Prevalence of Steatotic Liver Disease Among US Adults with Rheumatoid Arthritis.

BACKGROUND: The prevalence of steatotic liver disease (SLD) among patients with rheumatoid arthritis (RA) remains largely unknown. AIMS: To investigate the prevalence of SLD and liver fibrosis among patients with RA. METHODS: We utilized data from the United States (US)-based National Health and Nutrition Examination Survey (NHANES) 2017-2020 cycle. After applying established sample weights, we estimated the age-adjusted prevalence of SLD and its subclassifications (CAP ≥ 285 dB/m), high-risk NASH (FAST score) and liver fibrosis (LSM) among participants with self-reported RA. Multivariable logistic regression was performed to identify independent risk factors for metabolic dysfunction associated SLD (MASLD), high-risk NASH and fibrosis, respectively, among participants with RA. We present adjusted odds ratios (aORs) and 95% confidence intervals (CIs). RESULTS: Age-adjusted prevalence of MASLD among US adults with RA was 34.91% (95% CI: 24.02-47.65%). We also found that the age-adjusted prevalence of high-risk NASH (FAST score > 0.35) and significant fibrosis (LSM > 8.6 kPa) was 12.97% (95% CI: 6.89-23.07%) and 10.35% (95% CI: 5.55-18.48%), respectively. BMI ≥ 30 kg/m2, (aOR 6.23; 95% CI: 1.95-19.88), diabetes (aOR 5.90; 95% CI: 1.94-17.94), and dyslipidemia (aOR 2.83; 95% CI: 1.12-7.11) were independently associated with higher odds of MASLD among participants with RA. Diabetes (aOR 19.34; 95% CI: 4.69-79.70) was also independently associated with high-risk NASH. CONCLUSIONS: The prevalence of MASLD, high-risk NASH, and liver fibrosis among patients with RA is equal or higher than the general population. Future studies of large cohorts are needed to substantiate the role of systemic inflammation in the pathophysiology of MASLD.

Prevalence and Characteristics of Nonalcoholic Fatty Liver Disease and Fibrosis in People Living With HIV Monoinfection: A Systematic Review and Meta-analysis.

BACKGROUND AND AIMS: Liver disease remains a leading cause of morbidity and mortality among people living with HIV (PLWH). Emerging data suggest that PLWH are at high risk for developing nonalcoholic fatty liver disease (NAFLD). The aim of this review is to examine the current literature and provide an accurate estimate of the prevalence of NAFLD, nonalcoholic steatohepatitis (NASH), and fibrosis, and identify potential risk factors for NAFLD in PLWH. METHODS: We searched PubMed and Embase databases to identify studies reporting the prevalence of NAFLD and/or fibrosis in PLWH monoinfection. We performed a random effects meta-analysis of proportions to estimate the pooled prevalence of NAFLD, NASH, and fibrosis among PLWH monoinfection. We also examined potential risk factors for NAFLD by comparing characteristics of PLWH monoinfection with and without NAFLD. RESULTS: A total of 43 studies, reporting data for 8230 patients, met our eligibility criteria and were included in the meta-analysis. Based on imaging studies the overall pooled prevalence of NAFLD and moderate liver fibrosis (METAVIR ≥ F2) among PLWH monoinfection was 33.9% (95% confidence interval [CI], 29.67%-38.39%), and 12.00% (95% CI, 10.02%-14.12%), respectively. Based on biopsy studies, prevalence of NASH and significant liver fibrosis (stage ≥F2 on histology) was 48.77% (95% CI, 34.30%-63.34%) and 23.34% (95% CI, 14.98%-32.75%), respectively. Traditional metabolic syndrome and HIV-related factors were associated with NAFLD in PLWH. CONCLUSIONS: Our study confirms that the burden of NAFLD, NASH, and fibrosis is high among PLWH monoinfection. Prospective longitudinal studies are needed to delineate NAFLD, NASH, and fibrosis risk factors, and identify early interventions and new therapies for NAFLD in this population.

Impact of prior HBV, HAV, and HEV infection on non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. The association between prior hepatitis B virus (HBV), hepatitis A virus (HAV), hepatitis E virus (HEV) infection and NAFLD remains unclear. We utilized the 2017-2020 National Health and Nutrition Examination Survey (NHANES) and performed multivariable logistic regression analyses to examine the association of prior HBV, HAV and HEV infection with NAFLD, as well as high risk non-alcoholic steatohepatitis (NASH) and liver fibrosis. Our analysis included 2565 participants with available anti-HBc serology results, 1480 unvaccinated participants with anti-HAV results, and 2561 participants with anti-HEV results. Among participants with NAFLD, the age-adjusted prevalence of prior HBV, HAV and HEV infection was 3.48%, 32.08% and 7.45%, respectively. Prior infection with HBV, HAV and HEV was not associated with NAFLD (cut-off 285 dB/m) [aOR: 0.99 (95% CI, 0.77-1.29), 1.29 (95% CI, 0.95-1.75), and 0.94 (95% CI, 0.70-1.27), respectively] or high-risk NASH [aOR 0.72 (95% CI, 0.45-1.17), 0.92 (95% CI, 0.55-1.52), and 0.89 (95% CI, 0.41-1.94), respectively]. Participants with anti-HBc and anti-HAV seropositivity were more likely to have significant fibrosis [aOR: 1.53 (95% CI, 1.05-2.23) and 1.69 (95% CI, 1.16-2.47), respectively]. The odds of significant fibrosis are 53%, and 69% greater for participants with prior history of HBV and HAV infection. Healthcare providers should prioritize vaccination efforts and employ a tailored approach to NAFLD in patients with prior viral hepatitis and especially HBV or HAV infection to limit disease-related outcomes.

Ten-Eleven Translocation 1 Promotes Malignant Progression of Cholangiocarcinoma With Wild-Type Isocitrate Dehydrogenase 1.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly lethal disease without effective therapeutic approaches. The whole-genome sequencing data indicate that about 20% of patients with CCA have isocitrate dehydrogenase 1 (IDH1) mutations, which have been suggested to target 2-oxoglutarate (OG)-dependent dioxygenases in promoting CCA carcinogenesis. However, the clinical study indicates that patients with CCA and mutant IDH1 have better prognosis than those with wild-type IDH1, further complicating the roles of 2-OG-dependent enzymes. APPROACH AND RESULTS: This study aimed to clarify if ten-eleven translocation 1 (TET1), which is one of the 2-OG-dependent enzymes functioning in regulating 5-hydroxymethylcytosine (5hmC) formation, is involved in CCA progression. By analyzing The Cancer Genome Atlas (TCGA) data set, TET1 mRNA was found to be substantially up-regulated in patients with CCA when compared with noncancerous bile ducts. Additionally, TET1 protein expression was significantly elevated in human CCA tumors. CCA cells were challenged with α-ketoglutarate (α-KG) and dimethyl-α-KG (DM-α-KG), which are cosubstrates for TET1 dioxygenase. The treatments with α-KG and DM-α-KG promoted 5hmC formation and malignancy of CCA cells. Molecular and pharmacological approaches were used to inhibit TET1 activity, and these treatments substantially suppressed 5hmC and CCA carcinogenesis. Mechanistically, it was found that knockdown of TET1 may suppress CCA progression by targeting cell growth and apoptosis through epigenetic regulation. Consistently, targeting TET1 significantly inhibited CCA malignant progression in a liver orthotopic xenograft model by targeting cell growth and apoptosis. CONCLUSIONS: Our data suggest that expression of TET1 is highly associated with CCA carcinogenesis. It will be important to evaluate TET1 expression in CCA tumors before application of the IDH1 mutation inhibitor because the inhibitor suppresses 2-hydroxyglutarate expression, which may result in activation of TET, potentially leading to CCA malignancy.

A Predictive Model for the Diagnostic and Therapeutic Yield of Colonoscopy Performed for Lower Gastrointestinal Bleeding.

BACKGROUND: Although colonoscopy is routinely performed for the management of lower gastrointestinal bleeding (LGIB), the quality of evidence supporting its use is poor and its yield for active bleeding or malignancy is low in practice. METHODS: We conducted a retrospective analysis of all adult patients who underwent colonoscopies for LGIB at our hospital system between January 1, 2015, and December 31, 2019. A statistical model was built on a cohort of 5195 cases using multiple logistic regressions to predict the detection of various colonoscopy findings and the use of different colonoscopy maneuvers. The model was converted into a risk scoring system, named the TYPICAL Index, and was validated against a separate cohort of 914 cases. RESULTS: Active bleeding was only seen in 3.8% of colonoscopies performed for LGIB and endoscopic hemostasis was applied in 43.7% of actively bleeding lesions. Malignant-appearing lesions were detected in 2.5% of LGIB procedures and all cases of proximal colonic mass were identified in subjects age above 60. The TYPICAL Index, derived from age, gender, hemoglobin, creatinine, international normalized ratio, and prior colonoscopy for LGIB has a concordance statistic of 0.71 and a negative predictive value of 93.8% for potential bleeding sources requiring hemostasis, malignant-appearing lesions, or active bleeding on colonoscopy when applied using a threshold of 6. CONCLUSIONS: Active bleeding and malignancies are rarely encountered and endoscopic hemostasis is seldom required during colonoscopy performed for LGIB. We developed and validated a risk scoring system to identify cases of low predicted diagnostic and therapeutic yield and to guide clinical decision-making.

Targeting Aspartate Beta-Hydroxylase with the Small Molecule Inhibitor MO-I-1182 Suppresses Cholangiocarcinoma Metastasis.

BACKGROUND: Cholangiocarcinoma is a devastating disease with a 2% 5-year survival if the disease has spread outside the liver. The enzyme aspartate beta-hydroxylase (ASPH) has been demonstrated to be highly expressed in cholangiocarcinoma but not in normal bile ducts and found to stimulate tumor cell migration. In addition, it was found that targeting ASPH inhibits cholangiocarcinoma malignant progression. However, it is not clear whether targeting ASPH with the small molecule inhibitor MO-I-1182 suppresses cholangiocarcinoma metastasis. The current study aims to study the efficacy of MO-I-1182 in suppressing cholangiocarcinoma metastasis. METHODS: The analysis was performed in vitro and in vivo with a preclinical animal model by using molecular and biochemical strategies to regulate ASPH expression and function. RESULTS: Knockdown of ASPH substantially inhibited cell migration and invasion in two human cholangiocarcinoma cell lines. Targeting ASPH with a small molecule inhibitor suppressed cholangiocarcinoma progression. Molecular mechanism studies demonstrated that knockdown of ASPH subsequently suppressed protein levels of the matrix metalloproteinases. The ASPH knockdown experiments suggest that this enzyme may modulate cholangiocarcinoma metastasis by regulating matrix metalloproteinases expression. Furthermore, using an ASPH inhibitor in a rat cholangiocarcinoma intrahepatic model established with BED-Neu-CL#24 cholangiocarcinoma cells, it was found that targeting ASPH inhibited intrahepatic cholangiocarcinoma metastasis and downstream expression of the matrix metalloproteinases. CONCLUSION: ASPH may modulate cholangiocarcinoma metastasis via matrix metalloproteinases expression. Taken together, targeting ASPH function may inhibit intrahepatic cholangiocarcinoma metastasis and improve survival.

Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

ASPH-notch Axis guided Exosomal delivery of Prometastatic Secretome renders breast Cancer multi-organ metastasis.

BACKGROUND: Aspartate ß-hydroxylase (ASPH) is silent in normal adult tissues only to re-emerge during oncogenesis where its function is required for generation and maintenance of malignant phenotypes. Exosomes enable prooncogenic secretome delivering and trafficking for long-distance cell-to-cell communication. This study aims to explore molecular mechanisms underlying how ASPH network regulates designated exosomes to program development and progression of breast cancer. METHODS: Stable cell lines overexpressing or knocking-out of ASPH were established using lentivirus transfection or CRISPR-CAS9 systems. Western blot, MTT, immunofluorescence, luciferase reporter, co-immunoprecipitation, 2D/3-D invasion, tube formation, mammosphere formation, immunohistochemistry and newly developed in vitro metastasis were applied. RESULTS: Through physical interactions with Notch receptors, ligands (JAGs) and regulators (ADAM10/17), ASPH activates Notch cascade to provide raw materials (especially MMPs/ADAMs) for synthesis/release of pro-metastatic exosomes. Exosomes orchestrate EMT, 2-D/3-D invasion, stemness, angiogenesis, and premetastatic niche formation. Small molecule inhibitors (SMIs) of ASPH's ß-hydroxylase specifically/efficiently abrogated in vitro metastasis, which mimics basement membrane invasion at primary site, intravasation/extravasation (transendothelial migration), and colonization/outgrowth at distant sites. Multiple organ-metastases in orthotopic and tail vein injection murine models were substantially blocked by a specific SMI. ASPH is silenced in normal adult breast, upregulated from in situ malignancies to highly expressed in invasive/advanced ductal carcinoma. Moderate-high expression of ASPH confers more aggressive molecular subtypes (TNBC or Her2 amplified), early recurrence/progression and devastating outcome (reduced overall/disease-free survival) of breast cancer. Expression profiling of Notch signaling components positively correlates with ASPH expression in breast cancer patients, confirming that ASPH-Notch axis acts functionally in breast tumorigenesis. CONCLUSIONS: ASPH-Notch axis guides particularly selective exosomes to potentiate multifaceted metastasis. ASPH's pro-oncogenic/pro-metastatic properties are essential for breast cancer development/progression, revealing a potential target for therapy.

NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands.

The submandibular salivary gland (SMG), a major site of persistent infection for many viruses, contains a large NK cell population. Using NFIL3-deficient mice, PLZF reporter/fate mapping mice, and mixed bone marrow chimeras, we identified two distinct populations of NK cells in the SMG. Although phenotypically unique, the main population relies on NFIL3, but not PLZF, for development and, therefore, is developmentally similar to the conventional NK cell subset. In contrast, we found that approximately one quarter of the SMG NK cells develop independently of NFIL3. Interestingly, NFIL3-independent SMG tissue-resident NK (trNK) cells are developmentally distinct from liver trNK cells. We also demonstrated that the SMG NK cell hyporesponsive phenotype during murine CMV infection is tissue specific and not cell intrinsic. In contrast, NFIL3-independent SMG trNK cells are intrinsically hyporesponsive. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK-like cells with distinct phenotypes.

Endocrine sensitivity of estrogen receptor-positive breast cancer is negatively correlated with aspartate-ß-hydroxylase expression.

Although prognostic markers for early estrogen receptor (ER)-positive breast cancer have been extensively developed, predictive markers for adjuvant endocrine therapy are still lacking. Focusing on the mechanisms underlying endocrine resistance, we investigated whether the endocrine sensitivity of ER-positive breast cancer cells was correlated with the expression of aspartate-ß-hydroxylase (ASPH), which is involved in the development of hepatocellular carcinoma. ASPH expression in ER-positive and tamoxifen-resistant breast cancer cells was upregulated by the MAPK and phosphoinositide-3 kinase (PI3K) pathways, which both play pivotal roles in endocrine resistance. In the clinical setting, ASPH expression was negatively correlated with recurrence-free survival of luminal B breast cancer patients that received adjuvant endocrine therapy, but not in patients that did not receive adjuvant endocrine therapy. Luminal B breast cancer is one of the intrinsic molecular subtypes identified by the Prediction Analysis of Microarray 50 (PAM50) multiple gene classifier, and because of its poor response to endocrine therapy, chemotherapy in addition to endocrine therapy is generally required after surgical resection. Our results suggest that the endocrine sensitivity of luminal B breast cancer can be assessed by examining ASPH expression, which promotes the consideration of a prospective study on the association between ASPH expression at the mRNA and protein levels in luminal B breast cancer and subsequent response to endocrine therapy.

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor.

UNLABELLED: Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG cells than in HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. A low HBsAg/HBeAg ratio by ccHBV-infected HepG2/NTCP cells was attributable to dimethyl sulfoxide (DMSO) in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells after HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the cytomegalovirus promoter, was markedly increased by DMSO treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates and will help identify additional factors on virion surface promoting attachment to hepatocytes. IMPORTANCE: Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen after infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethyl sulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethyl sulfoxide could increase NTCP protein level despite transcriptional control by a cytomegalovirus promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.

Aspartate ß-hydroxylase modulates cellular senescence through glycogen synthase kinase 3ß in hepatocellular carcinoma.

UNLABELLED: Aspartate ß-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second-generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3ß and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3ß and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3ß, enhanced p16 expression in tumor cells, and promoted cellular senescence. CONCLUSIONS: We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC.

Restoration of Wnt/ß-catenin signaling attenuates alcoholic liver disease progression in a rat model.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is characterized by the development of fatty liver, alcoholic hepatitis, fibrosis and cirrhosis. However, the underlying mechanism(s) associated with progression remains elusive. Pro-inflammatory cytokines have been implicated in ALD progression due to pro-apoptotic effects on hepatocytes. Wnt/ß-catenin signaling recently has been shown to promote inflammation and apoptosis, suggesting that activation of this signaling pathway may modulate ALD progression. The current study was designed to test whether pharmacological activation of Wnt/ß-catenin signaling altered ALD development and progression in a rat model. METHODS: Adult male Long Evans rats were fed with isocaloric liquid diets containing 0% or 37% ethanol for 8 weeks, and also treated with Wnt agonist during the last 3 weeks of the feeding regimen. Liver and blood samples were subjected to histology, TUNEL assay, immunoblot analysis, real-time quantitative PCR, and alanine transaminase (ALT) assay. RESULTS: Wnt/ß-catenin signaling was negatively correlated with Foxo3A expression and reduced steatosis, cellular injury and apoptosis in ALD rats. Mutation experiments demonstrated that Foxo3A was critical for modulating these effects. Activation of Wnt/ß-catenin signaling suppressed Foxo3A-induced apoptosis through upregulation of serum/glucocorticoid regulated kinase 1 (SGK1). Moreover, pharmacological restoration of Wnt/ß-catenin signaling reduced ALD progression in vivo. CONCLUSIONS: Wnt/ß-catenin signaling plays a protective role in ALD progression via antagonizing Foxo3A-induced apoptosis, and activation of the Wnt/ß-catenin signaling cascade attenuates ALD progression.

A cell-surface ß-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-ß-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of ß-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. CONCLUSIONS: These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced ß-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

Impaired virion secretion by hepatitis B virus immune escape mutants and its rescue by wild-type envelope proteins or a second-site mutation.

Hepatitis B virus immune escape mutants have been associated with vaccine failure and reinfection of grafted liver despite immune prophylaxis, but their biological properties remain largely unknown. Transfection of 20 such mutants in a human hepatoma cell line identified many with severe impairment in virion secretion, which can be rescued to various extents by coexpression of wild-type envelope proteins or introduction of a novel glycosylation site. Consistent with their role in maintaining intra- or intermolecular disulfide bonds, cysteine residues within the "a" determinant are critical for virion secretion.

Therapeutic reversal of chronic alcohol-related steatohepatitis with the ceramide inhibitor myriocin.

Alcohol-related liver disease (ALD) is associated with steatohepatitis and insulin resistance. Insulin resistance impairs growth and disrupts lipid metabolism in hepatocytes. Dysregulated lipid metabolism promotes ceramide accumulation and oxidative stress, leading to lipotoxic states that activate endoplasmic reticulum (ER) stress pathways and worsen inflammation and insulin resistance. In a rat model of chronic alcohol feeding, we characterized the effects of a ceramide inhibitor, myriocin, on the histopathological and ultrastructural features of steatohepatitis, and the biochemical and molecular indices of hepatic steatosis, insulin resistance and ER stress. Myriocin reduced the severity of alcohol-related steatohepatitis including the abundance and sizes of lipid droplets and mitochondria, inflammation and architectural disruption of the ER. In addition, myriocin-mediated reductions in hepatic lipid and ceramide levels were associated with constitutive enhancement of insulin signalling through the insulin receptor and IRS-2, reduced hepatic oxidative stress and modulation of ER stress signalling mechanisms. In conclusion, ceramide accumulation in liver mediates tissue injury, insulin resistance and lipotoxicity in ALD. Reducing hepatic ceramide levels can help restore the structural and functional integrity of the liver in chronic ALD due to amelioration of insulin resistance and ER stress. However, additional measures are needed to protect the liver from alcohol-induced necroinflammatory responses vis-à-vis continued alcohol abuse.

Strain-dependent differences for suppression of insulin-stimulated glucose uptake in skeletal and cardiac muscle by ethanol.

BACKGROUND: Chronic ethanol (EtOH) consumption impairs the ability of insulin to suppress hepatic glucose production in a strain-dependent manner, with hepatic insulin resistance being greater in Long-Evans (LE) than Sprague-Dawley (SD) rats. We assessed whether strain differences exist for whole-body and tissue glucose uptake under basal and insulin-stimulated conditions and whether they were associated with coordinate strain-dependent elevations in muscle cytokines. METHODS: Male rats (160 g) were provided the Lieber-DeCarli EtOH-containing (36% total energy) diet or pair-fed a control diet for 8 weeks. Rats were studied in the basal state or during a euglycemic hyperinsulinemic clamp, and whole-body glucose flux assessed using (3) H-glucose and in vivo tissue glucose uptake by (14) C-2-deoxyglucose. RESULTS: EtOH impaired whole-body insulin-mediated glucose uptake (IMGU) more in SD than LE rats. This difference was due to impaired IMGU by gastrocnemius and heart in EtOH-fed SD versus LE rats. However, decreased IMGU in adipose tissue (epididymal and perirenal) produced by EtOH was comparable between strains. EtOH-induced insulin resistance in muscle from SD rats was associated with reduced AKT and AS160 phosphorylation and plasma membrane-localized GLUT4 protein as well as enhanced phosphorylation of c-Jun N-terminal kinase (JNK) and IRS-1 (S307), changes which were absent in muscle from LE rats. EtOH increased tumor necrosis factor alpha (TNFα) mRNA in gastrocnemius and fat under basal conditions in both SD and LE rats; however, hyperinsulinemia decreased TNFα in skeletal muscle from LE, but not SD rats. Interleukin (IL)-6 mRNA in gastrocnemius was increased under basal conditions and increased further in response to insulin in SD rats, but no EtOH- or insulin-induced change was detected in muscle IL-6 of LE rats. CONCLUSIONS: These data indicate strain-dependent differences in EtOH-induced IMGU in skeletal and cardiac muscle, but not fat, associated with sustained increases in TNFα and IL-6 mRNA and JNK activation and decreased plasma membrane GLUT4 in response to insulin.

Chronic ethanol diet increases regulatory T-cell activity and inhibits hepatitis C virus core-specific cellular immune responses in mice.

AIM: Chronic ethanol consumption is associated with persistent hepatitis C viral (HCV) infection. This study explores the role of the host cellular immune response to HCV core protein in a murine model and how chronic ethanol consumption alters T-cell regulatory (Treg) populations. METHODS: BALB/c mice were fed an isocaloric control or ethanol liquid diet. Dendritic cells (DC) were isolated after expansion with a hFl3tL-expression plasmid and subsequently transfected with HCV core protein. Core-containing DC (1 × 10(6) ) were s.c. injected (×3) in mice every 2 weeks. Splenocytes from immunized mice were isolated and stimulated with HCV core protein to measure generation of viral antigen-specific Treg, as well as secretion of interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-4. Cytotoxicity was measured by lactate dehydrogenase release from HCV core-expressing syngeneic SP2/19 myeloma cells. RESULTS: Splenocytes from mice immunized with ethanol-derived and HCV core-loaded DC exhibited significantly lower in vitro cytotoxicity compared to mice immunized with HCV core-loaded DC derived from isocaloric pair-fed controls. Stimulation with HCV core protein triggered higher IL-2, TNF-α and IL-4 release in splenocytes following immunization with core-loaded DC derived from controls as compared to chronic ethanol-fed mice. Splenocytes derived from mice immunized with core-loaded DC isolated from ethanol-fed mice exhibited a significantly higher CD25(+) FOXP3(+) and CD4(+) FOXP3(+) Treg population. CONCLUSION: These results suggest that immunization with HCV core-containing DC from ethanol-fed mice induces an increase in the CD25(+) FOXP3(+) and CD4(+) FOXP3(+) Treg population and may suppress HCV core-specific CD4(+) and CD8(+) T-cell immune responses.

Elevated 2-oxoglutarate antagonizes DNA damage responses in cholangiocarcinoma chemotherapy through regulating aspartate beta-hydroxylase.

Cholangiocarcinoma (CCA) is resistant to systemic chemotherapies that kill malignant cells mainly through DNA damage responses (DDRs). Recent studies suggest that the involvement of 2-oxoglutarate (2-OG) dependent dioxygenases in DDRs may be associated with chemoresistance in malignancy, but how 2-OG impacts DDRs in CCA chemotherapy remains elusive. We examined serum 2-OG levels in CCA patients before receiving chemotherapy. CCA patients are classified as progressive disease (PD), partial response (PR), and stable disease (SD) after receiving chemotherapy. CCA patients classified as PD showed significantly higher serum 2-OG levels than those defined as SD and PR. Treating CCA cells with 2-OG reduced DDRs. Overexpression of full-length aspartate beta-hydroxylase (ASPH) could mimic the effects of 2-OG on DDRs, suggesting the important role of ASPH in chemoresistance. Indeed, the knockdown of ASPH improved chemotherapy in CCA cells. Targeting ASPH with a specific small molecule inhibitor also enhanced the effects of chemotherapy. Mechanistically, ASPH modulates DDRs by affecting ATM and ATR, two of the major regulators finely controlling DDRs. More importantly, targeting ASPH improved the therapeutic potential of chemotherapy in two preclinical CCA models. Our data suggested the impacts of elevated 2-OG and ASPH on chemoresistance through antagonizing DDRs. Targeting ASPH may enhance DDRs, improving chemotherapy in CCA patients.

Role of NADPH oxidase NOX5-S, NF-κB, and DNMT1 in acid-induced p16 hypermethylation in Barrett's cells.

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.