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Background & Aims Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. Methods We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. Results We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. Conclusions Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. Lay summary This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.

Current literature highlights the need for precise histological quantitative assessment of fibrosis which cannot be achieved by conventional scoring systems, inherent to their discontinuous values and reader-dependent variability. Here we used an automated image analysis software to quantitate fibrosis deposition in two relevant preclinical models of liver fibrosis, and established correlation with other quantitative fibrosis descriptors. Longitudinal quantification of liver fibrosis was carried out during progression of post-necrotic (CCl4-induced) and metabolic (HF-CDAA feeding) models of chronic liver disease in mice. Whole slide images of picrosirius red-stained liver sections were analyzed in fully automated, unsupervised manner by our software. Fibrosis was characterized by a significant increase of collagen proportionate area (CPA) at weeks 3 (CCl4) and 8 (HF-CDAA) with a progressive increase up to week 18 and 24, respectively. CPA was compared to collagen content assessed biochemically by hydroxyproline assay (HYP) and standard histological staging systems. CPA showed a high correlation with HYP content for CCl4 (r = 0.8268) and HF-CDAA (r = 0.6799) models. High correlations were also found with Ishak score or its modified version (r = 0.9705) for CCl4 and HF-CDAA (r = 0.9062) as well as with NASH CRN for HF-CDAA (r = 0.7937). Such correlations support the use of automated digital analysis as a reliable tool to evaluate the dynamics of liver fibrosis and efficacy of antifibrotic drug candidates in preclinical models.

Background EDP‐305 is a novel and potent farnesoid X receptor (FXR) agonist, with no/minimal cross‐reactivity to TGR5 or other nuclear receptors. Herein we report therapeutic efficacy of EDP‐305, in direct comparison with the first‐in‐class FXR agonist obeticholic acid (OCA), in mouse models of liver disease. Methods EDP‐305 (10 and 30 mg/kg/day) or OCA (30mg/kg/day) was tested in mouse models of pre‐established biliary fibrosis (BALBc.Mdr2‐/‐, n = 9‐12/group) and steatohepatitis induced by methionine/choline‐deficient diet (MCD, n = 7‐12/group). Effects on biliary epithelium were evaluated in vivo and in primary EpCAM + hepatic progenitor cell (HPC) cultures. Results In a BALBc.Mdr2‐/‐ model, EDP‐305 reduced serum transaminases by up to 53% and decreased portal pressure, compared to untreated controls. Periportal bridging fibrosis was suppressed by EDP‐305 at both doses, with up to a 39% decrease in collagen deposition in high‐dose EDP‐305. In MCD‐fed mice, EDP‐305 treatment reduced serum ALT by 62% compared to controls, and profoundly inhibited perisinusoidal ‘chicken wire’ fibrosis, with over 80% reduction in collagen deposition. In both models, treatment with 30mg/kg OCA reduced serum transaminases up to 30%, but did not improve fibrosis. The limited impact on fibrosis was mediated by cholestasis‐independent worsening of ductular reaction by OCA in both disease models; OCA but not EDP‐305 at therapeutic doses promoted ductular proliferation in healthy mice and favoured differentiation of primary HPC towards cholangiocyte lineage in vitro. Conclusions EDP‐305 potently improved pre‐established liver injury and hepatic fibrosis in murine biliary and metabolic models of liver disease, supporting the clinical evaluation of EDP‐305 in fibrotic liver diseases including cholangiopathies and non‐alcoholic steatohepatitis.

We identified that Fibroblast Growth Factor 14 (FGF14) was preferentially methylated in colorectal cancer (CRC). In this study, we aimed to investigate the epigenetic regulation, biological function and molecular mechanism of FGF14 in CRC. The expression of FGF14 in CRC cell lines, normal human colon epithelial cell line, CRC tissues and paired adjacent normal tissues was detected by PCR and Western blot. The biological function of FGF14 in CRC was interrogated by cell viability assay, colony formation, flow cytometry, cell invasion and migration assay, as well as in vivo study. We found FGF14 was downregulated or silenced in all (10/10) CRC cell lines, while it was expressed in normal colonic tissues and normal human colon epithelial cell line. The expression of FGF14 was lower in primary CRCs as compared to their adjacent normal tissues. Significant higher methylation of FGF14 was observed in CRCs than that in normal tissues based on the data from TCGA database. The loss of FGF14 gene expression was restored by treatment with DNA methyltransferase inhibitor 5-Aza. Re-expression of FGF14 in CRC cell lines inhibited cell viability and colony formation, and induced cell apoptosis. FGF14 induced mitochondrial apoptosis and inhibited PI3K/AKT/mTOR pathway. In xenograft mouse model, overexpression of FGF14 significantly reduced tumor growth (P

Background Limited effective intervention for advanced hepatocellular carcinoma (HCC) is available. This study aimed to investigate the potential clinical utility of apatinib, a highly selective inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine kinase, as a radiosensitizer in the treatment of HCC. Methods Four human HCC cell lines SMMC-7721, MHCC-97H, HCCLM3 and Hep-3B were treated with apatinib, irradiation or combination treatment. Colony formation assay, flow cytometry and nuclear γ-H2AX foci immunofluorescence staining were performed to evaluate the efficacy of combination treatment. RNA sequencing was conducted to explore the potential mechanism. The impact of combination treatment on tumor growth was assessed by xenograft mice models. Results Colony formation assay revealed that apatinib enhanced the radiosensitivity of HCC cell lines. Apatinib suppressed repair of radiation-induced DNA double-strand breaks. Flow cytometry analysis showed that apatinib increased radiation-induced apoptosis. Apatinib radiosensitized HCC via suppression of radiation-induced PI3K/AKT pathway. Moreover, an in vivo study indicated apatinib combined with irradiation significantly decreased xenograft tumor growth. Conclusions Our results indicate that apatinib has therapeutic potential as a radiosensitizer in HCC, and PI3K/AKT signaling pathway plays a critical role in mediating radiosensitization of apatinib.

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. Methods: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4−/− mice. Results: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4−/− mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. Conclusions: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3., Lay summary: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice., Graphical abstrac

Background To compare the efficacy of low dose (27 mg) Bacillus Calmette-Guérin (BCG) and a full dose (81 mg) BCG immunotherapy for patients with intermediate and high-risk non-muscle invasive bladder cancer (NMIBC) after a typical transurethral bladder resection. Methods We constructed a Markov model for a 20-year simulation of the disease to compare the overall survival of patients with intermediate and high-risk of NMIBC between the full-dose therapy (FD group) and the low-dose therapy (LD group). Base case analysis, one-way and two-way sensitivity analysis and a second-order Monte Carlo analysis were performed based on data from 15 published articles. Results The expected overall survivals were 9.56 (9.55–9.57) years for FD group and 9.63 (9.61–9.64) years for LD group(P

The pathogenesis of primary sclerosing cholangitis (PSC) and the mechanistic link to inflammatory bowel disease remain ill-defined. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1)/clusters of differentiation (CD) 39, the dominant purinergic ecto-enzyme, modulates intestinal inflammation. Here, we have explored the role of CD39 in biliary injury and fibrosis. The impact of CD39 deletion on disease severity was studied in multidrug resistance protein 2 (Mdr2)-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse models of sclerosing cholangitis and biliary fibrosis. Antibody-mediated CD8+ T-cell depletion, selective gut decontamination, experimental colitis, and administration of stable adenosine triphosphate (ATP) agonist were performed. Retinoic acid-induced gut imprinting on T cells was studied in vitro. Over half of Mdr2-/-;CD39-/- double mutants, expected by Mendelian genetics, died in utero. Compared to Mdr2-/-;CD39+/+, surviving Mdr2-/-;CD39-/- mice demonstrated exacerbated liver injury, fibrosis, and ductular reaction. CD39 deficiency led to a selective increase in hepatic CD8+ T cells and integrin α4β7, a T-cell gut-tropism receptor. CD8+ cell depletion in Mdr2-/-;CD39-/- mice diminished hepatobiliary injury and fibrosis. Treatment with antibiotics attenuated, whereas dextran sulfate sodium-induced colitis exacerbated, liver fibrosis in Mdr2-/- mice. Colonic administration of αβ-ATP into CD39-sufficient Mdr2-/- mice triggered hepatic CD8+ cell influx and recapitulated the severe phenotype observed in Mdr2-/-;CD39-/- mice. In vitro, addition of ATP promoted the retinoic acid-induced imprinting of gut-homing integrin α4β7 on naive CD8+ cells. CD39 expression was relatively low in human normal or PSC livers but abundantly present on immune cells of the colon and further up-regulated in samples of patients with inflammatory bowel disease. Conclusion: CD39 deletion promotes biliary injury and fibrosis through gut-imprinted CD8+ T cells. Pharmacological modulation of purinergic signaling may represent a promising approach for the treatment of PSC. (Hepatology Communications 2017;1:957-972).

Progressive fibrosis, functional liver failure, and cancer are the central liver-related outcomes of nonalcoholic steatohepatitis (NASH) but notoriously difficult to achieve in mouse models. We performed a direct, quantitative comparison of hepatic fibrosis progression in well-defined methionine- and choline-deficient (MCD) and choline-deficient, amino-acid defined (CDAA) diets with increasing fat content (10–60% by calories) in C57Bl/6J and BALB/cAnNCrl mice. In C57Bl/6J mice, MCD feeding resulted in moderate fibrosis at week 8 (up to twofold increase in total hepatic collagen content) and progressive weight loss irrespective of dietary fat. In contrast, CDAA-fed mice did not lose weight and developed progressive fibrosis starting from week 4. High dietary fat in the CDAA diet model induced the lipid metabolism genes for sterol regulatory element-binding protein and stearoyl-CoA desaturase-2 and increased ductular reaction and fibrosis in a dose-dependent manner. Longitudinal analysis of CDAA with 60% fat (HF-CDAA) feeding revealed pronounced ductular reaction and perisinusoidal bridging fibrosis, with a sevenfold increase of hepatic collagen at week 12, which showed limited spontaneous reversibility. At 24 wk, HF-CDAA mice developed signs of cirrhosis with pan-lobular “chicken wire” fibrosis, 10-fold hydroxyproline increase, regenerative nodules, portal hypertension and elevated serum bilirubin and ammonia levels; 80% of mice (8/10) developed multiple glypican-3- and/or glutamine synthetase-positive hepatocellular carcinomas (HCC). High-fat (60%) supplementation of MCD in C57Bl/6J or feeding the HF-CDAA diet fibrosis-prone BALB/cAnNCrl strain failed to result in increased fibrosis. In conclusion, HF-CDAA feeding in C57Bl/6J mice was identified as an optimal model of steatohepatitis with robust fibrosis and ductular proliferations that progress to cirrhosis and HCC within 24 wk. This robust model will aid the testing of interventions and drugs for severe NASH. NEW & NOTEWORTHY Via quantitative comparison of several dietary models, we report HF-CDAA feeding in C57Bl/6 mice as an excellent model recapitulating several key aspects of fibrotic NASH: 1) robust, poorly reversible liver fibrosis, 2) prominent ductular reaction, and 3) progression to cirrhosis, portal hypertension, and liver cancer within 24 wk. High fat dose-dependently activates SREBP2/SCD2 genes and drives liver fibrosis in e HF-CDAA model. These features qualify the model as a robust and practical tool to study mechanisms and novel treatments addressing severe human NASH.

Background Colorectal cancer (CRC) is one of the most common malignancies worldwide. Emerging evidence indicates that promoter methylation of genes associated with gene silencing, plays an important role in the development and progression of CRC. In this study, we aimed to investigate the epigenetic regulation, biological function and molecular mechanism of FGF14 in CRC. Methods The expression of FGF14 in CRC cell lines, CRC tissues and paired adjacent normal tissues was detected by PCR and Western blot. The biological function of FGF14 in CRC was interrogated by cell viability assay, colony formation, flow cytometry, cell invasion and migration assay, as well as in vivo study. Results We found FGF14 was downregulated or silenced in all (10/10) CRC cell lines, while it was expressed in normal colonic tissues. The expression of FGF14 was lower in primary CRCs as compared to their adjacent normal tissues. Significant higher methylation of FGF14 was observed in CRCs than that in normal tissues based on the data from TCGA database. The loss of FGF14 gene expression was restored by treatment with DNA methyltransferase inhibitor 5-Aza. Re-expression of FGF14 in CRC cell lines inhibited cell viability and colony formation, and induced cell apoptosis. In xenograft mouse model, overexpression of FGF14 significantly reduced tumor growth (P Conclusions In conclusion, FGF14 is a novel tumor suppressor, which suppresses cell proliferation and induces cell apoptosis via mediating PI3K/AKT/mTOR pathway.

Background To investigate the role of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) on cell growth of intrahepatic cholangiocarcinoma (ICC). Methods Western blotting was performed to detect the expression of VEGF in tumour tissues and paired normal tissues from twelve patients with ICC. ICC cell line Huh28 was treated with exogenous recombinant human VEGF (rhVEGF). Cell growth was evaluated by cell counting, proliferation was detected by BrdU cell proliferation assay, and apoptosis was detected by flow cytometry. The expression of VEGF receptors VEGFR1/VEGFR2 in Huh28 cells after rhVEGF treatment was detected by Western blotting. VEGFR1 and VEGFR2 were blocked by specific antibodies, and cell apoptosis was examined by apoptosis-ELISA assay. The stably knockdown VEGFR2 cell line Huh28-shVEGFR2 (experimental group) and the control cell line Huh28-shNC (control group) were established using shRNA lentivirus. Subcutaneous tumour models in ten nude mice with Huh28-shVEGFR2 and Huh28-shNC were used to observe tumour growth. Results The protein expression of VEGF was up-regulated in ICC tumour tissues than in matched normal tissues (P Conclusions VEGF promotes ICC cells growth through inhibiting apoptosis of ICC cells in a VEGFR2-dependent signalling pathway.

Background Progressive fibrosis, functional liver failure and liver cancer are the central liver-related outcomes of nonalcoholic steatohepatitis (NASH). However, these pathologies are notoriously difficult to achieve in mouse models. We, therefore, performed a direct, quantitative comparison of hepatic fibrosis progression in well-defined methionine- and choline-deficient (MCD) and choline-deficient, amino-acid defined (CDAA) diets with various fat content (10–60% by calories) in C57Bl/6J and BALB/c mice. Methods MCD or CDAA diets were formulated with a fat content ranging from 10 to 60% by calories, and fed C57Bl6 and BALB/c mice for up to 24 weeks. Hepatic fibrosis progression was assessed using standardized histological and biochemical methods. Results MCD feeding resulted in moderate fibrosis with an up to 2-fold increase in total hepatic collagen content as determined via hydroxyproline (HYP) at week 8 and progressive weight loss, irrespective of dietary fat. In contrast, CDAA-fed mice did not lose weight and developed fibrosis starting from week 4. Dietary fat intake dose-dependently increased the ductular reaction and fibrosis in C57Bl/6J mice on the CDAA diet. Longitudinal analysis of CDAA with 60% fat (HF-CDAA) feeding revealed pronounced ductular reaction and perisinusoidal bridging fibrosis (C57Bl/6J>BALB/c), with a 7-fold HYP increase at week 12, that showed limited spontaneous reversibility. At 24 weeks, HF-CDAA mice developed signs of cirrhosis with severe pan-lobular ‘chicken wire’ fibrosis, 10-fold HYP increases, regenerative nodules, portal hypertension and elevated serum bilirubin and ammonia levels; 80% of mice (8/10) developed multiple glypican-3- and/or glutamine synthetase-positive hepatocellular carcinomas (HCC). Conclusions In conclusion, the 60% HF-CDAA feeding in C57Bl/6J mice was identified as an optimal model of steatohepatitis with robust fibrosis and ductular proliferations that progress to cirrhosis and HCC within 24 weeks. This robust model will aid the testing of interventions and drugs for severe NASH.

Incomplete radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) could initiate malignant transition. Patient-derived xenograft (PDX) mice model was established to investigate the effect of VEGF pathway in incomplete RFA of HCC with high fidelity. Cancer stem cell markers and metastatic markers were increased after incomplete RFA, with increased VEGFR1 and decreased VEGFR2 expression. In vitro experiments revealed sublethal heat treatment promoted migration ability of HepG2, HCCLM3, and SMMC7721 cells, which coincided with enhanced ability of sphere formation and up-regulation of VEGFR1, CD133, CD44, and EpCAM. Moreover, HCC cells secreted more VEGF after heat-treatment. VEGF promoted migration and enhanced stemness of HCC cells, which could not be suppressed by VEGFR2 inhibitor. PIGF, the ligand of VEGFR1, significantly increased migration and stemness of HCC cells. Blocking VEGFR1 reduced heat-induced enhancement of migration and stemness, whereas inhibition of VEGFR2 could not. In conclusion, VEGFR1 plays a critical role in sublethal heat treatment-induced enhancement of migration and stemness in HCC, suggesting that VEGFR1 may serve as a potential and promising therapeutic target for preventing recurrence after RFA.

Additional file 2: Figure S1. The effect of apatinib plus irradiation on cell cycle progression. SMMC-7721, MHCC-97H, HCCLM3 and Hep-3B cells were treated with or without apatinib for 24 h prior to exposure to 4 Gy irradiation. After 12 h, cells were collected for cell cycle analysis through flow cytometry. The radiation-induced G2/M-phase arrest was further enhanced by combination treatment in SMMC-7721 cell line, while such effect didn’t exist in other three cell lines.

Figure S1. One-way sensitivity analysis of the rate of disease recurrence with disease worsening in the FD group (a) and the LD group (b). The line above represents the therapy has a better expected value. The rate of the crossover point in (a) is 0.0271, and the crossover point in (b) is 0.0371. (DOCX 11978 kb)

Stress-induced phosphoprotein 1 (STIP1) is an adaptor protein that bridges between HSP70 and HSP90 folding and a secretory protein which regulates malignant cell growth. However, the role of STIP1 in hepatocellular carcinoma (HCC) remains unknown. Here, we found high expression of STIP1 in tumors was associated with worse overall survival (41.3 vs 62.7 months, P 0.001) in 231 HCC patients. STIP1 was overexpressed in HCC tissues compared to adjacent non-tumor liver tissue (64.9% vs 4.0% P 0.001), and serum STIP1 levels of HCC patients were elevated compared to healthy controls (P 0.001). Mechanistically, STIP1 promoted HCC growth through PI3K-AKT-dependent anti-apoptotic pathway. STIP1 mediated cell growth in an autocrine fashion, which could be suppressed either by neutralizing extracellular STIP1 or by knocking down intracellular STIP1. In xenograft mouse model, knockdown of STIP1 significantly reduced tumor growth (P 0.001). In conclusion, STIP1 is upregulated in HCC and associated with poor clinical prognosis. Blocking STIP1 activity suppresses HCC cell growth, providing the rationale for STIP1 as a potential therapeutic target in HCC.

An interlaboratory study was conducted in China to validate the modified AOAC Official Method 2001.03 for the determination of total dietary fiber (TDF) in foods containing resistant maltodextrin (RMD), which will be adopted as the National Standard Method of China. The kind of buffer solution, the volume of filtrate evaporation, the volume of eluent for desalting and residual solution after evaporation, etc. were modified, which had been proved to have acceptable accuracy and precision in the routine assay. TDF contents in 3 representative foods and 2 kinds of RMD ingredient (i.e., NUTRIOSE 06 and NUTRIOSE 10) were measured using the modified method in 6 eligible laboratories representing commercial, industrial, and governmental laboratories in China. The results of the interlaboratory study indicated that the intralaboratory repeatability, interlaboratory reproducibility, and precision of the modified method are adequate for reliable analysis of TDF in food containing RMD, as well as resistant dextrin. Compared to AOAC Official Method 2001.03, the modified method is time- and cost-saving.