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Objectives The aim of this meta-analysis was to conduct a contemporary systematic review of high quality non-randomised controlled trials to determine the effect of pre-liver transplantation (LT) transarterial chemoembolisation (TACE) on long-term survival and complications of hepatocellular carcinoma (HCC) patients. Background TACE is used as a neoadjuvant therapy to mitigate waitlist drop-out for patients with HCC awaiting LT. Previous studies have conflicting conclusions on the effect of TACE on long-term survival and complications of HCC patients undergoing LT. Methods CINAHL, Cochrane Controlled Register of Trials, Embase, PubMed, and Web of Science were systematically searched. Baseline characteristics included number of patients outside Milan criteria, tumour diameter, MELD score, and time on the waiting list. Primary outcomes included 3- and 5-year overall and disease-free survival. Secondary outcomes included tumour recurrence, 30-day postoperative mortality, and hepatic artery and biliary complications. Results Twenty-one high-quality NRCTs representing 8242 patients were included. Tumour diameter was significantly larger in TACE patients (3.49 cm vs 3.15 cm, P = 0.02) and time on the waiting list was significantly longer in TACE patients (4.87 months vs 3.46 months, P = 0.05), while MELD score was significantly higher in non-TACE patients (10.81 vs 12.35, P = 0.005). All primary and secondary outcomes displayed non-significant differences. Conclusion Patients treated with TACE had similar survival and postoperative outcomes to non-TACE patients, however, they had worse prognostic features compared to non-TACE patients. These findings strongly support the current US and European clinical practice guidelines that neoadjuvant TACE can be used for patients with longer expected waiting list times (specifically >6 months). Randomised controlled trials would be needed to increase the quality of evidence.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with a high mortality rate. Epithelial-to-mesenchymal transition (EMT) confers cancer cells with immune evasive ability by modulating the expression of immune checkpoints in many cancers. Thus, the aim of our study is to examine the interplay between EMT and immune checkpoint molecules in HCC. A reversible EMT model was utilised with transforming growth factor (TGF)-β1 as an EMT inducer for HCC cell lines Hep3B and PLC/PRF/5. HCC cells were treated with TGF-β1 for 72 h and the EMT status and immune checkpoint expression were examined. In addition, the migratory ability of HCC cells were examined using wound healing and transwell migration assays in the reversible EMT model. siRNA-mediated knockdown of immune checkpoint molecule, B7-H3, was further utilised to validate the association between TGF-β1-mediated EMT and immune checkpoint expression in HCC. In addition, a web-based platform, SurvExpress, was utilised to evaluate the association between expression of TGF-β1 in combination with immune checkpoint molecules and overall survival in HCC patients. We observed induction of EMT upon treatment of HCC cells with TGF-β1 revealed by reduced expression of epithelial markers along with increased expression of mesenchymal markers. Withdrawal of TGF-β1 reversed the process of EMT with elevated expression of epithelial markers and reduced expression of mesenchymal markers. TGF-β1 treatment elevated the migratory potential of HCC cells which was reversed following reversal assay. Notably, during TGF-β1-induced EMT, there was upregulation of immune checkpoint molecules PD-L1 and B7-H3. However, the reversal of EMT decreased the expression of PD-L1 and B7-H3. In addition, TGF-β1 driven EMT was reversed following knockdown of B7-H3 in both HCC cells further validating the interplay between TGF-β1-mediated EMT and immune checkpoint expression in HCC. Furthermore, the coordinate expression of TGF-β1 with PD-L1 (p=0.01487) and B7-H3 (p=0.009687) was correlated with poor overall survival in 422 HCC patients. Our study has demonstrated a close association between TGF-β1-mediated EMT and regulation of immune checkpoints in HCC.

Introduction: The Diagnostic and Statistical Manual of Mental Disorders (5th ed.; DSM–5) brought a change to the symptom clusters of posttraumatic stress disorder (PTSD). In line with the DSM–5 changes, an updated version of the Clinician-Administered PTSD Scale (CAPS–5) was released. The CAPS–5 is considered to be the gold-standard measure of PTSD; however, examinations of the psychometric properties and optimal factor structure of this scale are underrepresented in PTSD studies. Methods: This study used confirmatory factor analysis (CFA) to assess the factor structure of the CAPS–5 using a sample of 267 male Australian Vietnam Veterans. Models drawn from the PTSD CFA literature were used to test the underlying dimensions of PTSD: the four-factor DSM–5 model, six-factor externalizing behaviour and anhedonia models, and seven-factor hybrid model. Results: The results found that the DSM–5 model showed slightly less than adequate fit (comparative fit index [CFI] = 0.90, Tucker–Lewis index [TLI] = 0.88, root mean square error of approximation [RMSEA] = 0.064), however, other models showed acceptable fit. The anhedonia model provided a significantly better fit than the other models (CFI = 0.92, TLI = 0.90, RMSEA = 0.059). Discussion: Overall, the results supported the anhedonia model. This result may indicate that the underlying dimensions of PTSD in Australian Vietnam Veterans may best be represented by six distinct factors.

Post-traumatic stress disorder (PTSD) is associated with extensive physical comorbidities, including lower gastrointestinal symptoms. Diagnostic uncertainty and poor therapeutic responses may result in more frequent colonoscopies than clinically necessary. Polypectomy is standard practice when polyps are identified, and if PTSD is a risk factor for polyp formation, one would expect a higher rate of polyp detection and removal in veterans with PTSD than those without PTSD.To determine the association between PTSD and the rate of colonoscopy and polypectomy in Australian veterans.Diagnostic and therapeutic colonoscopy rates in Australian male Veterans aged ≥50 years were examined by reviewing case records of veterans who accessed Department of Veterans' Affairs funded health services between 1 January 2013 and 31 December 2018.A total of 138 471 veterans was included, of whom 28 018 had a diagnosis of PTSD; 56.4% were aged ≥65 years. Twenty-one percent of the entire cohort underwent at least one colonoscopy during the study period. Increased rates of diagnostic colonoscopy and polypectomy were associated with the presence of PTSD across all age brackets. The effect was empirically large as veterans with PTSD experience colonoscopy rates 76-81% greater than those without PTSD. Similarly, veterans with PTSD experienced polypectomy rates 76-81% greater than veterans without PTSD, and this increase persisted when controlling for the increased number of diagnostic colonoscopies they undergo.The presence of PTSD has a marked impact on colonoscopy rates in Australian veterans. The increased polypectomy rate independent of increased colonoscopy rate suggests that PTSD is a risk factor for colonic polyp formation.

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.

Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer‑related deaths globally. Epithelial‑to‑mesenchymal transition (EMT) is a cellular process that confers HCC tumor cells with the ability to evade the immune system. Immune escape in most tumors, including HCC, is controlled by immune checkpoint molecules. The aim of the present study was to investigate the association between EMT and immune checkpoint in HCC, and identify novel therapeutic targets for HCC. An in vitro model of reversible EMT was utilized based on cytokine tumor necrosis factor (TNF)‑α treatment of HCC cell lines Hep3B and PLC/PRF/5. Hep3B and PLC/PRF/5 cells were treated with TNF‑α, and the EMT status and the expression of immune checkpoint molecules was assessed by reverse transcription‑quantitative PCR, western blotting and immunofluorescence. To confirm an association between EMT and immune modulators, cells were exposed to culture medium with TNF‑α for 3 days to induce EMT, following which a reversal assay was performed. The expression of immune modulators and mesenchymal‑to‑epithelial transition (MET) status was investigated upon reversal of EMT. Furthermore, SurvExpress, a web‑based platform was utilized to analyze survival and recurrence in a dataset of patients with HCC. TNF‑α treatment for 3 days induced EMT in Hep3B and PLC/PRF/5 cells, as demonstrated by the downregulation of epithelial markers along with upregulation in mesenchymal markers. An EMT reversal assay was able to induce MET by increasing epithelial markers and decreasing mesenchymal markers. TNF‑α‑induced EMT led to the upregulation of immune modulators, including programmed death receptor ligand (PD‑L)1, PD‑L2, CD73 and B7‑H3. In contrast, reversal of EMT suppressed the expression of PD‑L1, PD‑L2, CD73 and B7‑H3. In addition, high expression of TNF‑α and PD‑L1 in 422 patients with HCC was associated with poor overall survival. The coordinate expression of TNF‑α with PD‑L2 in this patient cohort was associated with increased HCC recurrence. In conclusion, the present study demonstrated a close association between immune modulator expression and EMT induction/reversal driven by TNF‑α.

In this study we investigated genome-wide sperm DNA methylation patterns in trauma-exposed Vietnam veterans. At the genome-wide level, we identified 3 CpG sites associated with PTSD in sperm including two intergenic and one CpG within the CCDC88C gene. Of those associated with PTSD in sperm at a nominal level, 1868 CpGs were also associated with PTSD in peripheral blood (5.6% overlap) including the RORA, CRHR1 and DOCK2 genes that have been previously implicated in PTSD. A total of 10 CpG sites were significantly associated with a reported history of a diagnosed mental health condition in children and reached genome-wide significance. CpGs associated with a history of a reported mental health condition in children were also enriched (90% of tested genes) for genes previously reported to be resistant to demethylation, making them strong candidates for transgenerational inheritance. In conclusion, our findings identify a unique sperm-specific DNA methylation pattern that is associated with PTSD.

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib.

The past decade has seen many advancements in the development of three-dimensional (3D) in vitro models in pharmaceutical sciences and industry. Specifically, organoids present a self-organising, self-renewing and more physiologically relevant model than conventional two-dimensional (2D) cell cultures. Liver organoids have been developed from a variety of cell sources, including stem cells, cell lines and primary cells. They have potential for modelling patient-specific disease and establishing personalised therapeutic approaches. Additionally, liver organoids have been used to test drug efficacy and toxicity. Herein we summarise cell sources for generating liver organoids, the advantages and limitations of each cell type, as well as the application of the organoids in modelling liver diseases. We focus on the use of liver organoids as tools for drug validation and toxicity assessment.

Introduction: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths both globally and in Australia. Surveillance for HCC in at-risk populations allows diagnosis at an early stage, when potentially curable. However, most Australians diagnosed with HCC die of the cancer or of liver disease. In the changing landscape of HCC management, unique challenges may lead to clinical practice variation. As a result, there is a need to identify best practice management of HCC in an Australian context. This consensus statement has been developed for health professionals involved in the care of adult patients with HCC in Australia. It is applicable to specialists, general medical practitioners, nurses, health coordinators and hospital administrators. Methods and recommendations: This statement has been developed by specialists in hepatology, radiology, surgery, oncology, palliative care, and primary care, including medical practitioners and nurses. The statement addresses four main areas relevant to HCC management: epidemiology and incidence, diagnosis, treatment, and patient management. A modified Delphi process was used to reach consensus on 31 recommendations. Principal recommendations include the adoption of surveillance strategies, use of multidisciplinary meetings, diagnosis, treatment options and patient management. Changes in management as a result of this statement: This consensus statement will simplify HCC patient management and reduce clinical variation. Ultimately, this should result in better outcomes for patients with HCC.

Introduction: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence.Material and methods: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes + sham), 1 mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes + LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI + LPS) and liposome treatment followed by IRI + LPS (liposomes + IRI + LPS). Following 6 h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP).Results: The presence of hepatic macrophages was reduced by 90% in LPS and IRI + LPS groups pre-treated with clodronate liposomes (P Conclusions: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS of LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marcc Marzioni, Nicholas LaRusso and Peter Jansen.

Chronic inflammation is a key factor in symptomology and comorbidities of post-traumatic stress disorder (PTSD). Levels of a proinflammatory marker, C-reactive protein (CRP) are increased in individuals with PTSD but it is not clear if this is due to trauma exposure or PTSD. Our study aimed to assess the relationship between serum CRP levels, CRP SNPs, methylation, mRNA expression and PTSD in a homogenous trauma exposed Australian Vietnam veteran cohort. We hypothesized that decreased DNA methylation would be associated with increased gene expression and increased peripheral CRP levels in PTSD patients and that this would be independent of trauma. Participants were 299 Vietnam veterans who had all been exposed to trauma and approximately half were diagnosed with PTSD. We observed higher levels of serum CRP in the PTSD group compared to the non-PTSD group but after controlling for BMI and triglycerides the association did not remain significant. No association was found between CRP SNPs and PTSD or CRP levels. Absent in Melanoma 2 (AIM2) which is a mediator of inflammatory response and a determinant of CRP levels was analysed for DNA methylation and mRNA expression. We observed a trend level association between PTSD and AIM2 methylation after controlling for age, smoking, triglycerides, BMI and cell types. There was no significant interaction between PTSD and CRP levels on AIM2 methylation after controlling for covariates. We observed that as AIM2 methylation levels decreased, AIM2 mRNA expression increased. Elevated CRP levels were associated with AIM2 mRNA in the trauma exposed cohort but there was no significant interaction effect with PTSD. Our results could not confirm that CRP is a marker of PTSD independent of trauma in this group of older veterans. CRP may be a broad marker of disease risk, or a marker of PTSD in younger cohorts than those in this study.

Mesenchymal stem/stromal cells (MSCs) present a promising tool in cell‐based therapy for treatment of various diseases. Currently, optimization of treatment protocols in clinical studies is complicated by the variations in cell dosing, diverse methods used to deliver MSCs, and the variety of methods used for tracking MSCs in vivo. Most studies use a dose escalation approach, and attempt to correlate efficacy with total cell dose. Optimization could be accelerated through specific understanding of MSC distribution in vivo, long‐term viability, as well as their biological fate. While it is not possible to quantitatively detect MSCs in most targeted organs over long time periods after systemic administration in clinical trials, it is increasingly possible to apply pharmacokinetic modeling to predict their distribution and persistence. This Review outlines current understanding of the in vivo kinetics of exogenously administered MSCs, provides a critical analysis of the methods used for quantitative MSC detection in these studies, and discusses the application of pharmacokinetic modeling to these data. Finally, we provide insights on and perspectives for future development of effective therapeutic strategies using pharmacokinetic modeling to maximize MSC therapy and minimize potential side effects. Stem Cells Translational Medicine 2018;7:78–86

The iron overload disorder hereditary haemochromatosis is most commonly caused by HFE p.Cys282Tyr homozygosity. In the absence of results from any randomised trials, current evidence is insufficient to determine whether individuals with hereditary haemochromatosis and moderately elevated serum ferritin, should undergo iron reduction treatment. This trial aimed to establish whether serum ferritin normalisation in this population improved symptoms and surrogate biomarkers.This study was a multicentre, participant-blinded, randomised controlled trial done at three centres in Australia. We enrolled people who were homozygous for HFE p.Cys282Tyr, aged between 18 and 70 years, with moderately elevated serum ferritin, defined as 300-1000 μg/L, and raised transferrin saturation. Participants were randomly assigned, via a computer-generated random number, to undergo either iron reduction by erythrocytapheresis (treatment group) or sham treatment by plasmapheresis (control group). Randomisation was stratified by baseline serum ferritin (600 μg/L or ≥600 μg/L), sex, and study site. Erythrocytapheresis and plasmapheresis were done every 3 weeks, the number of procedures and volume of red cells or plasma removed determined on the basis of each patient's haemoglobin, haematocrit, and serum ferritin concentration, as well their height and weight. In the erythrocytapheresis group, the target was to reduce serum ferritin to less than 300 μg/L. The number of procedures for the control group was based on the initial serum ferritin and prediction of decrease in serum ferritin of approximately 120 μg/L per treatment. The primary outcome was patient-reported Modified Fatigue Impact Scale (MFIS) score, measured at baseline and before unblinding. Analyses were by intention to treat, including the safety analysis. The trial is registered with ClinicalTrials.gov, number NCT01631708, and has been completed.Between Aug 15, 2012, and June 9, 2016, 104 participants were randomly assigned to the treatment (n=54) and control (n=50) groups, of whom 94 completed the study (50 in the treatment group and 44 in the control group). Improvement in MFIS score was greater in the treatment group than in the control group (mean difference -6·3, 95% CI -11·1 to -1·4, p=0·013). There was a significant difference in the cognitive subcomponent (-3·6, -5·9 to -1·3, p=0·0030), but not in the physical (-1·90 -4·5 to 0·63, p=0·14) and psychosocial (-0·54, -1·2 to 0·11, p=0·10) subcomponents. No serious adverse events occurred in either group. One participant in the control group had a vasovagal event and 17 participants (14 in the treatment group and three in the control group) had transient symptoms assessed as related to hypovolaemia. Mild citrate reactions were more common in the treatment group (32 events [25%] in 129 procedures) compared with the control group (one event [1%] in 93 procedures).To our knowledge, this study is the first to objectively assess the consequences of iron removal in individuals with hereditary haemochromatosis and moderately elevated serum ferritin. Our results suggest that serum ferritin normalisation by iron depletion could be of benefit for all individuals with hereditary haemochromatosis and elevated serum ferritin levels.National Health and Medical Research Council (Australia).

AIM To investigate the synergistic hepato-protective properties of curcumin and vitamin E in an Hfe-/- high calorie diet model of steatohepatitis. METHODS Hfe-/- C57BL/6J mice were fed either a high calorie diet or a high calorie diet with 1 mg/g curcumin; 1.5 mg/g vitamin E; or combination of 1 mg/g curcumin + 1.5 mg/g vitamin E for 20 wk. Serum and liver tissue were collected at the completion of the experiment. Liver histology was graded by a pathologist for steatosis, inflammation and fibrosis. RNA and protein was extracted from liver tissue to examine gene and protein expression associated with fatty acid oxidation, mitochondrial biogenesis and oxidative stress pathways. RESULTS Hfe-/- mice fed the high calorie diet developed steatohepatitis and pericentral fibrosis. Combination treatment with curcumin and vitamin E resulted in a greater reduction of percent steatosis than either vitamin E or curcumin therapy alone. Serum alanine aminotransferase and non-alcoholic fatty liver disease (NAFLD) activity score were decreased following combination therapy with curcumin and vitamin E compared with high calorie diet alone. No changes were observed in inflammatory or fibrosis markers following treatment. Epididymal fat pad weights were significantly reduced following combination therapy, however total body weight and liver weight were unchanged. Combination therapy increased the mRNA expression of AdipoR2, Ppar-α, Cpt1a, Nrf-1 and Tfb2m suggesting enhanced fatty acid oxidation and mitochondrial biogenesis. In addition, combination treatment resulted in increased catalase activity in Hfe-/- mice. CONCLUSION Combination curcumin and vitamin E treatment decreases liver injury in this steatohepatitis model, indicating that combination therapy may be of value in NAFLD.

Acetaminophen (APAP) is the foremost cause of drug-induced liver injury in the Western world. Most studies of APAP hepatotoxicity have focused on the hepatocellular injury, but current hepatocyte-related biomarkers have delayed presentation time and a lack of sensitivity. APAP overdose can induce hepatic microvascular congestion, which importantly precedes the injury of hepatocytes. However, the underlying molecular mechanisms remain unclear. It is imperative to discover and validate sensitive and specific translational biomarkers of APAP-induced liver injury. Methods: In this study, we assessed APAP toxicity in sinusoidal endothelial cells and hepatocytes in mice treated with overdose APAP at different time points. The underlying mechanisms of APAP overdose induced sinusoidal endothelial cell injury were investigated by RT2 Profiler PCR arrays. The impact of APAP overdose on endothelial cell function was assessed by pseudovessel formation of endothelial cells in 2D Matrigel and in vivo hepatic vascular integrity using multiphoton microscopy. Finally, the effects of APAP overdose on oxygen levels in the liver and hepatic microcirculation were evaluated by contrast enhanced ultrasonography. Potential imaging-based vascular-related markers for early detection of APAP induced liver injury were assessed. Results: Our study confirmed that hepatic endothelial cells are an early and direct target for APAP hepatotoxicity. ICAM1-related cellular adhesion pathways played a prominent role in APAP-induced endothelial cell injury, which was further validated in primary human sinusoidal endothelial cells and human livers after APAP overdose. APAP overdose impacted pseudovessel formation of endothelial cells and in vivo hepatic vascular integrity. Use of ultrasound to detect APAP-induced liver injury demonstrated that mean transit time, an imaging-based vascular-related biomarker, was more sensitive and precise for early detection of APAP hepatotoxicity and monitoring the treatment response in comparison with a conventional blood-based biomarker. Conclusion: Imaging-based vascular-related biomarkers can identify early and mild liver injury induced by APAP overdose. With further development, such biomarkers may improve the assessment of liver injury and the efficacy of clinical decision-making, which can be extended to other microvascular dysfunction of deep organs.

Background & aims Iron has been proposed as influencing the progression of liver disease in subjects with non-alcoholic fatty liver disease (NAFLD). We have previously shown that, in the Hfe−/− mouse model of hemochromatosis, feeding of a high-calorie diet (HCD) leads to increased liver injury. In this study we investigated whether the feeding of an iron deficient/HCD to Hfe−/− mice influenced the development of NAFLD. Methods Liver histology was assessed in Hfe−/− mice fed a standard iron-containing or iron-deficient diet plus or minus a HCD. Hepatic iron concentration, serum transferrin saturation and free fatty acid were measured. Expression of genes implicated in iron regulation and fatty liver disease was determined by quantitative real-time PCR (qRT-PCR). Results Standard iron/HCD-fed mice developed severe steatosis whereas NAS score was reduced in mice fed iron-deficient HCD. Mice fed iron-deficient HCD had lower liver weights, lower transferrin saturation and decreased ferroportin and hepcidin gene expression than HCD-fed mice. Serum non-esterified fatty acids were increased in iron-deficient HCD-fed mice compared with standard iron HCD. Expression analysis indicated that genes involved in fatty-acid binding and mTOR pathways were regulated by iron depletion. Conclusions Our results indicate that decreasing iron intake attenuates the development of steatosis resulting from a high calorie diet. These results also suggest that human studies of agents that modify iron balance in patients with NAFLD should be revisited.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-β and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes.

Background & Aims Ferritin is purported to have proinflammatory and profibrogenic effects on hepatic stellate cells. Thus, rather than acting as a passive indicator of hepatic iron concentration (HIC) in hemochromatosis, ferritin may directly influence fibrosis. This study evaluated whether serum ferritin is a better predictor of hepatic fibrosis compared to variables previously associated with increased fibrosis risk in hemochromatosis. Methods We identified 291 C282Y HFE-homozygous patients who had undergone liver biopsy for histological fibrosis staging and measurement of HIC. Ordinal logistic regression determined the best model for fibrosis stage not including serum ferritin. Then, serum ferritin was introduced into this model to assess whether the predictive power of the model was significantly increased and to evaluate the effect on other predictors of fibrosis. Results Ordinal logistic regression analyses without serum ferritin demonstrated that log HIC (OR 2.89; P

The mechanisms that promote liver injury in nonalcoholic fatty liver disease (NAFLD) are yet to be thoroughly elucidated. As such, effective treatment strategies are lacking and novel therapeutic targets are required. Iron has been widely implicated in the pathogenesis of NAFLD and represents a potential target for treatment. Relationships between serum ferritin concentration and NAFLD are noted in a majority of studies, although serum ferritin is an imprecise measure of iron loading. Numerous mechanisms for a pathogenic role of hepatic iron in NAFLD have been demonstrated in animal and cell culture models. However, the human data linking hepatic iron to liver injury in NAFLD is less clear, with seemingly conflicting evidence, supporting either an effect of iron in hepatocytes or within reticulo-endothelial cells. Adipose tissue has emerged as a key site at which iron may have a pathogenic role in NAFLD. Evidence for this comes indirectly from studies that have evaluated the role of adipose tissue iron with respect to insulin resistance. Adding further complexity, multiple strands of evidence support an effect of NAFLD itself on iron metabolism. In this review, we summarise the human and basic science data that has evaluated the role of iron in NAFLD pathogenesis.

Multiphoton microscopy (MPM) has become increasingly popular and widely used in both basic and clinical liver studies over the past few years. This technology provides insights into deep live tissues with less photobleaching and phototoxicity, which helps us to better understand the cellular morphology, microenvironment, immune responses and spatiotemporal dynamics of drugs and therapeutic cells in the healthy and diseased liver. This review summarizes the principles, opportunities, applications and limitations of MPM in hepatology. A key emphasis is on the use of fluorescence lifetime imaging (FLIM) to add additional quantification and specificity to the detection of endogenous fluorescent species in the liver as well as exogenous molecules and nanoparticles that are applied to the liver in vivo. We anticipate that in the near future MPM-FLIM will advance our understanding of the cellular and molecular mechanisms of liver diseases, and will be evaluated from bench to bedside, leading to real-time histology of human liver diseases. Refereed/Peer-reviewed

The Asian Pacific Association for the Study of the Liver (APASL) convened an international working party on “APASL consensus statements and recommendations for management of hepatitis C” in March 2015 to revise the “APASL consensus statements and management algorithms for hepatitis C virus infection” (Hepatol Int 6:409–435, 2012). The working party consisted of expert hepatologists from the Asian–Pacific region gathered at the Istanbul Congress Center, Istanbul, Turkey on 13 March 2015. New data were presented, discussed, and debated during the course of drafting a revision. Participants of the consensus meeting assessed the quality of the cited studies. The finalized recommendations for hepatitis C prevention, epidemiology, and laboratory testing are presented in this review. Electronic supplementary material The online version of this article (doi:10.1007/s12072-016-9736-3) contains supplementary material, which is available to authorized users.

The Asian-Pacific Association for the Study of the Liver (APASL) convened an international working party on the “APASL consensus statements and recommendation on management of hepatitis C” in March, 2015, in order to revise “APASL consensus statements and management algorithms for hepatitis C virus infection (Hepatol Int 6:409–435, 2012)”. The working party consisted of expert hepatologists from the Asian-Pacific region gathered at Istanbul Congress Center, Istanbul, Turkey on 13 March 2015. New data were presented, discussed and debated to draft a revision. Participants of the consensus meeting assessed the quality of cited studies. Finalized recommendations on treatment of hepatitis C are presented in this review. Electronic supplementary material The online version of this article (doi:10.1007/s12072-016-9717-6) contains supplementary material, which is available to authorized users.

Nonalcoholic steatohepatitis (NASH) is characterized by adipose tissue dysfunction with insulin resistance and the dysregulation of adipokines.1 Recent data indicate repartitioning of iron from the liver to adipocytes in obesity and a role for iron in the development of adipose tissue dysfunction.2, 3 However, the molecular mechanisms have not been established. To test the hypothesis that iron modulates adipokine release, we performed a quantitative proteomics analysis of the human Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte secretome after 48 hours of treatment with ferric ammonium citrate (FAC). We used stable isotope-labeled amino acids in cell culture (SILAC) to characterize changes in the adipocyte secretome in response to iron. This technique has enabled direct comparison of quantities of individual proteins in the adipocyte secretome in response to iron using a proteomics approach as a tool for the identification of novel treatment targets in NASH. Detailed methodology is described in Supplementary Methods...

International audience; Background: Steatosis in donor livers poses a major risk of organ dysfunction due to their susceptibility to ischaemia-reperfusion (I/R) injury during transplant. Necroptosis, a novel form of programmed cell death, is orchestrated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL), has been implicated in I/R injury. Here we investigated the mechanisms of cell death pathways in an in vitro model of hepato-steatotic ischaemia. Methods: Free fatty acid (FFA) treated alpha mouse liver 12 (AML-12) cells were incubated in oxygen-glucose-deprivation (OGD) conditions as seen during ischaemia. Results: We found that OGD triggered upregulation of insoluble fraction of RIPK3 and MLKL in FFA + OGD cells compared to FFA control cells. We report that intervention with small interfering (si) MLKL and siRIPK3 significantly attenuated cell death in FFA + OGD cells. Absence of activated CASPASE8 and cleaved-CASPASE3, no change in the expression of CASPASE1 and prostaglandin-endoperoxide synthase 2 (Ptgs2) in FFA + OGD treated cells compared to FFA control cells indicated that apoptosis, pyroptosis and ferroptosis, respectively, are unlikely to be active in this model.Conclusion: Our findings indicate that RIPK3-MLKL dependent necroptosis contributed to cell death in our in vitro model. Both MLKL and RIPK3 are promising therapeutic targets to inhibit necroptosis during ischaemic injury in fatty liver.

This study reviews the evidence for the use of Bortezomib (BZB), a first-in-class proteasome inhibitor in advanced Hepatocellular carcinoma (HCC). This review aims to delineate the role of BZB within the management of non-surgical and metastatic HCC, either as an alternative or as an adjunct to the current treatment paradigm.In addition to BZB pharmacology and mechanism of action, safety and tolerance profiles of the drug obtained from clinical trials are explored. The utility of BZB as a therapeutic agent either alone or in combination with other therapies against HCC, including its application in both preclinical and clinical settings has been reviewed. In particular, we highlight the importance of preclinical evaluation of BZB as a combinatorial agent in synergism with other therapies for the use in the management of HCC.There has been much interest surrounding the use of BZB, a first-in-class proteasome inhibitor for HCC therapy. The discernment of outcomes of BZB clinical trials for HCC need to take into consideration the disease-specific factors that can affect survival outcomes including patient selection and aetiological differences. Further preclinical testing of BZB in combination with other therapeutic modalities can be important for eliciting enhanced anti-HCC effects.

STUDY OBJECTIVES: Recent results from the PTSD Initiative, a cross-sectional cohort study in Australian Vietnam veterans (VV) with and without posttraumatic stress disorder (PTSD), demonstrated an increased prevalence of self-reported sleep disturbances in those with PTSD. This study aimed to objectively assess the prevalence of sleep disorders in the same cohort using detailed polysomnography (PSG). METHODS: Participants from the PTSD Initiative were recruited to undergo PSG. PTSD status was determined with the Clinician Administered PTSD Scale for DSM-5 (CAPS-5). Subjective sleep information was attained via structured questionnaires. Data from single night PSG were compared between trauma-exposed VV with and without PTSD. RESULTS: A total of 74 trauma-exposed male VV (40 with PTSD) underwent PSG (prospective n = 59, retrospective n = 15). All PSG parameters were similar between groups. No difference was seen in PSG-diagnosed obstructive sleep apnea (OSA) or periodic limb movements of sleep (PLMS). VV with PTSD showed a trend toward increased duration of sleep with oxygen saturations < 90% (10% versus 1.8%; P = .07). VV with PTSD reported increased sleep onset latency (42.4 versus 13.3 minutes; P < .01); were less likely to report sleeping well (32.5% versus 67.5%; P < .01); had higher OSA risk using Berlin Questionnaire (BQ) (70% versus 38.2%; P < .01); and had higher rates of partner-reported limb movements (56.4% versus 17.6%; P < .01). No association between PSG-diagnosed OSA and PTSD severity was evident. CONCLUSIONS: In Australian VV with and without PTSD, no difference was seen across all PSG parameters including the diagnosis and severity of OSA and PLMS. However, VV with PTSD demonstrated an increased perception of sleep disturbances. CITATION: Baird T, Theal R, Gleeson S, McLeay S, O'Sullivan R; PTSD Initiative. Detailed polysomnography in Australian Vietnam veterans with and without posttraumatic stress disorder. J Clin Sleep Med. 2018;14(9):1577–1586.

Background Atypical iron overload without variation in the five clinically associated hereditary hemochromatosis genes is now recognized; however, their etiology remains unknown. Since the identification of iron overload in the bone morphogenetic protein 6 (Bmp6) knockout mouse, the search has been on for clinically pathogenic variants in the BMP6 gene. A recent report proposes that variants in the pro-peptide region of BMP6 are the underlying cause of several cases of iron overload. We performed targeted next-generation sequencing on three cases of atypical iron overload with Asian ethnicity and identified a p.Q118dup (aka p.E112indelEQ, p.Q115dup, p.Q118_L119insQ) variant in BMP6. The purpose of this study was to characterize the molecular function of the identified BMP6 variant. Molecular characterization by immunofluorescence microscopy and Western blotting of transfected cells, bioinformatics, and population analyses was performed. Results In contrast to reports for other BMP6 pro-peptide variants in this region, our data indicates that this variant does not affect the function of the mature BMP6 protein. Conclusions Our data suggest that assignment of disease causation in clinical cases of iron overload to pro-peptide variants in BMP6 should thus be treated with caution and requires biological characterization.

Iron is implicated in the pathogenesis of liver injury and insulin resistance (IR) and thus phlebotomy has been proposed as a treatment for nonalcoholic fatty liver disease (NAFLD). We performed a prospective 6-month randomized, controlled trial examining the impact of phlebotomy on the background of lifestyle advice in patients with NAFLD. Primary endpoints were hepatic steatosis (HS; quantified by magnetic resonance imaging) and liver injury (determined by alanine aminotransaminase [ALT] and cytokeratin-18 [CK-18]). Secondary endpoints included insulin resistance measured by the insulin sensitivity index (ISI) and homeostasis model of assessment (HOMA), and systemic lipid peroxidation determined by plasma F2-isoprostane levels. A total of 74 subjects were randomized (33 phlebotomy and 41 control). The phlebotomy group underwent a median (range) of 7 (1-19) venesection sessions and had a significantly greater reduction in ferritin levels over 6 months, compared to controls (−148 ± 114 vs. −38 ± 89 ng/mL; P

Background and AimDevelopment of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on -thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron-chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects.

Liver diseases, particularly viral hepatitis, cirrhosis and hepatocellular carcinoma, are common in clinical practice with high morbidity and mortality worldwide. Many substances for diagnostic imaging and therapy of liver diseases may have either severe adverse effects or insufficient effectiveness in vivo because of their nonspecific uptake. Therefore, by targeting the delivery of drugs into the liver or specific liver cells, drug efficiency may be largely improved. This review summarizes the up-to-date research progress focusing on nanoparticles targeting the liver for both diagnostic and therapeutic purposes.Targeting strategies, mechanisms of enhanced effects, and clinical applications of nanoparticles are discussed specifically. We believe that new targeting nanotechnology such as nanoprobes for multimodality imaging and multifunctional nanoparticles would facilitate significant advancements in this active research area in the near future. Refereed/Peer-reviewed

Rodent and cell-culture models support a role for iron-related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum samples at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein-4, tumor necrosis factor α, and interleukin-6, using a Quantibody, customized, multiplexed enzyme-linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36; P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo-IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD. (Hepatology Communications 2018;2:644-653).

This unit describes a protocol for elucidating the in vivo disposition of administered mesenchymal stem cells (MSCs). Specifically, direct visualization of donor cell spatiotemporal distribution and assessment of donor cell quantity in recipient organs are described. Protocols for data analysis are suggested, with the goal of developing a model to characterize and predict the physiological kinetics of administered MSCs. The use of this model is described, suggesting that it can be applied to abnormal conditions and has potential interspecies and inter-route predictive capability. These universal methods can be employed, regardless of the type of stem cell or disease, to guide future experiments and design treatment protocols. © 2017 by John Wiley & Sons, Inc.

Background and Aim: While genetic polymorphisms upstream of the interleukin-28B (IL28B) gene are associated with necroinflammatory activity grade in chronic hepatitis C virus genotype 1 (HCV-1) infection, any association with fibrosis is less definitive. Pretreatment liver biopsies in a cohort of treatment-naive patients with HCV-1 were analyzed to evaluate associations between liver histology, and the rs12979860 and rs8099917 IL28B single nucleotide polymorphisms.

On-treatment anemia is associated with higher sustained virological response (SVR) rates during peginterferon plus ribavirin (RBV) therapy. Inosine triphosphatase (ITPA) variants causing ITPase deficiency have been shown to protect against RBV-induced anemia. However, ITPase activity has not been associated with SVR. To study this discrepancy, we examined the relationships between ITPase activity, on-treatment anemia, SVR, and RBV levels in hepatitis C virus genotype 1 (HCV-1) patients from the CHARIOT study. ITPA genotype (rs7270101, rs1127354) was used to define ITPase activity in 546 patients. Plasma RBV levels were measured using high-performance liquid chromatography (HPLC). Relationships between ITPase activity, on-treatment hemoglobin (Hb) levels, RBV levels, and SVR were tested using regression modeling, survival analysis, and locally weighted scatterplot smoothing (LOWESS) plot analysis. Hb decline was independently associated with SVR (P

Transplanting donor livers with severe macrosteatosis is associated with increased risk of primary non-function (PNF). The purpose of this study was to identify steatosis-driven biomarkers as a predisposition to severe liver damage and delayed recovery following ischemia reperfusion injury. Wistar rats were fed a methionine- and choline-deficient (MCD) diet for up to three weeks to achieve severe macrosteatosis (>90%). Animals underwent diet withdrawal to control chow and/or underwent ischemia reperfusion and partial hepatectomy injury (I/R-PHx) and reperfused out to 7 days on control chow. For animals with severe macrosteatosis, hepatic levels of IL-33 decreased while Cyclin D1 levels increased in the absence of NF-κB p65 phosphorylation. Animals with high levels of nuclear Cyclin D1 prior to I/R-PHx either did not survive or had persistent macrosteatosis after 7 days on control chow. Survival 7 days after I/R-PHx fell to 57% which correlated with increased Cyclin D1 and decreased liver IL-33 levels. In the absence of I/R-PHx, withdrawing the MCD diet normalized IL-33, Cyclin D1 levels, and I/R-PHx survival back to baseline. In transplanted grafts with macrosteatosis, higher Cyclin D1 mRNA expression was observed. Shifts in Cyclin D1 and IL-33 expression may identify severely macrosteatotic livers with increased failure risk if subjected to I/R injury. Clinical validation of the panel in donor grafts with macrosteatosis revealed increased Cyclin D1 expression corresponding to delayed graft function. This pre-surgical biomarker panel may identify the subset of livers with increased susceptibility to PNF.

Mammalian target of rapamycin and angiotensin-converting enzyme inhibition has been shown to have antifibrotic activity in models of liver fibrosis. The aim of our study was to determine the efficacy of rapamycin, everolimus, irbesartan and captopril, alone and in combination, as antifibrotic agents in the Mdr2(-/-) model of cholestasis both in early injury and established disease.Mdr2(-/-) mice were treated for 4 weeks with vehicle, rapamycin (1 mg/kg) or everolimus (5 mg/kg) every second day or with captopril (30 mg/kg/day), irbesartan (10 mg/kg/day) or vehicle. Further groups of 3-week-old Mdr2(-/-) mice were treated with rapamycin and irbesartan in combination (1 mg/kg/day and 10 mg/kg/day) or with rapamycin (2 mg/kg/day) for 4 weeks. Liver injury and fibrosis were compared between treated and untreated animals.There were no significant improvements in liver injury, histology, hepatic hydroxyproline or profibrogenic gene expression following treatment with rapamycin, everolimus, captopril or irbesartan at any time point studied. Likewise, there were no improvements in liver histology or profibrogenic gene expression following combination therapy or high-dose rapamycin treatment.The antifibrotic effects of rapamycin, everolimus, captopril and irbesartan seen in other models of fibrosis were not replicated in the Mdr2(-/-) model in this study. This highlights the clear need to test specific antifibrotic agents in a number of different animal models. We believe this animal model is ideal to study usefulness of antifibrotic agents in cholestatic liver disease because of the similarity in genetics and hepatic histopathology to human cholestatic liver disease.

Background and AimsThe aim of this study was to examine the distribution of interferon lambda-3 (IFN-3) gene polymorphisms in previously untreated Australian patients with genotype 1 (Gt1) chronic hepatitis C (CHC) and to compare the IFN-3 genotype frequency among the different ethnic populations.

Endoplasmic reticulum (ER) stress is an important pathogenic mechanism for alcoholic (ALD) and nonalcoholic fatty liver disease (NAFLD). Iron overload is an important cofactor for liver injury in ALD and NAFLD, but its role in ER stress and associated stress signaling pathways is unclear. To investigate this, we developed a murine model of combined liver injury by co-feeding the mildly iron overloaded, the hemochromatosis gene-null (Hfe(-/)) mouse ad libitum with ethanol and a high-fat diet (HFD) for 8 weeks. This co-feeding led to profound steatohepatitis, significant fibrosis, and increased apoptosis in the Hfe(-/-) mice as compared with wild-type (WT) controls. Iron overload also led to induction of unfolded protein response (XBP1 splicing, activation of IRE-1α and PERK, as well as sequestration of GRP78) and ER stress (increased CHOP protein expression) following HFD and ethanol. This is associated with a muted autophagic response including reduced LC3-I expression and impaired conjugation to LC3-II, reduced beclin-1 protein, and failure of induction of autophagy-related proteins (Atg) 3, 5, 7, and 12. As a result of the impaired autophagy, levels of the sequestosome protein p62 were most elevated in the Hfe(-/-) group co-fed ethanol and HFD. Iron overload reduces the activation of adenosine monophosphate protein kinase associated with ethanol and HFD feeding. We conclude that iron toxicity may modulate hepatic stress signaling pathways by impairing adaptive cellular compensatory mechanisms in alcohol- and obesity-induced liver injury.