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Chronic liver disease can lead to liver fibrosis and ultimately cirrhosis, which is a significant health burden and a major cause of death worldwide. Reliable in vitro models are lacking and thus mono-cultures of cell lines are still used to study liver disease and evaluate candidate anti-fibrotic drugs. We established functional multicellular liver spheroid (MCLS) cultures using primary mouse hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and Kupffer cells. Cell-aggregation and spheroid formation was enhanced with 96-well U-bottom plates generating over ±700 spheroids from one mouse. Extensive characterization showed that MCLS cultures contain functional hepatocytes, quiescent stellate cells, fenestrated sinusoidal endothelium and responsive Kupffer cells that can be maintained for 17 days. MCLS cultures display a fibrotic response upon chronic exposure to acetaminophen, and present steatosis and fibrosis when challenged with free fatty acid and lipopolysaccharides, reminiscent of non-alcoholic fatty liver disease (NAFLD) stages. Treatment of MCLS cultures with potential anti-NAFLD drugs such as Elafibranor, Lanifibranor, Pioglitazone and Obeticholic acid shows that all can inhibit steatosis, but only Elafibranor and especially Lanifibranor inhibit fibrosis. Therefore, primary mouse MCLS cultures can be used to model acute and chronic liver disease and are suitable for the assessment of anti-NAFLD drugs.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFβ-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects. ispartof: BIOMATERIALS vol:276 ispartof: location:Netherlands status: published

Liver fibrosis is the result of persistent liver injury, and is characterized by sustained scar formation and disruption of the normal liver architecture. The extent of fibrosis is considered as an important prognostic factor for the patient outcome, as an absence of (early) treatment can lead to the development of liver cirrhosis and hepatocellular carcinoma. Till date, the most sensitive and specific way for the diagnosis and staging of liver fibrosis remains liver biopsy, an invasive diagnostic tool, which is associated with high costs and discomfort for the patient. Over time, non-invasive scoring systems have been developed, of which the measurements of serum markers and liver stiffness are validated for use in the clinic. These tools lack however the sensitivity and specificity to detect small changes in the progression or regression of both early and late stages of fibrosis. Novel non-invasive diagnostic markers with the potential to overcome these limitations have been developed, but often lack validation in large patient cohorts. In this review, we will summarize novel trends in non-invasive markers of liver fibrosis development and will discuss their (dis-)advantages for use in the clinic.

Animal testing is still the most popular preclinical assessment model for liver fibrosis. To develop efficient anti-fibrotic therapies, robust and representative in vitro models are urgently needed. The most widely used in vitro fibrosis model is the culture-induced activation of primary rodent hepatic stellate cells. While these cultures have contributed greatly to the current understanding of hepatic stellate cell activation, they seem to be inadequate to cover the complexity of this regenerative response. This review summarizes recent progress towards the development of 3D culture models of liver fibrosis. Thus far, only a few hepatic culture systems have successfully implemented hepatic stellate cells (or other non-parenchymal cells) into hepatocyte cultures. Recent advances in bioprinting, spheroid- and precision-cut liver slice cultures and the use of microfluidic bioreactors will surely lead to valid 3D in vitro models of liver fibrosis in the near future.

Background Platelet Derived Growth Factor Receptor beta (PDGFRβ) has been associated to hepatic stellate cell activation and has been the target of multiple therapeutic studies. However, little is known concerning its use as a diagnostic agent. Methods Circulating PDGFRβ levels were analysed in a cohort of patients with liver fibrosis/cirrhosis due to chronic alcohol abuse, viral hepatitis, or non-alcoholic fatty liver disease (NAFLD). The diagnostic performance of PDGFRβ as individual blood parameter, or in combination with other metabolic factors was evaluated. Findings sPDGFRβ levels are progressively increased with increasing fibrosis stage and the largest difference was observed in patients with significant fibrosis, compared to no or mild fibrosis. The accuracy of sPDGFRβ-levels predicting fibrosis could be increased by combining it with albumin levels and platelet counts into a novel diagnostic algorithm, the PRTA-score, generating a predictive value superior to Fib-4, APRI, and AST/ALT. The sPDGFRβ levels and the PRTA-score are independent of liver disease aetiology, thus overcoming one of the major weaknesses of current non-invasive clinical and experimental scores. Finally, we confirmed the diagnostic value of sPDGFRβ levels and the PRTA-score in an independent patient cohort with NAFLD which was staged for fibrosis by liver biopsy. Interpretation The PRTA-score is an accurate tool for detecting significant liver fibrosis in a broad range of liver disease aetiologies. Fund Vrije Universiteit Brussel, the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Flanders) (HILIM-3D; SBO140045), and the Fund of Scientific Research Flanders (FWO).

Background & Aims Hepatic stellate cell activation is a wound-healing response to liver injury. However, continued activation of stellate cells during chronic liver damage causes excessive matrix deposition and the formation of pathological scar tissue leading to fibrosis and ultimately cirrhosis. The importance of sustained stellate cell activation for this pathological process is well recognized, and several signalling pathways that can promote stellate cell activation have been identified, such as the TGFβ-, PDGF-, and LPS-dependent pathways. However, the mechanisms that trigger and drive the early steps in activation are not well understood. Methods and Results We identified the Hippo pathway and its effector YAP as a key pathway that controls stellate cell activation. YAP is a transcriptional co-activator and we found that it drives the earliest changes in gene expression during stellate cell activation. Activation of stellate cells in vivo by CCl 4 administration to mice or activation in vitro caused rapid activation of YAP as revealed by its nuclear translocation and by the induction of YAP target genes. YAP was also activated in stellate cells of human fibrotic livers as evidenced by its nuclear localization. Importantly, knockdown of YAP expression or pharmacological inhibition of YAP prevented hepatic stellate cell activation in vitro and pharmacological inhibition of YAP impeded fibrogenesis in mice. Conclusions YAP activation is a critical driver of hepatic stellate cell activation and inhibition of YAP presents a novel approach for the treatment of liver fibrosis.

Chronic liver disease is the result of long term exposure to viruses or toxins such as alcohol, fat and drugs, and forms the basis for the development of liver fibrosis and primary liver cancer. In vitro and in vivo models are key to study the pathways involved in chronic liver disease and for the development of therapeutics. 3D co-culture systems are becoming the in vitro standard, which requires freshly isolated primary hepatic cells. We developed a novel isolation method to simultaneously isolate liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs). The method exploits the scavenging activity of LSECs, the phagocytic capacity of KCs and the retinoid content of HSCs in vivo to enable direct processing by fluorescence-activated cell sorting without additional antibody binding and washing steps. UFACS3, for UV-FACS-based isolation of 3 non-parenchymal liver cell types, yields functional and pure LSECs (98 ± 1%), KCs (98 ± 1%) and HSCs (97 ± 3%), with less hands-on time from healthy and diseased rodent livers. This novel approach allows a fast and effective combined isolation of sinusoidal cells for further analysis.

Background & Aims: HEV genotype (gt) 3 infections are prevalent in high-income countries and display a wide spectrum of clinical presentations. Host - but not viral - factors are reported to be associated with worse clinical outcomes.Methods: Demographic, clinical, and biochemical data laboratory-confirmed HEV infections (by PCR and/or a combination of IgM and IgG serology) at the Belgian National Reference Centre between January 2010 and June 2018 were collected using stand-ardised case report forms. Genotyping was based on HEV open reading frame 2 sequences. Serum CXCL10 levels were measured by a magnetic bead-based assay. H&E staining was performed on liver biopsies.Results: A total of 274 HEV-infected individuals were included. Subtype assignment was possible for 179/218 viraemic cases, confirming gt3 as dominant with an almost equal representation of clades abchijklm and efg. An increased hospitalisation rate and higher peak serum levels of alanine aminotransferase, bilirubin, and alkaline phosphatase were found in clade efg-infected in-dividuals in univariate analyses. In multivariable analyses, clade efg infections remained more strongly associated with severe disease presentation than any of the previously identified host risk factors, being associated with a 2.1-fold higher risk of hos-pitalisation (95% CI 1.1-4.4, p = 0.034) and a 68.2% higher peak of bilirubin levels (95% CI 13.3-149.9, p = 0.010), independently of other factors included in the model. In addition, acute clade efg infections were characterised by higher serum CXCL10 levels (p = 0.0005) and a more pronounced liver necro-inflammatory activity (p = 0.022). Conclusions: In symptomatic HEV gt3 infections, clade efg is associated with a more severe disease presentation, higher serum CXCL10 levels, and liver necro-inflammatory activity, irrespective of known host risk factors.Clinical Trial Registration: The protocol was submitted to clinicaltrials.gov (NCT04670419).(c) 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. This study is a retrospective analysis of routinely collected data, within the Belgian National Reference Centre (NRC) programme funded by the National Institute for Health and Disability Insurance (RIZIV-INAMI, Belgium). TV is supported by a senior clinical investigator grant of the Research Foundation Flanders (FWO) (number 18B2821N, Belgium). JS and NH acknowledge funding from the European Research Council (ERC) Horizon 2020 research and innovation program (grant agreement 682540 — TransMID, European Union). The funding sources did not have any role in study design, in the collection, analysis, and interpretation of data, in the manuscript writing, or in the decision to submit the paper for publication. We thank Harry Dalton for helpful discussions on the national data collection and clinical outcome analysis and Christophe Conrad for his help in collecting patients data. The authors also especially thank all physicians from the following centres who participated in the study: Algemeen Ziekenhuis Alma, Eeklo; Algemeen Medisch Laboratorium, Antwerpen; Algemeen Stedelijk Ziekenhuis, Aalst; Algemeen Ziekenhuis Delta, Roeselare; Algemeen Ziekenhuis Delta, Torhout; Algemeen Ziekenhuis Nikolaas, Sint-Niklaas; Algemeen Ziekenhuis Sint-Elisabeth, Zottegem; Algemeen Ziekenhuis, Turnhout; Centre Hospitalier Universitaire Brugmann, Brussels; Centre Hospitalier de l’Ardenne, Libramont; Hôpital de Braine-l’Alleud-Waterloo, Braine-l’Alleud; Hôpital Delta, Brussels; Centre Hospitalier de Mouscron, Mouscron; Centre Hospitalier Régional Haute Senne, Soignies; Centre Hospitalier Epicura, Hornu; Centre Hospitalier Universitaire de Liège, Liège; Centre Hospitalier de la Wallonie Picarde, Tournai; Clinique CHC, Waremme; Grand Hôpital de Charleroi, Charleroi; Gasthuiszusters Antwerpen, Antwerpen; Hôpital Princesse Paola, Marche-en-Famenne; Hôpital Iris Sud, Brussels; Instituut voor Tropische Geneeskunde, Antwerpen; Hôpital de Jolimont, La Louvière; Centre Hospitalier de Jolimont, Lobbes; Centre Hospitalier de Jolimont, Nivelles; Centre Hospitalier de Jolimont, Tubize; Eurofins Labo Van Poucke, Kortrijk; Medisch Labo Medina, Dendermonde; Algemeen Ziekenhuis Maria Middelares, Gent; Ziekenhuis Netwerk Antwerpen Middelheim, Antwerpen; Cliniques Universitaires Saint-Luc, Brussels; Centre Hospitalier Universitaire UCL, Dinant; Centre Hospitalier Universitaire UCL, Namur; Centre Hospitalier Universitaire de MontGodinne, Yvoir; Clinique Saint-Luc, Bouge; Clinique Notre-Dame de Grâce, Gosselies; Sint-Andriesziekenhuis, Tielt; Algemeen Ziekenhuis West, Veurne; Algemeen Ziekenhuis Sint Blasius, Dendermonde; Algemeen Ziekenhuis SintJan, Brugge; Klinik Sankt-Joseph, Sankt-Vith; Algemeen Ziekenhuis SintLucas, Gent; Algemeen Ziekenhuis Sint-Maarten, Mechelen; Centre Hospitalier Universitaire Saint-Pierre, Brussels; Sint-Trudo Ziekenhuis, Sint-Truiden; Centre Hospitalier Universitaire Tivoli, La Louvière; Hôpital Erasme – Cliniques Universitaires de Bruxelles, Brussels; Universitair Ziekenhuis Antwerpen, Antwerpen; Universitair Ziekenhuis Gent, Gent; Cassiman David, Laleman Wim, Van Malenstein Hannah, Verslype Chris and Van der Merwe Schalk from Universitair Ziekenhuis Leuven, Leuven; Virga Jesse Ziekenhuis, Hasselt; Universitair Ziekenhuis Brussel, Brussels; Jan Yperman Ziekenhuis, Ieper.

Despite the enormous advances that have been made in the treatment of the plasma cell malignancy multiple myeloma (MM) this disease remains most often fatal. RAS, a family of GTPase genes, is frequently mutated in MM patients (20-45%). It has been shown that RAS oncoproteins, next to their classical roles in stimulating growth and survival, also have tumor suppressive effects. These effects are partially mediated through the Ras-Association Domain Family (RASSF), a group of 10 proteins and numerous isoforms. These tumor suppressors are often silenced in cancer cells due to methylation of the promotor. So far, however, no data about RASSF proteins is available in MM. Here, we investigated the expression and role of RASSF4 in MM. Using gene expression profiling data of 2 independent patient cohorts, we found that RASSF4 expression is significantly downregulated in MM cells compared to healthy bone marrow plasma cells and that a low expression at time of diagnosis associates with a bad prognosis. In addition, treatment with the histone deacetylase inhibitor quisinostat and/or the DNA methyltransferase inhibitor decitabine significantly increased RASSF4 expression both in vitro and in vivo, thus indicating that epigenetic modifications are involved in this silencing. Next, to elucidate the biological role of RASSF4 in MM we used a conditional lentiviral delivery system. Tree human myeloma cell lines (HMCL), namely AMO1, JJN3 and the IL-6 dependent cell line XG7, were transduced with either the empty pLenti 6.3 vector (control) or pLenti6.3 huRASSF4. Overexpression of RASSF4 in the HMCL revealed that RASSF4 has a strong negative effect on MM cell viability and potently induces apoptosis. This induction of apoptosis was found to be caspase-3 mediated. In addition, RASSF4 was found to inhibit the clonogenic capacity of the HMCL. Mechanistically, immunofluorescence stainingfor RASSF4 showed that RASSF4 is co-localized with the centrosome. In addition, RASSF4 overexpression was found to downregulate the expression of the anti-apoptotic protein Bcl-2. In conclusion, we have identified RASSF4 as a target for epigenetic silencing in MM and demonstrated its tumor suppressive function in MM cells. Further insights into the molecular mechanisms of RASSF4 in MM will continue to be explored both in vitro and in vivo.

Summary The epidemiology of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is continuously evolving. Updated data on dual HBV and HCV infection are still needed. Aims To assess the main characteristics of patients with HBV and HCV dual infection, to compare these with those of patients infected with either HBV or HCV and, among patients with dual infection, to assess fibrosis according to HCV replication. Methods Data of 23 patients with dual infection were compared to data from 92 age and sex-matched HBV or HCV monoinfected patients. Results Patients with dual infection were more often immigrants from Africa or Asia than HCV or HBV patients (52% vs. 20% and 22%, respectively, P = 0.01). Intravenous drug use was the route of transmission in 22% of patients with dual infection, which was less frequent than in HCV patients (41%) but more frequent than in HBV patients (0%). Extensive fibrosis or cirrhosis was as frequent among dual-infected patients as among those with HCV or chronic hepatitis B infection (19% vs. 29% vs. 14%, respectively, P = 0.4), even when fibrosis stage was reported considering the duration of infection. In dual-infected patients, the prevalence of extensive fibrosis or cirrhosis was similar in patients with and without detectable HCV RNA (18% vs. 20%). Conclusions Patients with HBV and HCV dual infection were more often immigrants from Africa or Asia and had similar fibrosis stages than HCV or HBV monoinfected patients. In patients with dual infection, extensive fibrosis or cirrhosis was not associated with HCV replication.

Alcoholic liver disease (ALD) is characterized by the development of steatosis, inflammation, hepatocyte necrosis, and apoptosis, with eventual development of fibrosis and cirrhosis [1–3]. Despite being one of the major causes of morbidity and mortality in the world, there are actually no current effective drug treatments for severe ALD [1–3]. Accumulating evidence suggests that ethanol metabolite acetaldehyde and its by-products (such as: 4hydroxy-2-nonenal (4-HNE), and malondialdehyde (MDA)) are highly toxic and even carcinogenic, contributing to the pathogenesis of various alcohol-associated disorders including ALD [1–3]. Over the last twenty years, researchers have been extensively exploring drugs that can activate aldehyde dehydrogenase (ALDH) enzyme and subsequently accelerate acetaldehyde clearance [4], and have demonstrated that these ALDH activators (e.g. ALDH activator-1: Alda-1) can protect against ethanol-induced cardiac injury, ischemic brain injury, radiation dermatitis etc. in animal models [4]. In this issue of the Journal of Hepatology, Zhong et al. [5] nicely demonstrated that treatment with Alda-1 reversed alcoholic steatosis and apoptosis by accelerating aldehyde clearance in an animal model of ALD. During lifespan, every organism receives toxic challenge from numerous harmful agents. Some are produced endogenously, while others accumulate after ingestion of food or exposure to environmental pollutants. Organisms have evolved effective protective mechanisms to prevent their propagation, such as: detoxification through enzymatic reactions. Among phase-I oxidizing enzymes, ALDH is a gene superfamily responsible for the detoxification of biogenic and xenogenic aldehydes [6], with the most abundant members, being ALDH2, which is involved in the metabolism of ethanol-derived acetaldehyde and other aldehydes [6], and ALDH1, which has been found to be highly expressed in many adult tissue stem cells or progenitor cells, including hematopoietic, neuron, muscle, hepatic, adipose stem cells, and

siRNA therapeutics are currently regarded as promising candidates to make a leap forward in the search for treatments of various hard to cure diseases. In order to exploit the full potential of siRNA based therapeutics, development of delivery systems that can efficiently guide the siRNA molecules to their target without major side effects will be the key to success. Lipid based delivery systems, originating from earlier research in the fields of gene delivery, are the most studied candidates for siRNA delivery. Here we discuss the requirements that need to be met by these siRNA delivery systems to ensure adequate stability after systemic application and subsequent deposition in the target tissue. The encountered hurdles in the blood stream and the solutions proposed in literature are discussed.

The DNA-binding transcription factor Smad-interacting protein-1 (Sip1) (also named Zfhx1b/ZEB2) plays essential roles in vertebrate embryogenesis. In Xenopus, XSip1 is essential at the gastrula stage for neural tissue formation, but the precise molecular mechanisms that underlie this process have not been fully identified yet. Here we show that XSip1 functions as a transcriptional repressor during neural induction. We observed that constitutive activation of BMP signaling prevents neural induction by XSip1 but not the inhibition of several epidermal genes. We provide evidence that XSip1 binds directly to the BMP4 proximal promoter and modulates its activity. Finally, by deletion and mutational analysis, we show that XSip1 possesses multiple repression domains and that CtBPs contribute to its repression activity. Consistent with this, interference with XCtBP function reduced XSip1 neuralizing activity. These results suggest that Sip1 acts in neural tissue formation through direct repression of BMP4 but that BMP-independent mechanisms are involved as well. Our data also provide the first demonstration of the importance of CtBP binding in Sip1 transcriptional activity in vivo.

BACKGROUND & AIMS: Severe alcoholic hepatitis (AH) is a life-threatening disease for which adequate oral nutritional support is recommended. We performed a randomized controlled trial to determine whether the combination of corticosteroid and intensive enteral nutrition therapy is more effective than corticosteroid therapy alone in patients with severe AH. METHODS: We enrolled 136 heavy consumers of alcohol (age, 18-75 y) with recent onset of jaundice and biopsy-proven severe AH in our study, performed at 18 hospitals in Belgium and 2 in France, from February 2010 through February 2013. Subjects were assigned randomly (1: 1) to groups that received either intensive enteral nutrition plus methylprednisolone or conventional nutrition plus methylprednisolone (controls). In the intensive enteral nutrition group, enteral nutrition was given via feeding tube for 14 days. The primary end point was patient survival for 6 months. RESULTS: In an intention-to-treat analysis, we found no significant difference between groups in 6-month cumulative mortality: 44.4% of patients died in the intensive enteral nutrition group (95% confidence interval [CI], 32.2%-55.9%) and 52.1% of controls died (95% CI, 39.4%-63.4%) (P = .406). The enteral feeding tube was withdrawn prematurely from 48.5% of patients, and serious adverse events considered to be related to enteral nutrition occurred in 5 patients. Regardless of group, a greater proportion of patients with a daily calorie intake less than 21.5 kcal/kg/day died (65.8%; 95% CI, 48.8-78.4) than patients with a higher intake of calories (33.1%; 95% CI, 23.1%-43.4%) (P < .001). CONCLUSIONS: In a randomized trial of patients with severe AH treated with corticosteroids, we found that intensive enteral nutrition was difficult to implement and did not increase survival. However, low daily energy intake was associated with greater mortality, so adequate nutritional intake should be a main goal for treatment. ClinicalTrials.gov number: NCT01801332.