Primary human hepatocyte spheroid generation and performance in different culture systems

Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes

Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with fatty liver, insulin resistance, and impaired suppression of hepatic glucose output. Endoplasmic reticulum stress-associated liver-specific transcription factor CREBH is emerging as a critical player in various hepatic metabolic pathways and regulates hepatic gluconeogenesis in diet-induced obese settings. In this study, we elucidated the critical role of CREBH in mediating CB1R signaling to regulate glucose homeostasis in primary rat and human hepatocytes. mRNA and protein levels and glucose production were analyzed in primary rat and human hepatocytes. ChIP assays were performed together with various transcriptional analyses using standard techniques. CB1R activation by 2-arachidonoylglycerol (2-AG) specifically induced CREBH gene expression via phosphorylation of the JNK signaling pathway and c-Jun binding to the AP-1 binding site in the CREBH gene promoter. 2-AG treatment significantly induced hepatic gluconeogenic gene expression and glucose production in primary hepatocytes, and we demonstrated that the CREBH binding site mutant significantly attenuated 2-AG-mediated activation of the gluconeogenic gene promoter. Endogenous knockdown of CREBH led to ablation of 2-AG-induced gluconeogenic gene expression and glucose production, and the CB1R antagonist AM251 or insulin exhibited repression of CREBH gene induction and subsequently inhibited gluconeogenesis in both rat and human primary hepatocytes. These results demonstrate a novel mechanism of action of activated CB1R signaling to induce hepatic gluconeogenesis via direct activation of CREBH, thereby contributing to a better understanding of the endocannabinoid signaling mechanism involved in regulating the hepatic glucose metabolism.

Hepatitis B virus X protein modulates cytosolic Ca2+ signaling in primary human hepatocytes

Nanofibrous PLGA electrospun scaffolds modified with type I collagen influence hepatocyte function and support viability in vitro

587 Micronuclei and response to in vitro bleomycin challenge in lymphocytes of workers exposed to low-levels of acrylonitrile

Aims Ribavirin is a nucleoside analogue and remains a necessary component of both interferon-based and directly acting anti-viral regimens for the treatment of hepatitis C virus infection. The achievable concentration of ribavirin within hepatocytes is likely to be an important determinant of therapeutic outcome. Invitro expression levels of equilibrative nucleoside transporter 1 (ENT1) has been shown to be a predictor of treatment response in patients receiving nucleoside-based chemotherapeutic agents. We therefore investigated whether a similar relationship existed between ENT1 expression and ribavirin uptake in freshly isolated primary hepatocytes. Methods Primary hepatocytes were cultured on collagen-coated plates and exposed to ribavirin. Parallel samples were taken for high-performance liquid chromatography to assess ribavirin uptake and for quantitative polymerase chain reaction to evaluate ENT1 expression. Similar assays were performed on the human hepatoma cell line (Huh7). ENT1 gene sequence was analysed by cloning of polymerase chain reaction amplified complementary DNA followed by direct sequencing. Results There was a strong direct correlation between expression of ENT1 in primary hepatocytes and ribavirin uptake at 24 hr. Huh7 cells expressed ENT1 at similar levels to the majority of primary hepatocytes, but did not take up ribavirin. Sequencing revealed that ENT1 in Huh7 cells is wild type. Conclusions In this study, we clearly demonstrate that ribavirin uptake in primary human hepatocytes is variable and correlates with ENT1 expression. This variation in ENT1 expression may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy.

Thromb Haemost 2008; 99: 799–800 Developing effective cell-based therapies to treat severe haemophilia B remains attractive for several reasons. Ondemand and prophylactic factor IX replacement using plasma-derived or recombinant factor IX are both safe and effective (1). However, the cost of either replacement product for lifelong therapy is a significant barrier for patients with limited access to health insurance to cover the ongoing cost of treatment. Further, long-term, cell-therapy-based factor IX replacement could probably materialize if a reliable delivery at ~ 10% of the normal factor IX plasma levels in treated patients could be assured, as consistent delivery in vivo of factor IX at this level would essentially prevent spontaneous bleeding in the individuals thus treated (2). Prior studies using viral vectors have shown long-term therapeutic efficacy in experimental animals (2–6). Thus far, however, viral vectors-based approaches have been essentially ineffective in patients due to both the very low levels and short-term delivery of factor IX in the patients thus treated (7, 8). Concerns associated with the short-term delivery of factor IX via viral vectors include antibody-mediated destruction of the delivery system and/or inactivation of factor IX (8, 9). Another concern associated with the use of viral vectors is the transient elevation of liver transaminases. Generation of humoral and cellular immune responses to both the delivery system and the factor IX delivered in vivo are potential concerns, regardless of the delivery system employed as an alternative to factor IX concentrate infusion. A proven effective and long-term cure of severe factor VIII or factor IX deficiency is successful liver transplantation (10–12). Since factor IX is synthesized by hepatocytes, effective transplantation and long-term survival of isolated hepatocytes have the potential to reduce or even eliminate the need for whole liver transplantation or periodic infusions of plasma-derived or recombinant factor IX into severe haemophilia B patients. Tatsumi et al. have demonstrated the therapeutic potential of hepatocyte transplantation in a patient with congenital factor VII deficiency and the therapeutic effectiveness of human hepatocytes transplanted under the kidney capsules, in delivering human factor VIII into the plasmas of severe haemophilia A mice (13, 14). The present study by Tatsumi et al. (15) in this issue of Thrombosis and Haemostasis is an extension of the authors’ previous work that has the long-term goal of ensuring both the reliability and robustness of hepatocytes engineered to deliver therapeutic levels of proteins ably synthesized by isolated primary hepatocytes. This study employed canine and human primary hepatocytes that were propagated into the well-described immunodeficient mouse model over-expressing urokinase (uPA) that confers an acquired selective growth disadvantage on the endogenous mouse hepatocytes of the uPA-SCID mice. The study demonstrates an effective propagation and engraftment of both canine and human hepatocytes in uPA/SCID mice. Importantly, the engrafted hepatocytes maintained their capacity to synthesize albumin and factor IX (canine or human as appropriate) at acceptable levels. One obvious advantage of primary hepatocyte-mediated factor IX delivery is that the factor IX delivered would likely have all the functional attributes of factor IX arising from all the post-translational modifications factor IX and other vitamin K-dependent clotting factors normally undergo. The limited volume of plasma available did not allow the authors to assess whether the transplanted hepatocytes also synthesized prothrombin, factor VII, factor X, protein C or protein S. The authors recognize several important drawbacks that must be tackled before hepatocyte transplantation could become an acceptable routine clinical procedure. Acceptable methods for conferring selective growth and engraftment advantages on the transplanted hepatocytes relative to the endogenous livers of the transplant recipients remain to be established. Immunosuppressive or immune modulation regimens will probably be mandatory to assure the long-term survival of engrafted hepatocytes, as well as the survival of the factor IX delivered. Use of SCID mice in this study clearly highlights this concern. In addition, acceptable methods for large scale culture of primary human hepatocytes in vitro or in vivo and the harvesting of pure human hepatocytes for transplantation must also be found. Possible engraftment of hepatocytes at sites other than the liver may have undesired clinical © 2008 Schattauer GmbH, Stuttgart

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival

Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis “RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease” (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208.

Herbal extracts of Chelidonium majus have been widely used in European countries and in Chinese herbal medicines for different applications, based on their anti‐viral, anti‐inflammatory and anti‐microbial properties and their use against various liver disorders including cancer. Recently, several cases of liver disease have been related to C. majus intake. Therefore, we have used primary hepatocyte cultures of human, rat, canine and monkey to elucidate the potential hepatotoxicity of C. majus.Water‐soluble extracts were prepared and incubated with primary hepatocytes. Cellular viability and functions were analyzed by means of lactate dehydrogenase (LDH) and urea release and via the MTT assay. The amounts of different alkaloids present in C. majus extracts were determined by HPLC analysis.The liquid extract showed a significant concentration‐dependent toxicity in human hepatocytes and at concentrations of 7.5 mg/ml for animal hepatocytes. In addition, C. majus lead to a decrease in urea release. The decline in urea production varied between the different species with regard to cellular sensitivity and to its maximal extent.The data suggest, that human hepatocytes seemed to be more sensitive to C. majus extracts than canine, rat and monkey hepatocytes, with rat hepatocytes being the most insensitive cells. In addition, these results demonstrate the necessity to use human hepatocytes in order to evaluate possible toxicological effects of complex plant extracts in humans.

THE OLIVACINE DERIVATIVE S 16020 (9-HYDROXY-5,6-DIMETHYL-N-[2-(DIMETHYLAMINO)ETHYL)-6H-PYRIDO(4,3-B)-CARBAZOLE-1-CARBOXAMIDE) INDUCES CYP1A AND ITS OWN METABOLISM IN HUMAN HEPATOCYTES IN PRIMARY CULTURE

Pre-incubation with OATP1B1 and OATP1B3 inhibitors potentiates inhibitory effects in physiologically relevant sandwich-cultured primary human hepatocytes

BackgroundMethylmalonic acidemia (MMA), a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT). Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition.MethodsTo assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction.ResultsPrimary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes.ConclusionThese experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

This paper describes Shell's multi-year safety improvement journey to eradicate serious injuries and fatalities, through embedding human and organization performance, following its refreshed and structurally changed approach to both Safety Leadership and Safety Management Systems. Sharing the end-to-end experience of this ‘Safety Refresh’ from inspiration to program design, change management and implementation, to evaluation of effectiveness, our journey is not complete, but leading and lagging indicators show promising sustained improvement. Safety Refresh is a multidimensional approach with four key elements, keeping Learner Mindset, Psychological Safety, and the Human Performance Principles at the heart of it. These elements are: Transition to the International Association of Oil and Gas Producers (IOGP) Life-Saving Rules and a change of approach when rules are not followed using Fair Event Handling from the Energy Institute's Making Compliance Easier Hearts and Minds tool, to help leaders respond to events with a learner mindset when errors happen. Frontline Work Execution – embedding human performance in how work is planned, briefed end debriefed through an environment of trust, supervisor coaching skills and improved start of work tools (e.g., IOGP Start Work Checks). A shift in risk management approach with our contractor partners. Strengthening our investigate and learn capability, including causally understanding systemic and human performance causes. Each part of the Business and Assets conducted Change Impact Assessments to understand their current position versus what needs to be different after adopting human performance principles. The planned changes were enabled through a global implementation toolkit to integrate within their existing improvement plans. A structured approach to both leadership capability building and process embedment, leads to changes in culture and lagging/leading performance indicators. Part of the impact was a shift in focus and metrics from Total Recordable Case Frequency (TRCF) to Fatalities and Permanent Impairment (FPI) events as a scorecard metric along with differentiating between high potential events that fail safe versus those which only fail lucky. The paper will share data on a significant reduction in FPI over a three-year period and a positive change in high potential incident reporting. Although TRCF increased, this was paired with a reduction in total injuries. This indicates that people are receiving better medical care (rather than a deteriorating safety performance). The journey is not complete with a focus on further embedment of Frontline Work Execution and partnering with our contractors on this human performance journey. The novelty lies in integration and implementation: the incorporation of behvioural science and industry practices into a global programme, integrating "soft" behavioural elements at all levels, with "hard" management system practices. Engaging the Businesses and Assets such that the change is not a bolt-on initiative but embedded in the fabric of local practices and procedures, most importantly enhancing the capability of our people – both staff and contractor.