliary injection of Compont gel is different from the classic obstructive jaundice animal model caused by classic bile duct ligation, which can provide an ideal rat model of chronic obstructive jaundice with liver fibrosis.

Elimination Pathways of Nanoparticles.

Understanding how nanoparticles are eliminated from the body is required for their successful clinical translation. Many promising nanoparticle formulations for in vivo medical applications are large (>5.5 nm) and nonbiodegradable, so they cannot be eliminated renally. A proposed pathway for these nanoparticles is hepatobiliary elimination, but their transport has not been well-studied. Here, we explored the barriers that determined the elimination of nanoparticles through the hepatobiliary route. The route of hepatobiliary elimination is usually through the following pathway: (1) liver sinusoid, (2) space of Disse, (3) hepatocytes, (4) bile ducts, (5) intestines, and (6) out of the body. We discovered that the interaction of nanoparticles with liver nonparenchymal cells ( e. g., Kupffer cells and liver sinusoidal endothelial cells) determines the elimination fate. Each step in the route contains cells that can sequester and chemically or physically alter the nanoparticles, which influences their fecal elimination. We showed that the removal of Kupffer cells increased fecal elimination by >10 times. Combining our results with those of prior studies, we can start to build a systematic view of nanoparticle elimination pathways as it relates to particle size and other design parameters. This is critical to engineering medically useful and translatable nanotechnologies.

Large Liver Blood Vessels and Bile Ducts Are Not Damaged by Electrochemotherapy with Bleomycin in Pigs.

The first clinical studies on the use of electrochemotherapy to treat liver tumours that were not amenable to surgery or thermal ablation techniques have recently been published. However, there is still a lack of data on the effects of electrochemotherapy on normal liver tissue. Therefore, we designed a translational animal model study to test whether electrochemotherapy with bleomycin causes clinically significant damage to normal liver tissue, with emphasis on large blood vessels and bile ducts. We performed electrochemotherapy with bleomycin or delivered electric pulses alone using a potentially risky treatment strategy in eight pigs. Two and seven days after treatment, livers were explanted, and histological analysis was performed. Blood samples were collected before treatment and again before euthanasia to evaluate blood biomarkers of liver function and systemic inflammatory response. We found no thrombosis or other clinically significant damage to large blood vessels and bile ducts in the liver. No clinical or laboratory findings suggested impaired liver function or systemic inflammatory response. Electrochemotherapy with bleomycin does not cause clinically significant damage to normal liver tissue. Our study provides further evidence that electrochemotherapy with bleomycin is safe for treatment of patients with tumours near large blood vessels in the liver.

Human-relevant preclinical in vitro models for studying hepatobiliary development and liver diseases using induced pluripotent stem cells.

IMPACT STATEMENT: In this review, we address the potential of human-induced pluripotent stem cell-based hepatobiliary differentiation technology as a means to study human liver development and cell fate determination, and to model liver diseases in an effort to develop a new human-relevant preclinical platform for drug development.

Stable gastric pentadecapeptide BPC 157 in the therapy of the rats with bile duct ligation.

Recently, stable gastric pentadecapeptide BPC 157 reversed the high MDA- and NO-tissue values to the healthy levels. Thereby, BPC 157 therapy cured rats with bile duct ligation (BDL) (sacrifice at 2, 4, 6, 8 week). BPC 157-medication (10 µg/kg, 10 ng/kg) was continuously in drinking water (0.16 µg/ml, 0.16 ng/ml, 12 ml/rat/day) since awakening from surgery, or since week 4. Intraperitoneal administration was first at 30 min post-ligation, last at 24 h before sacrifice. Local bath BPC 157 (10 µg/kg) with assessed immediate normalization of portal hypertension was given immediately after establishing portal hypertension values at 4, 6, 8 week. BPC 157 therapy markedly abated jaundice, snout, ears, paws, and yellow abdominal tegmentum in controls since 4th week, ascites, nodular, steatotic liver with large dilatation of main bile duct, increased liver and/or cyst weight, decreased body weight. BPC 157 counteracts the piecemeal necrosis, focal lytic necrosis, apoptosis and focal inflammation, disturbed cell proliferation (Ki-67-staining), cytoskeletal structure in the hepatic stellate cell (α-SMA staining), collagen presentation (Mallory staining). Likewise, counteraction includes increased AST, ALT, GGT, ALP, total bilirubin, direct and indirect and decreased albumin serum levels. As the end-result appear normalized MDA- and NO-tissue values, next to Western blot of NOS2 and NOS3 in the liver tissue, and decreased IL-6, TNF-α, IL-1ß levels in liver tissue. Finally, although portal hypertension is sustained in BDL-rats, with BPC 157 therapy, portal hypertension in BDL-rats is either not even developed or rapidly abated, depending on the given BPC 157's regimen. Thus, BPC 157 may counteract liver fibrosis and portal hypertension.

Effects of Endotoxin on Type 3 Inositol 1,4,5-Trisphosphate Receptor in Human Cholangiocytes.

Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll-like receptor 4 (TLR4), which links to activation of nuclear factor-κB (NF-κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-κB, and promoter activity was inhibited by p65/p50. Nested 0.5- and 1.0-kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF-κB to down-regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.

Directing the growth and alignment of biliary epithelium within extracellular matrix hydrogels.

Three-dimensional (3D) printing of decellularized extracellular matrix (dECM) hydrogels is a promising technique for regenerative engineering. 3D-printing enables the reproducible and precise patterning of multiple cells and biomaterials in 3D, while dECM has high organ-specific bioactivity. However, dECM hydrogels often display poor printability on their own and necessitate additives or support materials to enable true 3D structures. In this study, we used a sacrificial material, 3D-printed Pluronic F-127, to serve as a platform into which dECM hydrogel can be incorporated to create specifically designed structures made entirely up of dECM. The effects of 3D dECM are studied in the context of engineering the intrahepatic biliary tree, an often-understudied topic in liver tissue engineering. Encapsulating biliary epithelial cells (cholangiocytes) within liver dECM has been shown to lead to the formation of complex biliary trees in vitro. By varying several aspects of the dECM structures' geometry, such as width and angle, we show that we can guide the directional formation of biliary trees. This is confirmed by computational 3D image analysis of duct alignment. This system also enables fabrication of a true multi-layer dECM structure and the formation of 3D biliary trees into which other cell types can be seeded. For example, we show that hepatocyte spheroids can be easily incorporated within this system, and that the seeding sequence influences the resulting structures after seven days in culture. STATEMENT OF SIGNIFICANCE: The field of liver tissue engineering has progressed significantly within the past several years, however engineering the intrahepatic biliary tree has remained a significant challenge. In this study, we utilize the inherent bioactivity of decellularized extracellular matrix (dECM) hydrogels and 3D-printing of a sacrificial biomaterial to create spatially defined, 3D biliary trees. The creation of patterned, 3D dECM hydrogels in the past has only been possible with additives to the gel that may stifle its bioactivity, or with rigid and permanent support structures that may present issues upon implantation. Additionally, the biological effect of 3D spatially patterned liver dECM has not been demonstrated independent of the effects of dECM bioactivity alone. This study demonstrates that sacrificial materials can be used to create pure, multi-layer dECM structures, and that strut width and angle can be changed to influence the formation and alignment of biliary trees encapsulated within. Furthermore, this strategy allows co-culture of other cells such as hepatocytes. We demonstrate that not only does this system show promise for tissue engineering the intrahepatic biliary tree, but it also aids in the study of duct formation and cell-cell interactions.