Diseased Liver Tissue Research Articles

Scaling-law mechanical marker for liver fibrosis diagnosis and drug screening through machine learning.

Studies of cell and tissue mechanics have shown that significant changes in cell and tissue mechanics during lesions and cancers are observed, which provides new mechanical markers for disease diagnosis based on machine learning. However, due to the lack of effective mechanic markers, only elastic modulus and iconographic features are currently used as markers, which greatly limits the application of cell and tissue mechanics in disease diagnosis. Here, we develop a liver pathological state classifier through a support vector machine method, based on high dimensional viscoelastic mechanical data. Accurate diagnosis and grading of hepatic fibrosis facilitates early detection and treatment and may provide an assessment tool for drug development. To this end, we used the viscoelastic parameters obtained from the analysis of creep responses of liver tissues by a self-similar hierarchical model and built a liver state classifier based on machine learning. Using this classifier, we implemented a fast classification of healthy, diseased, and mesenchymal stem cells (MSCs)-treated fibrotic live tissues, and our results showed that the classification accuracy of healthy and diseased livers can reach 0.99, and the classification accuracy of the three liver tissues mixed also reached 0.82. Finally, we provide screening methods for markers in the context of massive data as well as high-dimensional viscoelastic variables based on feature ablation for drug development and accurate grading of liver fibrosis. We propose a novel classifier that uses the dynamical mechanical variables as input markers, which can identify healthy, diseased, and post-treatment liver tissues.

HepScope: CNN-based single-cell discrimination of malignant hepatocytes

Background: Hepatocellular carcinoma (HCC) poses a significant global health challenge. This study aims to identify robust single-cell biomarkers specifically designed to distinguish between malignant and non-malignant hepatocytes, thereby advancing precision medicine strategies for HCC. Methods: At the single-cell level, we investigated the reliability of established marker genes for malignant hepatocytes in distinguishing them from their non-malignant counterparts. Leveraging one of the largest integrated single-cell transcriptomics (scRNA-seq) datasets of healthy and diseased liver tissues, alongside independent scRNA-seq, spatial transcriptomics (stRNA-seq), bulk RNA-seq, and bulk proteome datasets, we defined a signature gene set, denoted as HepScope. Five other publicly available gene sets related to HCC were used for comparison with HepScope. The development of machine learning (ML) and 1-dimensional convolutional neural network (1D-CNN) models involved utilizing normalized expression of signature genes from scRNA-seq datasets. The HepScope-1D-CNN model was developed to assess differentiation performance by considering the expression values of each signature gene across different tissues and within the same tissue. Finally, we calculated a risk score based on the signature genes to evaluate its prognostic capability. Result: We demonstrated the lack of specificity and consistency among current HCC marker genes in distinguishing malignant hepatocytes from non-malignant hepatocytes. The differential expression (DEG) analysis identified 113 genes upregulated in malignant hepatocytes, forming the HepScope. In contrast to HepScope, the expression values of the five other publicly available gene sets did not exhibit the ability to discriminate between malignant and non-malignant hepatocytes. Utilizing the genes from HepScope, the Gaussian Naive Bayes ML model proficiently distinguished between malignant and non-malignant hepatocytes. The HepScope-CNN model showed the highest performance and effciency compared to other 1D-CNN models trained with different gene sets. Conclusion: In conclusion, our study reveals a novel gene set designed to precisely identify malignant hepatocytes at the single-cell level. This research holds promise for advancing precision medicine in HCC, providing valuable insights into the development of targeted therapies and personalized treatment strategies. We acknowledge support provided by the National Research Foundation (NRF) of Korea (2020R1A6A1A03043539, 2020M3A9D8037604, and 2022R1C1C1004756) and the Korea Health Technology R&D Project of the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HR22C1734). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells

The presence of CD8+ T cells in the cytoplasm of biliary epithelial cells (BEC) has been correlated with biliary damage associated with primary biliary cholangitis (PBC). Here, we characterise the mechanism of CD8+ T cell invasion into BEC. CD8+ T cells observed within BEC were large, eccentric, and expressed E-cadherin, CD103 and CD69. They were also not contained within secondary vesicles. Internalisation required cytoskeletal rearrangements which facilitated contact with BEC. Internalised CD8+ T cells were observed in both non-cirrhotic and cirrhotic diseased liver tissues but enriched in PBC patients, both during active disease and at the time of transplantation. E-cadherin expression by CD8+ T cells correlated with frequency of internalisation of these cells into BEC. E-cadherin+ CD8+ T cells formed β-catenin-associated interactions with BEC, were larger than E-cadherin- CD8+ T cells and invaded into BEC more frequently. Overall, we unveil a distinct cell-in-cell structure process in the liver detailing the invasion of E-cadherin+ CD103+ CD69+ CD8+ T cells into BEC.

7 Gene expression analysis of diseased and control liver tissue to explore pathogenic pathways in inflammation

7 Gene expression analysis of diseased and control liver tissue to explore pathogenic pathways in inflammation

Farnesoid X receptor in chronic liver diseases: an immunohistochemical study

ABSTRACT Chronic hepatitis C virus (HCV) related liver diseases are still an ongoing cause of hepatic failure despite the effective role of direct-acting anti-viral agents. Farnesoid X receptor (FXR) agonists have a potential therapeutic effect on the management of chronic liver diseases (CLD). However, data regarding FXR protein expression in human CLDs are limited and conflicting. We aimed to assess the immunohistochemical expression of FXR in HCV-related chronic hepatitis and cirrhosis in comparison with metabolic-associated fatty liver disease (MAFLD) and normal liver tissue. The expression of FXR was low both in hepatocytes and bile ducts of HCV-related chronic hepatitis and cirrhosis (p = .001, respectively). In addition, a significantly low expression of FXR was observed in HCV-related hepatitis and cirrhosis groups compared to MAFLD in hepatocytes (p < .001, for both) and bile ducts (p = .004 and p = .018). FXR expression in HCV-related cirrhosis was significantly associated with compensated liver function (p = .032) and low inflammatory activity (p = .022). FXR expression decreases in HCV-related CLDs. There was some evidence that FXR expression could protect against post-hepatitis cirrhosis.

Imaging of Liver Tissues Using a Combination of Silver Impregnation and Low-Vacuum Scanning Electron Microscopy; A Simple Method for the High-Resolution Visualization of Reticulin Structures with Applicability to a Quantitative Analysis.

Low-vacuum scanning electron microscopy (LVSEM) enables the detailed three-dimensional imaging of archival tissues without special pretreatments. The clinical utility of LVSEM in the assessment of liver diseases has not yet been defined. So, we investigated the utility of LVSEM imaging in morphological assessments of normal and diseased liver tissues, with a focus on reticulin structures. Formalin-fixed tissue samples of two normal livers and two hepatocellular carcinomas with background regenerative nodules/areas were stained with platinum blue stain or silver-impregnated using Watanabe's method and then comparatively observed under LVSEM. We also evaluated the applicability of LVSEM imaging of liver tissues to a quantitative analysis using a digital image analysis technique. Optimal high-resolution images of reticulin structures were obtained using 10-μm-thick silver-impregnated sections. Reticulin fibers were clearly observed to run dendritically around sinusoids in normal livers, and markedly increased in regenerative nodules/areas. Normal reticulin frameworks were lost in hepatocellular carcinoma, leaving a few fragments of reticulin fibers within tumors. Moreover, when a quantitative analysis was applied to these images, we successfully demonstrated a significantly higher reticulin fiber density in regenerative nodules/areas than in the normal liver (P < 0.05). We not only obtained detailed three-dimensional images of reticulin structures in various liver tissues by LVSEM combined with silver impregnation but also showed their applicability to a quantitative analysis. The method presented herein may be applied to future studies for the more accurate diagnosis and better classification/risk stratification of various liver diseases.

The study of a key molecule Caspase-1 of inflammasome in hepatitis B virus-related diseases

Objective: To investigate the expression and role of asparte-specific cysteine protease (Caspase)-1, inflammasomes key molecule, in hepatitis B virus (HBV)-related diseases. Methods: HBV-related liver disease patients' serum (438 cases) and liver tissue (82 cases) samples were collected from Beijing You'an Hospital affiliated with Capital Medical University. The mRNA expression level of caspase-1 in liver tissue was detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein expression level of Caspase-1 in liver tissue was detected by the immunofluorescence method. The activity of Caspase-1 was detected using the Caspase-1 colorimetric assay kit. The level of Caspase-1 in the serum was detected by an ELISA kit. Results: The results of qRT-PCR showed that the mRNA level of Caspase-1 was downregulated in patients with chronic hepatitis B (CHB), cirrhosis (LC), and hepatocellular carcinoma (HCC), while up-regulated in patients with acute-on-chronic liver failure (ACLF) (P＜0.01) compared with normal subjects. Immunofluorescence assays showed that Caspase-1 protein levels were elevated in ACLF patients, decreased in HCC and LC patients, and slightly elevated in CHB patients. The activity of Caspase-1 was slightly higher in liver tissue from CHB, LC, and HCC patients than in the normal control group, and there was no statistically significant difference between the groups. Additionally, compared with the control group, Caspase-1 activity was significantly reduced in the ACLF group (P＜0.01). Serum Caspase-1 levels were significantly lower in patients with CHB, ACLF, LC, and HCC than in normal subjects, and serum Caspase-1 levels were lowest in patients with ACLF (P＜0.001). Conclusion: Caspase-1, a key molecule of inflammasomes, plays an important role in HBV-related diseases and has significant differences, showing distinct features for ACLF than other HBV-related diseases.

Histopathological characteristics of striped catfish (Pangasianodon hypophthalmus) in challenge with Edwardsiella ictaluri

The present study attempted to understand the histopathological index of striped catfish (Pangasianodon hypophthalmus) in challenge with Edwardsiella ictaluri, the causative agent of bacillary necrosis of Pangasius. Total of 355 healthy striped catfish juveniles was challenged with E. ictaluri (25 control; 330 infected). After the challenge, a total of 355 samples of the trunk kidney, liver, and spleen were collected at five different time points including just prior to infection, 24, 48, 264 and 312 hours post-infection (hpi) to measure the represented tissue damage of the fish. Results showed that many areas of the diseased liver, kidney, and fish organ tissues have been congested and hemorrhaged early at 24 hpi. The number of macrophage centers in the kidney and spleen increased at 24 - 48 hpi. Tissue damage (multifocal hemorrhages; necrosis) increased sharply from the period 48 - 256 hpi. The increased number of dead fish throughout infection, particularly the period from 48 to 200 hpi. At 312 hpi, the histopathological index was the highest, so the mortality rate was the highest during the experimental period.

Proteomic quantification of perturbation to pharmacokinetic target proteins in liver disease

Model-based assessment of drug pharmacokinetics in liver disease requires quantification of abundance and disease-related changes in hepatic enzymes and transporters. This study aimed to assess performance of three label-free methods [high N (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA)] against QconCAT-based targeted data in healthy and diseased (cancer and cirrhosis) liver tissue. Measurements were compared across methods and disease-to-control ratios provided a ‘disease perturbation factor’ (DPF) for each protein. Mean label-free measurements of targets correlated well (Pearson's coefficient, r = 0.91–0.98 p < 0.001) and with targeted data (r = 0.65–0.95, p < 0.001). Concordance with targeted data was generally moderate (Lin's concordance coefficient, ρc = 0.46–0.92), depending on methodology. Moderate precision and accuracy were observed for label-free methods (average fold error, AFE = 1.44–1.68; absolute average fold error, AAFE = 2.44–3.23). The DPF reconciled the data and indicated downregulated expression in cancer and cirrhosis, consistent with an inflammatory effect. HiN estimated perturbation consistently with targeted data (AFEHiN = 1.07, AAFEHiN = 1.57), whereas iBAQ overestimated (AFEiBAQ = 0.81, AAFEiBAQ = 1.67) and TPA underestimated (AFETPA = 1.37, AAFETPA = 1.65) disease effect. Progression from mild to severe cirrhosis was consistent with progressive decline in expression, reproduced by HiN but overestimated by iBAQ and underestimated by TPA (AFEHiN = 0.98, AFEiBAQ = 0.60, AFETPA = 1.24). DPF data confirmed non-uniform disease effect on drug-elimination pathways and progressive impact of disease severity. SignificanceThis study demonstrated good correlation and moderate concordance between intensity-based label-free proteomic methods (HiN, iBAQ and TPA) and targeted data. Label-free measurements tended to overestimate abundance, but differences were reconciled using a disease perturbation factor (DPF) for each protein. With targeted data as a reference, HiN defined disease perturbation and the impact of disease progression consistently, indicating that the use of ‘razor’ peptides for quantification against an exogenous standard provides biologically sensible quantitative fingerprints of disease. Disease-driven perturbations in expression relative to healthy baseline are incorporated into drug kinetic models used to predict drug exposure in disease populations where clinical studies may not be feasible.

Response of Human Liver Tissue to Innate Immune Stimuli.

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

Comparative Histological Study of Therapeutic Effect of Mesenchymal Stem Cells versus Mesenchymal Stem Cells Co-Cultured with Liver Tissue on Carbon Tetrachloride-Induced Hepatotoxicity in Adult Male Albino Rats.

Liver diseases are major causes of morbidity and mortality. Mesenchymal stem cells (MSCs) have immunomodulatory, anti-inflammatory, and antifibrotic effects, so they can be used in the treatment of liver diseases. MSCs co-cultured with diseased liver tissue improve the homing capacity, survival rate, and paracrine effects of the MSCs, as well as the ability to enhance liver function. This work aimed to study the therapeutic effect of MSCs versus MSCs co-cultured with liver tissue on carbon tetrachloride (CCl4)-induced hepatotoxicity in adult male albino rats. Twenty adult male albino rats were divided into four equal groups; Group I (control group), Group II received CCl4 intraperitoneally (i.p.), Group III received CCl4 i.p. and then injected with MSCs intravenously (i.v.), and Group IV received CCl4 i.p. and then injected with co-cultured MSCs i.v. Finally, liver specimens were processed for light microscopy (LM) and electron microscopy (EM). Statistical analysis was carried out to assess histological scoring, area percentage of collagen fibers, number of glial fibrillary acidic protein-positive cells, and biochemical analysis of alanine aminotransferase and aspartate aminotransferase. Statistical analysis of (histological scoring, area % of collagen fibers, and biochemical analysis) was done by using one-way analysis of variance (ANOVA) test using graphpad software (SanDiego, CA, USA). The means ± standard deviations were used for statistical analysis. LM of Group II revealed loss of hepatic architecture and diffuse fibrosis with dilated congested blood vessels, bile ductular proliferation, and cellular infiltrations. Vacuolated cytoplasm with or without pyknotic nuclei was observed in addition to micro- and macro-steatosis. EM demonstrated disfigured hepatocytes with abnormal organelles surrounding atypical nucleus. Group III showed restoration of the normal liver architecture with greater extent in Group IV. Statistical analysis confirmed the microscopic findings. Co-cultured MSCs with diseased liver tissue augmented the therapeutic effects of MSCs in treating hepatotoxicity induced by CCl4 in adult male albino rats.

Evaluation of the solubility of 11-keto-β-boswellic acid and its histological effect on the diabetic mice liver using a novel technique

Background and Aim:The literature is scant on the effect of 11-keto-β-boswellic acid (KBA) on the liver of diabetes-induced mice. This study was designed to develop a rapid, sensitive, accurate, and inexpensive detection technique for evaluating the solubility of KBA obtained from the gum resin of Omani frankincense (Boswellia sacra) in the liver of streptozotocin-induced diabetic mice using Fourier transform infrared (FTIR) reflectance spectroscopy coupled with principal components analysis (PCA). It also aimed to investigate the effect of KBA on histological changes in the hepatocytes of diabetic mice.Materials and Methods:Eighteen mice were assigned to the healthy control group, the diabetic control group, or the KBA-treated diabetic group. Liver tissue samples from all groups were scanned using an FTIR reflectance spectrophotometer in reflection mode. FTIR reflectance spectra were collected in the wavenumber range of 400-4000 cm−1 using an attenuated total reflectance apparatus.Results:FTIR reflectance spectra were analyzed using PCA. The PCA score plot, which is an exploratory multivariate data set, revealed complete segregation among the three groups’ liver samples based on changes in the variation of wavenumber position in the FTIR reflectance spectra, which indicated a clear effect of KBA solubility on treatments. Histological analysis showed an improvement in the liver tissues, with normal structures of hepatocytes exhibiting mild vacuolation in their cytoplasm.Conclusion:KBA improved the morphology of liver tissues in the diabetic mice and led to complete recovery of the damage observed in the diabetic control group. FTIR reflectance spectroscopy coupled with PCA could be deployed as a rapid, low-cost, and non-destructive detection method for evaluating treatment effects in diseased liver tissue based on the solubility of KBA.

Evaluation of Ultrasonic Attenuation in Primary and Secondary Human Liver Tumors and Its Potential Effect on High-Intensity Focused Ultrasound Treatment

Primary and secondary liver tumors are completely different diseases but are usually treated similarly using high-intensity focused ultrasound (HIFU). However, the acoustic parameters of these tissues are not well documented. In this study, attenuation coefficients were evaluated in fresh primary (N = 8) and secondary (N = 13) human liver tumor samples recovered by hepatectomy. The average attenuation coefficients of the primary and secondary liver tumors were 0.10 ± 0.03 and 0.20 ± 0.04 Np/cm/MHz, respectively. The average attenuation coefficients of the liver tissue surrounding the primary and secondary tumors were 0.16 ± 0.07 and 0.07 ± 0.02 Np/cm/MHz, respectively. Numerical simulations performed using these values revealed that completely different HIFU ablation patterns were created in primary and secondary liver tumors using the same exposure parameters. The dimensions of a typical HIFU lesion were two times larger in secondary liver tumors than in primary tumors. HIFU treatment parameters should be set properly according to the acoustic properties of the diseased liver tissue.

Thymoquinone Potentiated the Anticancer Effect of Cisplatin on Hepatic Tumorigenesis by Modulating Tissue Oxidative Stress and Endoplasmic GRP78/CHOP Signaling

Thymoquinone (TQ) combined with Cisplatin may augment its anticancer effect on hepatocellular carcinoma (HCC), through oxidative stress mitigation and endoplasmic reticulum (ER) protein modulation. Fifty adult male Wistar albino rats were assigned into five equal experimental groups (n = 10); 1) Control, 2) diethylnitrosamine/carbon tetrachloride-induced liver tumorigenesis model (HCC), 3) Cisplatin (2 mg.kg−1ip) treated rats, 4) Thymoquinone treated group (20 mg.kg−1oral), and 5) group treated with both drugs as in Groups 3 and 4. Treatment regimens started following model confirmation and continued for 4 weeks. In the HCC model, we detected elevated ER chaperone glucose-regulated protein-78 (GRP78) and reduced C/EBP-homologous protein (CHOP)-mediated apoptosis that was accompanied by the elevated alpha-fetoprotein (AFP) marker and deteriorated liver functions. Our original results indicated that Thymoquinone potentiated the pro-apoptotic effect of cisplatin by modulating GRP78/CHOP signaling. Cisplatin/TQ reduced the elevated GRP78 and induced CHOP-mediated apoptosis in the diseased liver tissues compared to the HCC and Cisplatin treated groups. Cisplatin/TQ combination normalized AFP levels and improved liver functions compared to both HCC and cisplatin groups alone. In conclusion, Thymoquinone enhanced the efficacy of Cisplatin in HCC treatment by modulating the GRP78/CHOP/caspase-3 pathway. Thymoquinone is recommended to achieve greater therapeutic benefits and reduce the cisplatin hepatotoxicity in HCC management.

Interleukin-37 Inhibits Colon Carcinogensis During Chronic Colitis.

Inflammatory bowel disease increases the risk of developing colon cancer. Interleukin (IL-) 37 is a fundamental inhibitor of innate immunity by reducing systemic and local inflammation. IL-37 protein is expressed in healthy and diseased bowel and liver tissue. Here, we tested whether transgenic expression of human IL-37 protects IL-10 deficient (IL-10KO) mice from chronic colitis. IL-37tg mice were crossbred with IL-10KO mice. Homozygous IL-10KO/IL-37tg and IL10KO drank 2% dextran sulfate sodium (DSS) in water for 5 days to induce mild colitis. Colon carcinogenesis was triggered by intragastric administration of celecoxib. Endpoints were clinical parameters of colitis, cytokine responses in LPS-stimulated whole blood and explanted colon specimen and qPCR analysis of colon biopsies. Colon inflammation and number of adenoma—carcinoma were analyzed by histology. During the DSS-induction phase IL-10KO and IL-10KO/IL-37tg mice had a similar weight loss due to mild acute colitis. From day 115 there was a significantly improved weight gain in IL-10KO/IL37-tg mice, though colon length was similar. After ex vivo LPS stimulation whole blood of IL-10KO/IL-37tg compared to IL-10KO mice released less IL-6, IL-17, IFNγ, and TNFα and ex vivo colon cultures showed reduced IL-6 production both indicative of reduced inflammatory conditions under the influence of IL-37. Six out of 10 IL-10KO mice developed colon adenoma and carcinoma. Only one adenoma but no carcinoma was detected in colons of IL-10KO/IL-37tg mice. In conclusion, IL-37 transgene expression protects IL-10KO mice from colon carcinogenesis. It remains unclear whether IL-37 has direct tumor suppressing properties.

Blended electrospinning with human liver extracellular matrix for engineering new hepatic microenvironments

Tissue engineering of a transplantable liver could provide an alternative to donor livers for transplant, solving the problem of escalating donor shortages. One of the challenges for tissue engineers is the extracellular matrix (ECM); a finely controlled in vivo niche which supports hepatocytes. Polymers and decellularized tissue scaffolds each provide some of the necessary biological cues for hepatocytes, however, neither alone has proved sufficient. Enhancing microenvironments using bioactive molecules allows researchers to create more appropriate niches for hepatocytes. We combined decellularized human liver tissue with electrospun polymers to produce a niche for hepatocytes and compared the human liver ECM to its individual components; Collagen I, Laminin-521 and Fibronectin. The resulting scaffolds were validated using THLE-3 hepatocytes. Immunohistochemistry confirmed retention of proteins in the scaffolds. Mechanical testing demonstrated significant increases in the Young’s Modulus of the decellularized ECM scaffold; providing significantly stiffer environments for hepatocytes. Each scaffold maintained hepatocyte growth, albumin production and influenced expression of key hepatic genes, with the decellularized ECM scaffolds exerting an influence which is not recapitulated by individual ECM components. Blended protein:polymer scaffolds provide a viable, translatable niche for hepatocytes and offers a solution to current obstacles in disease modelling and liver tissue engineering.

Hepatocellular Carcinoma Risk Stratification by Genetic Profiling in Patients with Cirrhosis.

Prediction of future hepatocellular carcinoma (HCC) risk in the sizable chronic liver disease population is an urgent unmet need to enable regular HCC screening for early detection. Germline deoxyribonucleic acid polymorphisms likely represent etiology-specific host factors that determine HCC susceptibility, including single nucleotide polymorphisms in EGF, IFNL3, MICA, and TLL1 in hepatitis C with or without active viral infection, and PNPLA3, TM6SF2, and MBOAT7 in metabolic liver diseases. Transcriptome-based prognostic liver signature in diseased liver tissue has been associated with long-term HCC risk in viral and metabolic etiologies. Transcriptomic signatures of hepatic injury and specific cell type such as aggregated lymphocytes also predict HCC development. Circulating factors such as proteins and their chemical modification, nucleotides, and metabolites may serve for less-invasive assessment of short- or long-term HCC risk. These biomarkers will enable individual HCC risk-based personalized clinical management for cost-effective early HCC detection and improvement of patient survival.

Genetic determinants of steatosis and fibrosis progression in paediatric non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents today. In comparison with adult disease, paediatric NAFLD may show a periportal localization, which is associated with advanced fibrosis. This study aimed to assess the role of genetic risk variants for histological disease pattern and severity in childhood NAFLD. We studied 14 single nucleotide polymorphisms (SNP) in a cohort of 70 adolescents with biopsy-proven NAFLD. Genotype was compared to an adult control cohort (n=200) and analysed in relation to histological disease severity and liver tissue proteomics. Three of the 14 SNPs were significantly associated with paediatric NAFLD after FDR adjustment, rs738409 (PNPLA3, P=2.80×10-06 ), rs1044498 (ENPP1, P=0.0091) and rs780094 (GCKR, P=0.0281). The severity of steatosis was critically associated with rs738409 (OR=3.25; 95% CI: 1.72-6.52, FDR-adjusted P=0.0070). The strongest variants associated with severity of fibrosis were rs1260326, rs780094 (both GCKR) and rs659366 (UCP2). PNPLA3 was associated with a portal pattern of steatosis, inflammation and fibrosis. Proteome profiling revealed decreasing levels of GCKR protein with increasing carriage of the rs1260326/rs780094 minor alleles and downregulation of the retinol pathway in rs738409 G/G carriers. Computational metabolic modelling highlighted functional relevance of PNPLA3, GCKR and UCP2 for NAFLD development. This study provides evidence for the role of PNPLA3 as a determinant of portal NAFLD localization and severity of portal fibrosis in children and adolescents, the risk variant being associated with an impaired hepatic retinol metabolism.

TLR7-let-7 Signaling Contributes to Ethanol-Induced Hepatic Inflammatory Response in Mice and in Alcoholic Hepatitis.

BackgroundToll‐like receptor 7 (TLR7) is an endosomal TLR that is activated by single‐stranded RNA, including endogenous microRNAs (e.g., let‐7b). Increased hepatic expression of TLRs, microRNAs, and inflammatory mediators is linked to ethanol (EtOH) exposure and to alcoholic liver disease (ALD). ALD invovles chronic hepatic inflammation that can progress to alcoholic hepatitis (AH), a particularly severe form of ALD. This study aimed to investigate TLR7 expression in patients with different liver disease phenotypes and in mouse liver following alcohol exposure.MethodsHepatic mRNA expression was determined by RNA sequencing of liver tissue from patients with liver disease or normal liver tissue. Mice were exposed to subchronic EtOH followed by administration of the TLR7 agonist imiquimod. Primary human hepatocytes were exposed to EtOH or imiquimod in vitro.Results RNAseq analysis revealed that hepatic expression of TLR7 and let‐7b microRNA, an endogenous TLR7 ligand, was significantly increased in AH patients. Hepatic expression of TLR7 and let‐7b positively correlated with hepatic IL‐8 mRNA expression. In mice, EtOH increased hepatic TLR7 mRNA expression and enhanced imiquimod‐induced expression of the pro‐inflammatory mediators TNF α, MCP‐1, and iNOS. In vitro, EtOH significantly increased hepatocyte TLR7 mRNA and the TLR7 agonist, imiquimod, induced hepatocyte expression of TNF α and IL‐8 mRNA. EtOH also increased the release of let‐7b in microvesicles from hepatocytes, suggesting that EtOH can increase the expression of both the receptor and its endogenous ligand.ConclusionsThese studies suggest that increased TLR7 signaling caused by increased expression of TLR7 and its endogenous ligand let‐7b may contribute to the enhanced inflammatory response associated with AH.

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.