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Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage. Hepatocytes grown in a dish are immature and do not metabolize compounds as a real liver would. Here, the authors supply stem cell-derived hepatocytes with amino acids at a higher concentration than nutritionally necessary, changing the metabolism of these cells, making them more mature and useful for drug screening and toxicity studies.

Background and study aim Indeterminate biliary strictures and difficult bile duct stones remain clinically arduous and challenging situations. We aimed to evaluate the utility of the single-operator cholangioscopy (SOC)-system SpyGlass in both conditions in a single-center biliopancreatic interventional unit and in perspective of available aggregated literature. Methods Usefulness of SOC was assessed for the above-mentioned indications by means of the combination of successful procedural completion, clinical success and incidence of procedure-related adverse events in our own prospective cohort from 3/2010 to 7/2014 and all available literature till 6/2015. Results Our single-center cohort constituted of 84 patients undergoing SpyGlass either for indeterminate strictures ( n  = 45) or difficult stones ( n  = 39). In addition, a comprehensive literature review yielded 851 patients (from 15 series) for either stenosis ( n  = 646, 75.9 %) and difficult stones ( n  = 205, 24.1 %). In our series, overall procedural success amounted to 85.7 % (with 88.9 % for stenosis or 82.1 % for stones) compared to 90.7, 91.5 and 88.3 % in overall literature, respectively. Sensitivity, specificity and accuracy for visual diagnosis in our cohort added up to 83.3, 82.9 and 82.9 % compared to 90.8, 90.9 and 90.8 % in the pooled analysis. Respective figures for SOC - directed biopsies totaled 85.7, 100 and 95.7 % in our cohort and 72.4, 100 and 84 % overall. Overall procedure-related complications varied between 9.4 and 21.4 %. Conclusions The SOC-platform SpyGlass can be considered useful in the context of indeterminate biliary strictures and difficult-to-remove biliary stones. In both, SpyGlass-assisted intervention is associated with high procedural success and alters clinical outcome compared to conventional approaches with an acceptable safety profile.

Background Acute hepatic porphyria (AHP) is a family of four rare genetic diseases, each involving deficiency in a hepatic heme biosynthetic enzyme. Resultant overproduction of the neurotoxic intermediates δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) leads to disabling acute neurovisceral attacks and progressive neuropathy. We evaluated the AHP disease burden in patients aged ≥ 12 years in a post hoc analysis of the Phase 3, randomized, double-blind, placebo-controlled ENVISION trial of givosiran (NCT03338816), an RNA interference (RNAi) therapeutic that targets the enzyme ALAS1 to decrease ALA and PBG production. We analyzed baseline AHP severity via chronic symptoms between attacks, comorbidities, concomitant medications, hemin-associated complications, and quality of life (QOL) and evaluated givosiran (2.5 mg/kg monthly) in patients with and without prior hemin prophylaxis on number and severity of attacks and pain scores during and between attacks. Results Participants (placebo, n = 46; givosiran, n = 48) included patients with low and high annualized attack rates (AARs; range 0–46). At baseline, patients reported chronic symptoms (52%), including nausea, fatigue, and pain; comorbidities, including neuropathy (38%) and psychiatric disorders (47%); concomitant medications, including chronic opioids (29%); hemin-associated complications (eg, iron overload); and poor QOL (low SF-12 and EuroQol visual analog scale scores). A linear relationship between time since diagnosis and AAR with placebo suggested worsening of disease over time without effective treatment. Givosiran reduced the number and severity of attacks, days with worst pain scores above baseline, and opioid use versus placebo. Conclusions Patients with AHP, regardless of annualized attack rates, have considerable disease burden that may partly be alleviated with givosiran.

Intestinal dysbiosis and elevated lipopolysaccharides (LPS) levels have been implicated in the development of obesity, insulin resistance and non-alcoholic steatohepatitis (NASH). In order to determine if LPS levels are elevated in patients with NASH compared to patients with non-alcoholic fatty liver (NAFL) and, if elevated LPS levels correlated with histological severity of non-alcoholic fatty liver disease (NAFLD) we compared LPS, markers of LPS bioactivity and pro-inflammatory cytokines/chemokines in patients undergoing bariatric surgery. At the time of surgery a liver biopsy was taken allowing the stratification into well-delineated subgroups including: No NAFL/NAFL; NASH; NASH with fibrosis and NASH cirrhotics, using the NAFLD Activity Score (NAS). Anthropometric data and plasma were collected for assessment of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14), intestinal-type fatty acid binding protein (iFABP), Toll-like receptors 2 and 4 (TLR2, 4) and a panel of cytokines/chemokines. Similar analysis was performed on plasma from a cohort of healthy controls. Our data indicate elevated levels of LPS, LBP, sCD14, iFABP and TLR2,4 in obese patients compared to healthy controls, however, these parameters remained unaltered within patients with limited liver disease (NAFL) compared to NASH/NASH with fibrosis subgroups. Hierarchic cluster analysis using endotoxin-related parameters failed to discriminate between lean controls, NAFLD. While similar cluster analysis implementing inflammation-related parameters clearly distinguished lean controls, NALFD subgroups and NASH cirrhotics. In addition, LPS levels was not associated with disease severity while TNFα, IL8, and CCL3 featured a clear correlation with transaminase levels and the histological severity of NALFD. In conclusion our data indicate a stronger correlation for circulating inflammatory- rather than endotoxin-related parameters in progression of NAFLD and highlights the need for additional larger studies in unravelling further mechanistic insights.

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase ( PSPH ), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH. The ribosomal protein RPL10 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). Here, the authors show that it promotes proliferation of T-ALL cells by upregulating the serine biosynthesis enzyme phosphoserine phosphatase which in turn modulates serine and glycine metabolism.

SLC35A2-CDG is an X-linked congenital disorder of glycosylation (CDG), characterized by defective UDP-galactose transport into the Golgi and endoplasmic reticulum and consequent insufficient galactosylation of glycans. Clinically, this translates into a range of predominantly neurological symptoms. Although the pathomechanism of this disorder is not fully understood, oral galactose supplementation has led to clinical and biochemical improvement in some patients. Here, we show that protein glycosylation (N- and O-linked) was only minimally disturbed in SLC35A2-CDG patient-derived fibroblasts. However, lipid glycosylation was significantly impaired, with accumulation of glucosylceramide and deficiency of digalactosylated glycosphingolipids (GSLs) and complex gangliosides. Galactose supplementation increased UDP-galactose, its transport into the Golgi, and improved deficient GSL synthesis through direct incorporation of the provided galactose. This improved GSL homeostasis in all patient-derived fibroblasts and in another SLC35A2 deficient cell model (CHO-Lec8). Additionally, SLC35A2-CDG serum analysis identified hydroxylated GSLs, particularly GM3, as potential disease biomarkers. Given the essential role of gangliosides in central nervous system function, their deficiency is likely a key factor in the neurological involvement of this disorder. These findings pave the way for new nutritional therapies with GSL supplements and highlight the importance of studying lipid glycosylation to better understand the complex pathophysiology of CDG.

De novo malignancy (DNM) is the primary cause of mortality after liver transplantation (LT) for alcohol-related liver disease (ALD). However, data on risk factors for DNM development after LT are limited, specifically in patients with ALD. Therefore, we retrospectively analyzed all patients transplanted for ALD at our center before October 2016. Patients with a post-LT follow-up of <12 months, DNM within 12 months after LT, patients not on tacrolimus in the 1st year post-LT, and unknown smoking habits were excluded. Tacrolimus drug exposure level (TDEL) was calculated by area under the curve of trough levels in the 1st year post-LT. 174 patients received tacrolimus of which 19 (10.9%) patients developed a DNM between 12 and 60 months post-LT. Multivariate cox regression analysis identified TDEL [HR: 1.710 (1.211–2.414); p = 0.002], age [1.158 (1.076–1.246); p < 0.001], number of pack years pre-LT [HR: 1.021 (1.004–1.038); p = 0.014] and active smoking at LT [HR: 3.056 (1.072–8.715); p = 0.037] as independent risk factors for DNM. Tacrolimus dose minimization in the 1st year after LT and smoking cessation before LT might lower DNM risk in patients transplanted for ALD.

Background Off-label use of (orphan) medicinal products for (rare) diseases is quite common but not underpinned by clinical studies to confirm efficacy and safety. No risk-analyses by regulatory agencies are carried out. The objective of this study was to map off-label use of orphan medicinal products in Belgium in terms of attitude towards off-label prescribing, factors influencing off-label prescribing, disclosure of information towards the patient, reporting of off-label use, risks and consequences. Most of the EMA authorized orphan drugs are fully reimbursed in Belgium under well-defined circumstances. Moreover, a “Special Solidarity Fund” takes care of some specific cases eventually prescribed off-label. Methods Semi-structured interviews with seven physicians with expertise in the treatment with and six experts in the reimbursement of orphan medicinal products in Belgium. This task was performed by five last-year pharmacy students after having studied profoundly the medical literature around off-label prescribing. They had no previous contact with the participants. Results Most participants do agree with the off-label use if the medicinal product is quite safe and well-tolerated, if the on-label indication is rather general and when all other options have failed in some specific, evidence-based indications, especially in children. Before starting off-label use, the patient/family needs to be fully and clearly informed. The treatment is not reimbursed but sometimes sponsored by the company or by charity funds. Reporting of the outcome is necessary to avoid losing valuable information. The prescriber is responsible and can be held accountable. Conclusions While there is support from physicians and reimbursement experts, there is also concern in case of off-label use, mainly for reasons of patient safety especially when medicinal products are prescribed off-label in the absence of medical or scientific justification and driven by cost-containment motives.

Background Acute hepatic porphyria (AHP) is a rare, debilitating disease characterized by potentially life-threatening attacks often resulting in chronic symptoms that negatively impact daily functioning and quality of life. Symptoms of AHP prevent many individuals from working and achieving lifetime work averages. The aim of this study was to apply a public economic framework to evaluate AHP in Belgium, taking into consideration a broad range of costs that are relevant to government in relation to social benefit payments and lifetime taxes paid. Methodology A public economic framework was developed exploring lifetime costs for government attributed to an individual with AHP and recurrent attacks in Belgium. Work-activity and lifetime direct taxes paid, indirect consumption taxes and requirements for public benefits were estimated based on established clinical pathways for AHP and compared to the general population (GP). The model includes AHP-related healthcare costs and non-AHP healthcare costs for the GP. Results Lifetime earnings are reduced in an individual with AHP by €347,802 per person (p.p.), translating to reduced lifetime taxes paid of €183,187 for an AHP individual compared to the GP. We estimate increased lifetime disability benefit support of €247,242 for an AHP individual compared to GP. Lifetime healthcare costs for a person with AHP were estimated to be €3,030,316 due to frequent hospitalisations associated with porphyria attacks compared to the GP. The lifetime costs for a person with 12 attacks per annum factoring in transfers, taxes and healthcare costs are estimated to be €3,460,745 p.p. Eliminating AHP attacks after 10 years of active disease, thus, enabling a person to return to work increases lifetime earnings by €224,575 p.p. Increased work activity in such individuals would generate an estimated €118,284 p.p. over their lifetime. The elimination of AHP attacks could also lead to reductions in disability payments of €179,184 p.p. and healthcare cost savings of €1,511,027 p.p. Conclusions Due to severe disability resulting from constant attacks, AHP patients with recurrent attacks incur significant public costs. Lifetime taxes paid are reduced as these attacks occur during peak earning and working years. In those patients, reducing AHP attacks can confer significant fiscal benefits for government, including reduced healthcare costs, reduced disability payments and improved tax revenue.