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The pathogenesis of viral infection is attributed to two folds: intrinsic cell death pathway activation due to the viral cytopathic effect, and immune-mediated extrinsic cellular injuries. The immune system, encompassing both innate and adaptive immunity, therefore acts as a double-edged sword in viral infection. Insufficient potency permits pathogens to establish lifelong persistent infection and its consequences, while excessive activation leads to organ damage beyond its mission to control viral pathogens. The innate immune response serves as the front line of defense against viral infection, which is triggered through the recognition of viral products, referred to as pathogen-associated molecular patterns (PAMPs), by host cell pattern recognition receptors (PRRs). The PRRs–PAMPs interaction results in the induction of interferon-stimulated genes (ISGs) in infected cells, as well as the secretion of interferons (IFNs), to establish a tissue-wide antiviral state in an autocrine and paracrine manner. Cumulative evidence suggests significant variability in the expression patterns of PRRs, the induction potency of ISGs and IFNs, and the IFN response across different cell types and species. Hence, in our understanding of viral hepatitis pathogenesis, insights gained through hepatoma cell lines or murine-based experimental systems are uncertain in precisely recapitulating the innate antiviral response of genuine human hepatocytes. Accordingly, this review article aims to extract and summarize evidence made possible with bona fide human hepatocytes-based study tools, along with their clinical relevance and implications, as well as to identify the remaining gaps in knowledge for future investigations. [ABSTRACT FROM AUTHOR]

The pathogenesis of viral infection is attributed to two folds: intrinsic cell death pathway activation due to the viral cytopathic effect, and immune-mediated extrinsic cellular injuries. The immune system, encompassing both innate and adaptive immunity, therefore acts as a double-edged sword in viral infection. Insufficient potency permits pathogens to establish lifelong persistent infection and its consequences, while excessive activation leads to organ damage beyond its mission to control viral pathogens. The innate immune response serves as the front line of defense against viral infection, which is triggered through the recognition of viral products, referred to as pathogen-associated molecular patterns (PAMPs), by host cell pattern recognition receptors (PRRs). The PRRs–PAMPs interaction results in the induction of interferon-stimulated genes (ISGs) in infected cells, as well as the secretion of interferons (IFNs), to establish a tissue-wide antiviral state in an autocrine and paracrine manner. Cumulative evidence suggests significant variability in the expression patterns of PRRs, the induction potency of ISGs and IFNs, and the IFN response across different cell types and species. Hence, in our understanding of viral hepatitis pathogenesis, insights gained through hepatoma cell lines or murine-based experimental systems are uncertain in precisely recapitulating the innate antiviral response of genuine human hepatocytes. Accordingly, this review article aims to extract and summarize evidence made possible with bona fide human hepatocytes-based study tools, along with their clinical relevance and implications, as well as to identify the remaining gaps in knowledge for future investigations.

Anamorelin, developed for the treatment of cancer cachexia, is an orally active medication that improves appetite and food intake, thereby increasing body mass and physical functioning. It is classified as a growth hormone secretagogue and strictly monitored by the World Anti-Doping Agency (WADA), owing to its anabolic enhancing potential. Identifying anamorelin and/or metabolite biomarkers of consumption is critical in doping controls. However, there are currently no data available on anamorelin human metabolic fate. The aim of this study was to investigate and identify biomarkers characteristic of anamorelin intake using in silico metabolite predictions with GLORYx, in vitro incubation with 10-donor-pooled human hepatocytes, liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis, and data processing with Thermo Scientific's Compound Discoverer. In silico prediction resulted in N-acetylation at the methylalanyl group as the main transformation (score, 88%). Others including hydroxylation at the indole substructure, and oxidation and N-demethylation at the trimethylhydrazino group were predicted (score, ≤36%). Hepatocyte incubations resulted in 14 phase I metabolites formed through N-demethylation at the trimethylhydrazino group, N-dealkylation at the piperidine ring, and oxidation at the indole and methylalanyl groups; and two phase II glucuronide conjugates occurring at the indole. We propose four metabolites detected as specific biomarkers for toxicological screening. [ABSTRACT FROM AUTHOR]

Human hepatocyte culture system represents by far the most physiologically relevant model for our understanding of liver biology and diseases; however, its versatility has been limited due to the rapid and progressive loss of genuine characteristics, indicating the inadequacy of in vitro milieu for fate maintenance. This study, therefore, is designed to define environmental requirements necessary to sustain the homeostasis of terminally differentiated hepatocytes. Our study reveals that the supplementation of dimethyl sulfoxide (DMSO) is indispensable in mitigating fate deterioration and promoting adaptation to the in vitro environment, resulting in the restoration of tight cell-cell contact, cellular architecture, and polarity. The morphological recovery was overall accompanied by the restoration of hepatocyte marker gene expression, highlighting the interdependence between the cellular architecture and the maintenance of cell fate. However, beyond the recovery phase culture, DMSO supplementation is deemed detrimental due to the potent inhibitory effect on a multitude of hepatocyte functionalities while its withdrawal results in the loss of cell fate. In search of DMSO substitute, our screening of organic substances led to the identification of dimethyl sulfone (DMSO2), which supports the long-term maintenance of proper morphology, marker gene expression, and hepatocytic functions. Moreover, hepatocytes maintained DMSO2 exhibited clinically relevant toxicity in response to prolonged exposure to xenobiotics as well as alcohol. These observations suggest that the stepwise culture configuration consisting of the consecutive supplementation of DMSO and DMSO2 confers the microenvironment essential for the fate and functional maintenance of terminally differentiated human hepatocytes. [ABSTRACT FROM AUTHOR]

Anamorelin, developed for the treatment of cancer cachexia, is an orally active medication that improves appetite and food intake, thereby increasing body mass and physical functioning. It is classified as a growth hormone secretagogue and strictly monitored by the World Anti-Doping Agency (WADA), owing to its anabolic enhancing potential. Identifying anamorelin and/or metabolite biomarkers of consumption is critical in doping controls. However, there are currently no data available on anamorelin human metabolic fate. The aim of this study was to investigate and identify biomarkers characteristic of anamorelin intake using in silico metabolite predictions with GLORYx, in vitro incubation with 10-donor-pooled human hepatocytes, liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis, and data processing with Thermo Scientific’s Compound Discoverer. In silico prediction resulted in N-acetylation at the methylalanyl group as the main transformation (score, 88%). Others including hydroxylation at the indole substructure, and oxidation and N-demethylation at the trimethylhydrazino group were predicted (score, ≤36%). Hepatocyte incubations resulted in 14 phase I metabolites formed through N-demethylation at the trimethylhydrazino group, N-dealkylation at the piperidine ring, and oxidation at the indole and methylalanyl groups; and two phase II glucuronide conjugates occurring at the indole. We propose four metabolites detected as specific biomarkers for toxicological screening.

Adeno-associated virus (AAV) vectors have been successfully used in patients with bleeding disorders and blindness. For human liver targeting, two major factors restrict effective AAV transduction after systemic administration of AAV vectors: human hepatocyte tropism and neutralizing antibodies (Nabs). In this study, we attempted to isolate AAV variants with the ability to transduce human hepatocytes and escape Nabs using a directed evolution approach in vivo. After four cycles of selection, 14 AAV capsid mutants were identified from a capsid shuffling library selected in the presence of human Intravenous Immunoglobulin (IVIG) and isolated from human hepatocytes xenografted into chimeric mice. AAV neutralization assays using IVIG showed that most of the mutants showed the Nab escape pattern in a manner similar to that of AAV8 or AAV9 and better than that of other AAV serotypes. Different mutants displayed varying capacities to escape Nab activity from individual serum samples collected from healthy subjects or hemophilia patients. The mutant AAV LP2-10 was found in 12 colonies out of 25, which was composed of capsids from AAV serotypes 2, 6, 8, and 9, with VP3 subunits derived from AAV8 swapped with AAV6 from residues 261 to 272. The mutant AAV LP2-10 manifested a higher ability than that of other serotypes to escape Nabs in IVIG and most human serum samples. After injection of AAV vectors encoding a self-complementary GFP cassette into chimeric mice, LP2-10 transduced human hepatocytes with efficiency similar to that of AAV8. In summary, AAV mutants can be isolated in humanized mice with both human hepatocyte tropism and the ability to evade Nab activity.

Viral hepatitis particularly Hepatitis B Virus (HBV) is still an ongoing health issue worldwide. Despite the vast technological advancements in research and development, only HBV vaccines, typically given during early years, are currently available as a preventive measure against acquiring the disease from a secondary source. In general, HBV can be cleared naturally by the human immune system if detected at low levels early. However, long term circulation of HBV in the peripheral blood may be detrimental to the human liver, specifically targeting human hepatocytes for cccDNA integration which inevitably supports HBV life cycle for the purpose of reinfection in healthy cells. Although there is some success in using nucleoside analogs or polyclonal antibodies targeting HBV surface antigens (HBsAg) in patients with acute or chronic HBV+ (CHB), majority of them would either respond only partially or succumb to the disease entirely unless they undergo liver transplants from a fully matched healthy donor and even so may not necessarily guarantee a 100% chance of survival. Indeed, in vitro/ex vivo cultures and various transgenic animal models have already provided us with a good understanding of HBV but they primarily lack human specificity or virus-host interactions in the presence of human immune surveillance. Therefore, the demand of utilizing humanized mice has increased over the last decade as a pre-clinical platform for investigating human-specific immune responses against HBV as well as identifying potential immunotherapeutic strategies in eradicating the virus. Basically, this review covers some of the recent developments and key advantages of humanized mouse models over other conventional transgenic mice platforms. [ABSTRACT FROM AUTHOR]

BACKGROUND: Oxidative stress injury plays an important role in the development of chronic hepatitis, and sulforaphane is a natural product with good antioxidant activity. OBJECTIVE: To investigate the protective effects of sulforaphane on oxidative stress injury of human hepatocytes (LO2) stimulated by H2O2, and to investigate its possible mechanism. METHODS: LO2 cells were cultured in vitro, followed by 24-hour treatment with 100 μg/L H2O2 to induce the oxidative stress model of the cells. The cells were divided into five groups: control group, model group, sulforaphane treatment groups (low-, middle-, high-dose groups: H2O2+10, 20, and 40 μmol/L sulforaphane, respectively). Cell counting kit-8 was used to measure the relative survival rate of cells, and analyze the levels of oxidative stress products in cell homogenate. Western blot assay was used to determine the expression levels of nuclear factor erythroid-2 related factor 2 (Nrf2), GCLC, NQO1 and heme oxygenase-1. The study protocol was in accordance with the related ethic requirements of Central Hospital of Enshi Autonomous Prefecture. RESULTS AND CONCLUSION: H2O2 could induce oxidative stress injury in LO2 cells. Compared with the model group, sulforaphane at various concentrations could significantly induce the proliferation of LO2 cells, inhibit the production of malondialdehyde and nitric oxide, and promote the activity of superoxide dismutase (P < 0.05). Western blot results revealed that sulforaphane could induce the transfer of Nrf2 into the nucleus and significantly increase the expression of GCLC, NQO1, and heme oxygenase-1 proteins (P < 0.05). To conclude, sulforaphane can significantly relieve the oxidative stress injury of LO2 cells induced by H2O2 and its underlying mechanism may be related to the activation of Nrf2 pathway. [ABSTRACT FROM AUTHOR]

Advancement in both bioengineering and cell biology of the liver led to the establishment of the first-generation humanized liver chimeric mouse (HLCM) model in 2001. The HLCM system was initially developed to satisfy the necessity for a convenient and physiologically representative small animal model for studies of hepatitis B virus and hepatitis C virus infection. Over the last two decades, the HLCM system has substantially evolved in quality, production capacity, and utility, thereby growing its versatility beyond the study of viral hepatitis. Hence, it has been increasingly employed for a variety of applications including, but not limited to, the investigation of drug metabolism and pharmacokinetics and stem cell biology. To date, more than a dozen distinctive HLCM systems have been established, and each model system has similarities as well as unique characteristics, which are often perplexing for end-users. Thus, this review aims to summarize the history, evolution, advantages, and pitfalls of each model system with the goal of providing comprehensive information that is necessary for researchers to implement the ideal HLCM system for their purposes. Furthermore, this review article summarizes the contribution of HLCM and its derivatives to our mechanistic understanding of various human liver diseases, its potential for novel applications, and its current limitations. [ABSTRACT FROM AUTHOR]

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl- l -lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/ C max) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/ C max>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI. [ABSTRACT FROM AUTHOR]

Drug-induced liver injury (DILI) diagnosis and classification (hepatocellular, cholestatic, and mixed) relies on traditional clinical biomarkers (eg ALT and ALP), despite limitations such as extrahepatic interferences, narrow dynamic ranges, and low mechanistic value. microRNAs may be very useful for complementing traditional DILI biomarkers but most studies in this direction have considered only paracetamol poisoning. Thus the value of microRNAs (miRNAs) as biomarkers for idiosyncratic DILI has not yet been demonstrated. In this study, we first examined the effect of model cholestatic drugs on the human hepatocyte miRNome by RNAseq and RT-qPCR. Results demonstrated that chlorpromazine, cyclosporin A, and ANIT induced (miR-21-3p, -21-5p, -22-3p, -27a-5p, -1260b, -34a-5p, and -98-5p) and repressed (-122-5p, -192-5p, -30c-5p, -424-5p, and -16-5p) specific miRNAs in sandwich-cultured upcyte hepatocytes. However, no common signature was found for cholestatic drugs. Next we investigated the levels of these miRNA in human serum and found that most were also significantly altered in cholestatic/mixed DILI patients upon hospital/ambulatory admission. However, miR-122-5p, -192-5p, -34a-5p, and -22-3p demonstrated a much more significant induction in patients with hepatocellular DILI, thus revealing better specificity for hepatocellular damage. Time-course analyses demonstrated that -1260b and -146 had a very similar profile to ALP, but with wider dynamic ranges, while -16-5p and -451a showed a negative correlation. Conversely, -122-5p and -192-5p correlated with ALT but with wider dynamic ranges and faster recoveries. Finally, the 122/451a and 122/16 ratios showed excellent prediction performances in both the study [area under the receiver operating characteristic curve (AUROC) >0.93] and the validation cohort (AUROC > 0.82), and can, therefore, be postulated for the first time as circulating miRNA biomarkers for idiosyncratic DILI. [ABSTRACT FROM AUTHOR]

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7. • LC-MS study on biotransformation of phytocannabinoids in human hepatocytes. • MS-fragmentation patterns for CBD, CBN, CBC, CBG and their metabolites reported. • The study screened for the activity of recombinant human cytochromes P450 and UDP-glucuronosyltransferases. • The susceptibility to transformations in hepatocytes: CBN > CBG > CBC > CBD. [ABSTRACT FROM AUTHOR]

Patients with end‐stage renal disease have increased plasma concentrations of statins, which is a risk factor for rhabdomyolysis, as well as elevated levels of uremic toxins (UTs). We investigated the effects of uremic serum residue and UTs on organic anion‐transporting peptide (OATP1B1)‐ and OATP1B3‐mediated pravastatin uptake. We evaluated the effects of normal serum residue with four UTs (hippuric acid, 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furan propionate, indole‐3‐acetic acid, and 3‐indoxyl sulfate) and uremic serum residue on pravastatin uptake by OATP1B1‐ or OATP1B3‐expressing HEK293 cells. Furthermore, we assessed the contribution of each transporter using cryopreserved human hepatocytes. Uremic serum residue and UTs significantly inhibited OATP1B1‐mediated pravastatin uptake. Uremic serum residue accelerated OATP1B3‐mediated pravastatin uptake, while UTs had no effect. There was no difference in pravastatin uptake between uremic‐ and normal serum residue‐treated hepatocytes. The results suggest that the effects of uremic serum on pravastatin hepatic uptake may be considered negligible in end‐stage renal disease patients. [ABSTRACT FROM AUTHOR]

Background/Aim: Emerging evidence suggests that microRNA (miRNA) mediated gene regulation influences the maintenance of metabolic homeostasis, particularly the states of obesity and insulin resistance, thereby providing a potential link between miRNAs and nonalcoholic fatty liver disease (NAFLD). Methods: Sprague-Dawley rats fed a high-fat diet (HFD) were used to establish a rat model of NAFLD. The miRNA expression profile of liver tissues was evaluated using Illumina HiSeq deep sequencing. Selected miRNAs were then validated by real-time PCR at both 4- and 12-week time points. Furthermore, the expression levels of these miRNAs were assessed in HepG2 cells and human hepatocytes treated with free fatty acids (FFAs) and proinflammatory factors (tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Results: Our results showed that consumption of a HFD for 4 weeks caused simple steatosis, which progressed to steatohepatitis at 12 weeks. miRNA deep sequencing analysis identified 44 known up-regulated miRNAs (fold change >1.5) and 12 down-regulated miRNAs (fold change in vitro and vivo. Interestingly, the expression levels of these six miRNAs were increased in HepG2 cells and human hepatocytes after treatment with FFAs and proinflammatory factors. Conclusion: These findings suggest a critical role for miRNAs in the pathogenesis of NAFLD.

25B-NBF, 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is a new psychoactive substance classified as a phenethylamine. It is a potent agonist of the 5-hydroxytryptamine receptor, but little is known about its metabolism and elimination properties since it was discovered. To aid 25B-NBF abuse screening, the metabolic characteristics of 25B-NBF were investigated in human hepatocytes and human cDNA-expressed cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes using liquid chromatography⁻high resolution mass spectrometry. At a hepatic extraction ratio of 0.80, 25B-NBF was extensively metabolized into 33 metabolites via hydroxylation, O-demethylation, bis-O-demethylation, N-debenzylation, glucuronidation, sulfation, and acetylation after incubation with pooled human hepatocytes. The metabolism of 25B-NBF was catalyzed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and UGT2B7 enzymes. Based on these results, it is necessary to develop a bioanalytical method for the determination of not only 25B-NBF but also its metabolites in biological samples for the screening of 25B-NBF abuse.

Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5 ′ -GGTTCA-(n)5-AGGTCA-3 ′) in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.