Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a liver tumor that usually arises in patients with cirrhosis. Hepatic stellate cells are key players in the progression of HCC, as they create a fibrotic micro-environment and produce growth factors and cytokines that enhance tumor cell proliferation and migration. We assessed the role of endoplasmic reticulum (ER) stress in the cross-talk between stellate cells and HCC-cells. Mice with a fibrotic HCC were treated with the IRE1α-inhibitor 4μ8C, which reduced tumor burden and collagen deposition. By co-culturing HCC-cells with stellate cells, we found that HCC-cells activate IREα in stellate cells, thereby contributing to their activation. Inhibiting IRE1α blocked stellate cell activation, which then decreased proliferation and migration of tumor cells in different in vitro 2D and 3D co-cultures. In addition, we also observed cell-line specific direct effects of inhibiting IRE1α in tumor cells

Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Background. Hepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA. Method. Blood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 ◦C and 25 ◦C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to −80 ◦C for storage. The effect of multiple cycles of freezing and thawing of plasma at −20 ◦C or −80 ◦C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log10 copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0. Results. The mean HBV pgRNA level in the initially collected plasma samples was 5.58 log10 copies/mL (ranging from 3.08 to 8.04 log10 copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by −0.05 log10 copies/mL (95% confidence interval (CI) −0.095 to −0.005, p = 0.03), −0.075 log10 copies/mL (95% CI [−0.12 to −0.03], p = 0.001), −0.084 log10 copies/mL (95% CI [−0.13 to −0.039], p =< 0.001), and −0.120 log10 copies/mL (95% CI [−0.17 to −0.076], p =< 0.001), respectively. However, these changes were below 0.5 log10 copies/mL and thus were not clinically significant. Compared with the samples stored at 4 ◦C, there were no significant differences in pgRNA levels in samples stored at 25 ◦C for any of the storage durations (−0.01 log10 copies/mL; 95% CI [−0.708 to 0.689], p = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at −20 ◦C and −80 ◦C. Conclusion. The plasma HBV pgRNA level was stable at 4 ◦C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable

Non-invasive tests for liver fibrosis assessment in patients with chronic liver diseases: a prospective study

There is an urgent need of non-invasive tests (NITs) for monitoring treatment response and disease progression in chronic liver disease. Liver stiffness (LS) evaluated by transient elastography (TE), shear wave elastography (SWE), and magnetic resonance elastography (MRE) and serum markers e.g. APRI and FIB-4 scores were assessed at baseline and the 1-year follow-up. In all, 89 chronic hepatitis C virus (HCV) patients with sustained virological response and 93 non-alcoholic fatty liver disease (NAFLD) patients were included. There was a significantly strong correlation among imaging techniques. Using MRE as the reference standard, the area under the receiver operating characteristics curves for TE, SWE, APRI, and FIB-4 in detecting stage1–4 fibrosis were 0.88–0.95, 0.87–0.96, 0.83–0.89, and 0.79–0.92, respectively. In chronic HCV patients, the values of TE, SWE, MRE, APRI and FIB-4 significantly decreased from baseline to the 1-year follow-up. Liver steatosis did not significantly change over time. In NAFLD, compared to obese patients, non-obese patients had less LS and steatosis at baseline, and these values did not show significant changes at the 1-year follow-up. Our study suggests that the current NITs have a good correlation and accuracy in monitoring the treatment outcomes in patients with chronic liver diseases

Validation and prognostic value of EZ-ALBI score in patients with intermediate-stage hepatocellular carcinoma treated with trans-arterial chemoembolization

Background: Heterogeneity of liver function and tumor burden in intermediate-stage hepatocellular carcinoma (HCC) results in different outcomes after transarterial chemoembolization (TACE). Easy albumin-bilirubin (EZ-ALBI), a simplified albumin-bilirubin (ALBI) score, has recently been proposed as a new prognostic score for HCC. This study aimed to validate the EZ-ALBI score and evaluate the impact of dynamic changes in patients with intermediate-stage HCC undergoing TACE. Methods: All patients with HCC treated with TACE at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between January 2015 and December 2019 were prospectively enrolled. Intermediate-stage HCC was defined as Barcelona Clinic Liver Cancer (BCLC) stage B or unresectable single HCC with size > 5 cm in BCLC stage A. EZ-ALBI and ALBI scores were calculated and stratified into three different grades. Overall survival (OS) and prognostic factors were assessed using the Kaplan–Meier curve and Cox proportional hazard model. Decision analysis curves were used to evaluate the clinical utility of the predictive scores. Results: Among 672 patients with HCC treated with TACE, 166 patients with intermediate-stage HCC who met the eligibility criteria were enrolled. The median OS of all patients in the cohort was 21 months. A good correlation between the EZ-ALBI and ALBI scores was observed (correlation coefficient 1.000, p 20 ng/ml were significantly associated with OS [hazard ratio (HR) 2.20 (95% confidence interval [CI] 1.24–3.88, p = 0.007), 3.26 (95% CI 1.24–8.57, p = 0.016), and 1.77 (95% CI 1.10–2.84, p = 0.018), respectively]. Following TACE, 42 (29.6%) patients had a worsening EZ-ALBI grade. However, the EZ-ALBI grade migration was not significantly correlated with OS. EZ-ALBI and ALBI score provided improved discriminatory ability (Harrell’s concordance index 0.599 and 0.602, respectively) and better net benefit compared with Child-Turcotte-Pugh and Model for End-stage Liver Disease scores. Conclusions: The baseline EZ-ALBI score demonstrated good predictive performance for survival and a strong correlation with conventional ALBI scores. Both the EZ-ALBI and ALBI scores outperformed other prognostic models in patients with intermediate-stage HCC receiving TACE. However, the dynamic change in the EZ-ALBI grade after TACE was not associated with postprocedural survival

Optimization and validation of a novel three-dimensional co-culture system in decellularized human liver scaffold for the study of liver fibrosis and cancer

The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib re-versed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvi-ronment and could be employed for anti-fibrotic and anti-HCC drug screening

The incidence, etiologies, outcomes, and predictors of mortality of acute liver failure in Thailand: a population-base study

Background: Acute liver failure (ALF) is uncommon but progresses rapidly with high mortality. We investigated the incidence, etiologies, outcomes, and predictive factors for 30-day mortality in patients with ALF. Methods: We conducted a population-based study of ALF patients hospitalized between 2009 and 2013 from the Thai Nationwide Hospital Admission database, which comprises 76% of all admissions from 858 hospitals across 77 provinces in Thailand. ALF was diagnosed using ICD-10 codes K72.0 and K71.11. Patients with liver cirrhosis were excluded. Results: There were 20,589 patients diagnosed with ALF during the study period with 12,277 (59.6%) males and mean age of 46.6 ± 20.7 years. The incidence of ALF was 62.9 per million population per year. The most frequent causes of ALF were indeterminate (69.4%), non-acetaminophen drug-induced (26.1%), and viral hepatitis (2.5%). Acetaminophen was the presumptive cause in 1.7% of patients. There were 5502 patients (26.7%) who died within 30 days after admission. One patient (0.005%) underwent liver transplantation. The average hospital stay was 8.7 ± 13.9 days, and the total cost of management was 1075.2 ± 2718.9 USD per admission. The most prevalent complications were acute renal failure (ARF)(24.2%), septicemia (18.2%), and pneumonia (12.3%). The most influential predictive factors for 30-day mortality were ARF (HR = 3.64, 95% CI: 3.43–3.87, p < 0.001), malignant infiltration of the liver (HR = 3.37, 95% CI: 2.94–3.85, p < 0.001), and septicemia (HR = 1.96, 95%CI: 1.84–2.08, p < 0.001). Conclusions: ALF patients have poor outcomes with 30-day mortality of 26.7% and high economic burden. Indeterminate etiology is the most frequent cause. ARF, malignant infiltration of the liver, and septicemia are main predictors of 30-day mortality

Changes in hepatic fibrosis and vitamin D levels after viral hepatitis C eradication using direct-acting antiviral therapy

Background: Vitamin D (VD) is important in hepatic fibrogenesis in animal models and human studies. VD deficiency is associated with liver fibrosis progression. Metabolic dysfunction of the liver, as an intermediate organ for VD metabolism, contributes partly to this deficiency. We hypothesized that improving hepatic fibrosis and inflammation in chronic hepatitis C (CHC) patients after eradication with direct-acting antivirals (DAA) would increase 25-hydroxyVD [25(OH)VD] levels. Methods: Eighty CHC patients (17 chronic hepatitis, and 63 cirrhosis) were enrolled. Baseline characteristics, hepatitis C viral load (VL), genotypes, liver enzymes and liver stiffness measurements (LSM) were assessed at baseline. Blood samples for 25(OH)VD and the procollagen type III N-terminal peptide (P3NP) were collected at baseline, 24 and 48 weeks. LSMs were re-evaluated at 48 weeks. Serum 25(OH)VD levels < 30 ng/mL were defined as VD insufficiency/deficiency. Paired t-tests were used for statistical analyses. Results: Among 80 patients, the mean age was 57.7 ± 10.5 years, and 52.5% were men. The mean VL was 6.1 ± 0.7 logIU/mL with genotype 1 predominance (55%). All patients achieved sustained virological response. The alanine aminotransferase levels decreased from 79.9 ± 53.3 U/L at baseline to 25.7 ± 17.2 and 22.3 ± 11.0 U/L at 24 and 48 weeks, respectively (p < 0.001). The mean LSM decreased from 19.2 ± 15.3 to 11.7 ± 8.0 kPa at 48 weeks (p < 0.001). The P3NP levels decreased from 43.6 ± 22.0 ng/mL before treatment to 35.7 ± 21.1 and 29.4 ± 15.0 ng/mL at 24 and 48 weeks, respectively (p < 0.001). The proportions of VD insufficiency/deficiency were 72.5%, 91.3%, and 86.5% at baseline, 24 and 48 weeks, respectively. The 25(OH)VD levels decreased from 26.3 ± 10.7 ng/mL at baseline to 20.8 ± 8.1 and 20.8 ± 8.5 ng/mL at 24 and 48 weeks, respectively (p < 0.001). Conclusions: Curative treatment with DAA attenuated the liver stiffness and inflammation but did not improve VD levels. Over 80% of patients remained VD insufficient/deficient. Whether VD replacement during and after DAA therapy can improve hepatic fibrosis remains unclear. Trial registration The Thai Clinical Trial Registry as TCTR20161025001 (31 October 2016). http://www.clinicaltrials.in.th/index.php?tp=regtrials&menu=trialsearch&smenu=fulltext&task=search&task2=view1&id=2136

Effect of vitamin D supplementation in patients with chronic hepatitis C after direct-Acting antiviral treatment a randomized, double-blind, placebo-controlled trial

Background Replacement of vitamin D (VD) among patients with chronic hepatitis C (CHC) before viral eradication has demonstrated a protective effect on serum markers associated with hepatic fibrogenesis. We therefore hypothesized that VD may facilitate further fibrosis amelioration following curative treatment with direct-Acting antivirals (DAA). Methods. This study was a randomized, double-blind, placebo-controlled trial con-ducted between February 2018 and August 2018. Patients with CHC and VD deficiency were randomized in a 1:1 ratio to either receive ergicalciferol or placebo over 6 weeks. Biochemical analysis indicators, including 25-hydroxyvitamin D (25(OH)D), fibrogenic markers [(transforming growth factor beta 1 (TGF-1) and tissue in-hibitors of matrix metalloproteinases 1 (TIMP-1)], and fibrolytic markers [matrix metalloproteinase 9 (MMP-9) and amino terminal type III procollagen peptide (P3NP)], were assessed at baseline and at 6 weeks. Serum 25(OH)D was analyzed by a chemiluminescence immunoassay. Serum hepatic fibrogenesis markers were measured using a quantitative sandwich enzyme-linked immunosorbent assay. Results. Seventy-five patients with CHC and VD deficiency were randomly assigned to VD (nD37) and placebo (nD38) groups. At the end of the study, the mean serum 25(OH)D level had risen to a normal level in the VD group, but was still deficient in the placebo group (41.8 9.1 vs. 18.1 4.6 ng/mL, p 0.001). Upon restoration of the VD level, there were no significant mean differences in the change from baseline for TGF-1 (0.6 ng/mL (95% confidence interval (95% CI) [2.81.7]), pD0:63), TIMP-1 (5.5 ng/mL (95% CI [26.4 15.3]), pD0:60), MMP-9 (122.9 ng/mL (95% CI [69.0 314.8]), pD0:21), and P3NP (0.1 ng/mL (95% CI [2.4 2.2]), pD0:92) between the VD and placebo groups. Conclusion. Short-Term VD supplementation after DAA treatment in patients with CHC does not improve serum fibrogenesis markers and may not expedite the residual liver fibrosis healing process. Future studies are warranted to evaluate the long-Term effect of VD supplementation on hepatic fibrosis regression

Transcriptomic Profiling of In Vitro Tumor-Stromal Cell Paracrine Crosstalk Identifies Involvement of the Integrin Signaling Pathway in the Pathogenesis of Mesenteric Fibrosis in Human Small Intestinal Neuroendocrine Neoplasms.

Aim: Analysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples. Methods: An indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry. Results: RT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway - related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs. Conclusion: This study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway - the integrin pathway - was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment