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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.

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.

While liver transplantation remains the sole treatment option for patients with end-stage liver disease, there are numerous limitations to liver transplantation including the scarcity of donor livers and a rise in livers that are unsuitable to transplant such as those with excess steatosis. Fatty livers are susceptible to ischaemia-reperfusion (IR) injury during transplantation and IR injury results in primary graft non-function, graft failure and mortality. Recent studies have described new cell death pathways which differ from the traditional apoptotic pathway. Necroptosis, a regulated form of cell death, has been associated with hepatic IR injury. Receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL) are thought to be instrumental in the execution of necroptosis. The study of hepatic necroptosis and potential therapeutic approaches to attenuate IR injury will be a key factor in improving our knowledge regarding liver transplantation with fatty donor livers. In this review, we focus on the effect of hepatic steatosis during liver transplantation as well as molecular mechanisms of necroptosis and its involvement during liver IR injury. We also discuss the immune responses triggered during necroptosis and examine the utility of necroptosis inhibitors as potential therapeutic approaches to alleviate IR injury.

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.

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 dosing regimen can be considered the Achilles' heel of mesenchymal stem/stromal cell (MSC)‐based therapies. The establishment of optimal dosage and route of administration of MSCs requires the ability to visualize and quantitatively determine their in vivo distribution. It is increasingly possible to apply pharmacokinetic modelling to predict their distribution and persistence.

The two most important advances in the field over the past 30 years have been the identification of the HFE gene (and the associated p.Cys282Tyr substitution), and the discovery of the hormone hepcidin, which is inappropriately low in this condition and is the pathophysiological basis of the increased iron absorption. Modified Classification of Hemochromatosis based on Recommendations of the International Society for the Study of Iron in Biology and Medicine (modified from [5]) Novel classification Molecular pattern Features HFE-related p.Cys282Tyr homozygosity compound heterozygosity p.Cys282Tyr/ His63 Asp compound heterozygosity of p.Cys282Tyr with other rare pathogenic variants 106-109or HFE deletion.110 Variable penetrance Always consider the presence of host or environmental cofactors for iron overload and co-toxic liver injury, e.g., alcohol, metabolic-associated fatty liver disease In subjects with, p.Cys282Tyr/His63Asp compound heterozygosity or p.His63Asp homozygosity and iron overload-related disease consider referral to a specialist center to determine the need for second-line genetic testing for rarer variants Non-HFE-related Rare pathogenic variants in “non-HFE” genes: HJV-related HAMP-related TFR2-related SLC40A1 (GOF)-related May be associated with severe iron loading in multiple organs in younger populations While mutations in any hepcidin-regulatory gene may be causative, the effects of novel mutations should be confirmed through functional and epidemiological studies Molecular subtypes characterization only at specialized centers, but the diagnosis of non-HFE-related hemochromatosis is sufficient to start treatment at non-specialized centers Digenic Double heterozygosity and/or double homozygosity/heterozygosity for mutations in 2 different genes involved in iron metabolism (HFE and/or non-HFE) More commonly, p.Cys282Tyr mutation in HFE gene might coexist with mutation in other genes; rarely, both mutations involve non-HFE genes Molecularly undefined Molecular characterization (still) not available after sequencing of known genes (provisional diagnosis) The case should be referred to specialized centers for further consideration Forms of both HFE-related and non-HFE-related hemochromatosis result in iron overload by diminished hepcidin. Selected population studies of the frequency of HFE p.Cys282Tyr homozygosity Country Study population Cohort size Frequency of HFE p.Cys282Tyr Australia [23] Workplace 11,307 1 in 221 Australia [24] Cohort enrolled through the electoral roll (enriched for Northern European ancestry) 29,676 1 in 146 USA [11] Primary care and blood drawing laboratories 99,711 1 in 333 United Kingdom [25] Postal invitation to individuals registered with the National Health Service 451,243 1 in 156 Norway [26] Hospitalized individuals (Caucasian only) 1900 1 in 136 Spain [27] Blood donors 5370 1 in 671 France [28] Attendees at health appraisal centers 9396 1 in 174 The highest prevalence of HFE p.Cys282Tyr homozygosity is among Northern Europeans, in particular, Ireland and Scandinavia, with a lesser prevalence in Southern Europe

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.

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 mechanisms responsible for disturbed iron homoeostasis in hereditary haemochromatosis are poorly understood. However, results of some studies indicate a link between hepcidin, a liver-derived peptide, and intestinal iron absorption, suggesting that this molecule could play a part in hepatic iron overload. To investigate this possible association, we studied the hepatic expression of the gene for hepcidin (HAMP) and a gene important in iron transport (IREG1) in patients with haemochromatosis, in normal controls, and in Hfe-knockout mice. We extracted total RNA from the liver tissue of 27 patients with HFE-associated haemochromatosis, seven transplant donors (controls), and Hfe-knockout mice. HAMP and IREG1 mRNA concentrations were examined by ribonuclease protection assays and expressed relative to the housekeeping gene GAPD. There was a significant decrease in HAMP expression in untreated patients compared with controls (5·4-fold, 95% CI 3·3–7·5; p<0·0001) despite significantly increased iron loading. Similarly, we noted a decrease in Hamp expression in iron-loaded Hfe-knockout mice. Hepatic IREG1 expression was greatly upregulated in patients with haemochromatosis (1·8-fold, 95% CI 1·5–2·2; p=0·002). There was a significant correlation between hepatic iron concentration and expression of HAMP (r=0·59, p=0·02) and IREG1 (r=0·67, p=0·007) in untreated patients. Lack of HAMP upregulation in HFE-associated haemochromatosis despite significant hepatic iron loading indicates that HFE plays an important part in the regulation of hepcidin expression in response to iron overload. Our results imply that the liver is important in the pathophysiology of HFE-associated haemochromatosis. Furthermore, the increase in hepatic IREG1 expression in haemochromatosis suggests that IREG1 could function to facilitate the removal of excess iron from the liver.

Oxidative stress reflects an imbalance between reactive oxygen species (ROS) and antioxidants, which has been reported as an early unifying event in the development and progression of various diseases and as a direct and mechanistic indicator of treatment response. However, highly reactive and short-lived nature of ROS and antioxidant limited conventional detection agents, which are influenced by many interfering factors. Here, we present a two-photon sensing platform for in vivo dual imaging of oxidative stress at the single cell-level resolution. This sensing platform consists of three probes, which combine the turn-on fluorescent transition-metal complex with different specific responsive groups for glutathione (GSH), hydrogen peroxide (H 2 O 2 ) and hypochlorous acid (HOCl). By combining fluorescence intensity imaging and fluorescence lifetime imaging, these probes totally remove any possibility of crosstalk from in vivo environmental or instrumental factors, and enable accurate localization and measurement of the changes in ROS and GSH within the liver. This precedes changes in conventional biochemical and histological assessments in two distinct experimental murine models of liver injury. The ability to monitor real-time cellular oxidative stress with dual-modality imaging has significant implications for high-accurate, spatially configured and quantitative assessment of metabolic status and drug response.