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OBJECTIVE: To determine trends and sexual risk behaviors associated with anal intercourse among college women over an 8-year period. METHODS: A sexual activity questionnaire was used to collect data from 813 students enrolled in a women's health course. RESULTS: Thirty-two percent of the women had engaged in anal intercourse, and this measure was consistent across time. Women who had engaged in anal intercourse were significantly younger at first intercourse and had a greater lifetime number of partners and more reported STIs. CONCLUSION: This study underscores the importance of expanding our understanding of sexual behaviors of college women and openly addressing anal intercourse as a part of the sexual repertoire of college women. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICI) have improved metastatic melanoma outcomes; however, toxicities, such as hepatitis, can be dose-limiting or even fatal. 1 Systemic glucocorticoids and antimetabolite immunosuppressive medications remain the mainstay of treatment for ICI-hepatitis, but options for patients refractory to these therapies are limited. 2 Herein we present 3 cases of glucocorticoid-refractory ICI-hepatitis treated with tofacitinib, an inhibitor of Janus kinase (JAK) 1 and 3. These patients represent consecutive patients referred to the Massachusetts General Hospital Severe Immunotherapy Complications service who were determined by our experts to have treatment failure with systemic glucocorticoid and antimetabolite combination therapy between August 2022 and September 2023. 3 These were the only 3 patients managed by the Severe Immunotherapy Complications service who were treated with tofacitinib for ICI-hepatitis during that time., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Hematology is a diagnostic tool used to evaluate the health status of horses. However, breed differences are often not considered., Objectives: The objective was to compare complete blood count variables among Warmbloods, Thoroughbreds, and stock horses (SH)., Methods: Ninety-six healthy horses were grouped by breed (Warmbloods, Thoroughbreds, and SH). Samples were collected through venipuncture for complete blood count analysis. One-way ANOVA with Tukey's tests or Kruskal-Wallis with Dunn's post hoc tests were used to compare hematologic variables among groups., Results: Warmbloods had a significantly lower total white blood cell (WBC) count (6.08 ± 1.11 × 10 9 /L) and lymphocyte count (1.76 ± 0.41 × 10 9 /L) than Thoroughbreds (7.28 ± 1.45; 2.28 ± 5.16 × 10 9 /L, respectively; P < .001) and SH (7.21 ± 1.18 × 10 9 /L, P < .01; 2.10 ± 5.17 × 10 9 /L; P < .05). Warmbloods had a significantly lower red blood cell count (7.7 ± 0.8 × 10 12 /L) and higher mean corpuscular volume (MCV, 49.4 ± 2.2 fL) than Thoroughbreds (8.42 ± 1.2 × 10 12 /L, P < .01; 47.3 ± 3.0 fL). Warmbloods had lower MCVs than SH (49.4 ± 2.2 vs 51.2 ± 2.6 fL). The mean cell hemoglobin concentration (MCHC) was higher in Warmbloods (35.0, 33.8-36.2 g/dL) and Thoroughbreds (34.9, 33.4-35.7 g/dL) than in SH breeds (34.0, 33.4-35.4 g/dL; P < .001, both). Total protein concentrations were significantly lower in Thoroughbreds (67, 59-80 g/L) compared with SH (71, 64-83 g/dL) (P < .05)., Conclusions: Warmbloods had decreased WBC and lymphocyte counts compared with Thoroughbreds and SH, and Thoroughbreds had increased red blood cell counts. Thoroughbreds had lower total protein concentrations than SH. Clinicians should consider breed differences when interpreting hematologic values., (© 2024 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of American Society for Veterinary Clinical Pathology.)

Diagnostic challenges continue to impede development of effective therapies for successful management of alcohol-associated hepatitis (AH), creating an unmet need to identify noninvasive biomarkers for AH. In murine models, complement contributes to ethanol-induced liver injury. Therefore, we hypothesized that complement proteins could be rational diagnostic/prognostic biomarkers in AH. Here, we performed a comparative analysis of data derived from human hepatic and serum proteome to identify and characterize complement protein signatures in severe AH (sAH). The quantity of multiple complement proteins was perturbed in liver and serum proteome of patients with sAH. Multiple complement proteins differentiated patients with sAH from those with alcohol cirrhosis (AC) or alcohol use disorder (AUD) and healthy controls (HCs). Serum collectin 11 and C1q binding protein were strongly associated with sAH and exhibited good discriminatory performance among patients with sAH, AC, or AUD and HCs. Furthermore, complement component receptor 1-like protein was negatively associated with pro-inflammatory cytokines. Additionally, lower serum MBL associated serine protease 1 and coagulation factor II independently predicted 90-day mortality. In summary, meta-analysis of proteomic profiles from liver and circulation revealed complement protein signatures of sAH, highlighting a complex perturbation of complement and identifying potential diagnostic and prognostic biomarkers for patients with sAH.

Background: Intrahepatic cholestasis of pregnancy is associated with a significant risk of stillbirth, which contributes to variation in clinical management. Recent Society for Maternal-Fetal Medicine guidance recommends delivery at 36 weeks of gestation for patients with serum bile acid levels of >100 μmol/L, consideration for delivery between 36 and 39 weeks of gestation stratified by bile acid level, and against preterm delivery for those with clinical features of cholestasis without bile acid elevation., Objective: This study aimed to investigate institutional practices before the publication of the new delivery timing recommendations to establish the maternal and neonatal effects of late preterm, early-term, and term deliveries in the setting of cholestasis., Study Design: This study examined maternal and neonatal outcomes of 441 patients affected by cholestasis delivering 484 neonates in a 4-hospital system over a 30-month period. Logistic and linear regression analyses were performed to assess neonatal outcomes concerning peak serum bile acid levels at various gestational ages controlling for maternal comorbidities, multiple pregnancies, and neonatal birthweight., Results: With the clinical flexibility afforded by the new guidelines, pregnancy prolongation to term may have been achieved in 91 patients (21%), and 286 patients (74%) with bile acid elevation could have delivered at a later gestational age. Preterm deliveries of patients with bile acid levels of >10 μmol/L were associated with higher rates of neonatal intensive care unit admission and adverse neonatal outcomes than early-term deliveries., Conclusion: Study data suggested an opportunity for education and practice change to reflect current Society for Maternal-Fetal Medicine guidelines in efforts to reduce potential neonatal morbidities associated with late preterm deliveries among pregnancies affected by cholestasis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Despite being a leading cause of advanced liver disease, alcohol-associated liver disease (ALD) has no effective medical therapies. The circulating proteome, which comprises proteins secreted by different cells and tissues in the context of normal physiological function or in the setting of disease and illness, represents an attractive target for uncovering novel biology related to the pathogenesis of ALD. In this work, we used the aptamer-based SomaScan proteomics platform to quantify the relative concentration of over 1300 proteins in a well-characterized cohort of patients with the spectrum of ALD. We found a distinct circulating proteomic signature that correlated with ALD severity, including over 600 proteins that differed significantly between ALD stages, many of which have not previously been associated with ALD to our knowledge. Notably, certain proteins that were markedly dysregulated in patients with alcohol-associated hepatitis were also altered, to a lesser degree, in patients with subclinical ALD and may represent early biomarkers for disease progression. Taken together, our work highlights the vast and distinct changes in the circulating proteome across the wide spectrum of ALD, identifies potentially novel biomarkers and therapeutic targets, and provides a proteomic resource atlas for ALD researchers and clinicians.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-β1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-β1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-β1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-β1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-β1-induced OCT4/Nanog-dependent pathway., (Copyright © 2022 by The American Association of Immunologists, Inc.)

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain., (© 2018 by the American Association for the Study of Liver Diseases.)

Background: Coinfection with human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV)-related liver fibrosis. Macrophages are triggered during both viral infections and are critical in liver inflammation/fibrogenesis. Liver fibrosis strongly associates with serum soluble CD163 (sCD163, a macrophage activation marker); comprehensive evaluation in HIV/HCV coinfection is lacking., Methods: We retrospectively analyzed sCD163 (enzyme-linked immunosorbent assay) and hepatic CD163 (immunofluorescent CD163/CD68 costaining) in patients infected with HIV/HCV, HCV, or HIV, pre- and post-antiviral therapy., Results: sCD163 was significantly higher in HIV/HCV compared to either monoinfection, and decreased following successful antiviral therapy, although did not fully normalize. In HIV/HCV, sCD163 was associated with necroinflammation, Ishak fibrosis scores, and noninvasive fibrosis scores. We observed a novel trend whereby sCD163 levels progressively increase with increasing Ishak fibrosis score, peaking at stage 4, above which levels plateaued. Periportal CD163+ macrophage frequency was also higher with increasing fibrosis score. When stratified by fibrosis stage, sCD163 levels were higher in HIV/HCV than HCV but only in individuals with mild to moderate fibrosis., Conclusions: In HIV/HCV, increasing sCD163 levels accompanied periportal CD163+ macrophage enrichment in mild to moderate fibrosis, but not in established cirrhosis, suggesting that sCD163 is a dynamic biomarker of fibrogenesis rather than accumulated fibrosis. Our findings implicate HIV-related macrophage activation in accelerated fibrosis progression in HIV/HCV coinfection.

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections., (Copyright © 2018 Duan et al.)

Doxycycline is an oral tetracycline antibiotic that has been associated with upper gastrointestinal (GI) mucosal injury. Recently, characteristic vascular degeneration has been reported in the stomach and duodenum in patients with doxycycline-induced injury. Fourteen patients who underwent upper GI endoscopy for nonspecific symptoms and were found to have doxycycline-induced gastric and esophageal injury are described. Most patients showed characteristic vascular injury. A control group of gastric erosions and esophageal ulcers showed no cases with the characteristic vascular changes. Clinical, endoscopic, and pathologic features of doxycycline-induced upper GI tract injury are reviewed, with an emphasis on vascular injury.

Aim: To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection., Methods: In this study, we utilize a gene editing tool, transcription activator-like effector nucleases (TALENs), to generate human hepatoma cells with a stable genetic deletion of APOB to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDA-approved antisense inhibitor of apoB100, on HCV using in vitro cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method., Results: We found that apoB100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALEN-mediated gene deletion of apoB ( APOB KO ), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apoB did not play a role in HCV entry or replication. However, the virus produced by APOB KO cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus., Conclusion: ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to declare.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells., Conclusion: HIV accentuates an HCV-driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFβ1 up-regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951-1968)., (© 2016 by the American Association for the Study of Liver Diseases.)

Background & Aims: Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response., Methods: We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection., Results: We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties., Conclusions: Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation., Lay Summary: In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB, and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

Unlabelled: The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway., Importance: Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background &/aims: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing., Methods: We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model., Results: We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect., Conclusions: The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV-infected cells the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B., Conclusion: The kinetics of gene induction are fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV., (© 2014 by the American Association for the Study of Liver Diseases.)

The hepatitis C virus (HCV) requires elements of host lipid metabolism for every step in the viral life cycle. Clinically, it has long been observed that patients with chronic hepatitis C have lower nonhigh-density lipoprotein cholesterol, and these levels rise after successful treatment. The HCV itself circulates as a highly lipidated lipoviral particle, which closely resembles very low-density lipoprotein (VLDL). Several required coentry factors for the virus to gain access to the hepatocytes have been described, and several, including SRB1, LDL-R, and the NPC1L1 receptors, are important receptors for lipoprotein and cholesterol uptake. Inside the cell, the virus induces lipogenesis, and specifically induces the master regulator sterol response element binding protein. Viral replication then requires the concerted efforts of viral proteins combined with several host factors involved in cholesterol synthesis. The virus is then packaged alongside the cellular machinery for VLDL production. The complex interplay highlights pathways of hepatic steatosis and unveils drug targets for the treatment of HCV., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background & Aims: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis., Methods: The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-β signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression., Results: Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-β signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-β signaling., Conclusions: Thus reciprocal regulation between HCV and SNARK promotes TGF-β signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

TRPA1, a member of the transient receptor potential (TRP) family of cation channels, has mainly been characterized as a chemosensory protein in neuronal cells. TRPA1 is activated by toxic or irritating volatile agents like allyl isothiocyanate (AITC), tear gas, formalin, or cigarette smoke. To date, little is known about a function of TRPA1 in non-neuronal cells in the respiratory system and even less regarding a possible role in cancer biology. Here, we show that TRPA1 is expressed in a panel of human small cell lung cancer (SCLC) cell lines. Of note, TRPA1 mRNA was also significantly higher expressed in tumor samples of SCLC patients as compared to non-SCLC tumor samples or non-malignant lung tissue. Stimulation of SCLC cells with AITC led to a rise of the intracellular calcium concentration. This calcium response was inhibited by TRPA1 antagonists. Furthermore, AITC or formalin stimulated ERK1/2 in TRPA1-expressing HEK293 cells and in SCLC cells via a Src- and calcium-dependent mechanism. More importantly, TRPA1 activation in SCLC cells prevented apoptosis induced by serum starvation and thus promoted cell survival, an effect which could be blocked by inhibition of TRPA1 or ERK1/2. Vice versa, down-regulation of TRPA1 severely impaired anchorage-independent growth of SCLC cells. Since TRPA1 appears to play a pivotal role for cell survival in SCLC cells we propose that this channel could represent a promising target for therapeutic interventions. Furthermore, our data suggest that exogenous, inhalable activators of TRPA1 could be able to exert tumor promoting effects in SCLC cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.

Development of robust cell culture models for hepatitis C viral infection has greatly increased our understanding of this virus and its life cycle. This knowledge has led to the development of many drugs that target specific elements of viral replication, including viral proteins and host factors required for replication. The NS3/4A serine protease inhibitors were the first of these to be used in the clinic, and reagents that target other elements of the viral lifecycle are in advanced stages of clinical development. These include new NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors, NS5A inhibitors, and host-directed antivirals, such as cyclophilin inhibitors. Alternative interferons with possibly improved tolerability, specifically interferon-λ1 (interleukin-29), are also under development. These new reagents against hepatitis C virus should lead to highly effective, well-tolerated, and likely interferon-sparing therapies in the next several years., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady-state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for antiviral design.

In recent years, some genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with hepatitis C viral containment, treatment response, and disease progression. IL28B is a gene on chromosome 19, coding for interferon-λ3, and two polymorphisms upstream of this the gene have been strongly associated with clinical outcomes after treatment for the hepatitis C virus (HCV). The IL28B polymorphisms have additionally been associated with spontaneous clearance. The mechanism has yet to be clearly defined, but appears to involve differential responsiveness to interferon signaling between the favorable and unfavorable genotypes. ITPA is a gene on chromosome 20, coding for inosine triphosphatase, and polymorphisms on this gene have been associated with ribavirin-induced hemolytic anemia. Functional variants of ITPA have been identified that have decreased enzymatic activity, which appear to protect against anemia. Finally, PNPLA3 polymorphisms were initially described as predictors of nonalcoholic fatty liver disease, but have recently been associated with disease progression in HCV., (© Thieme Medical Publishers.)

Background: Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library., Methods: To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatin's anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication., Conclusions: Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.

To examine the validity of the deuterium (D) incorporation technique for measurement of human cholesterol synthesis rates, D uptake from D2O into cholesterol was compared to sterol balance in 13 subjects each under three controlled diet settings. Subjects (age 62 +/- 3.6 yr, body weight 74 +/- 4.0 kg, BMI 27 +/- 1.4) consumed weight maintenance diets enriched in either corn oil, beef tallow, or stick corn oil margarine over a 5-week period. During the final week of the study period, subjects were given 1.2 g/D2O per kg body water. D enrichment was measured in plasma water and total cholesterol over 24 h. Also, during the final week, dietary intake and fecal elimination rates of cholesterol were assessed over one 6-day period to calculate sterol balance. There was no significant difference (t = 0.858, P = 0.397) between D incorporation into cholesterol (1,183 +/- 92 mg/day) and sterol balance (1,316 +/- 125 mg/day). Among diets, net cholesterol biosynthesis measured by D incorporation agreed (r = 0.745, P = 0.0001) with values derived from sterol balance. The degree of association between methods was not influenced by the wide range of fatty acid composition of the diet fat. These data demonstrate the utility of the simple, non-restrictive deuterium incorporation method as a reliable means of determining cholesterol biosynthesis in free-living humans.

The coulometric electrolytic principle is utilized for the measurement of moisture sorbed on sintered thorium dioxide. Moisture is removed from the sample by heating and is swept by dry argon gas through a hygrometer. The resulting hygrometer output is converted into frequency pulses and counted cumulatively. The instrument is calibrated by use of standard hydrated chemicals. The number of counts/mu;g of water obtained from standards compares favourably with the theoretical number of counts/mu;g calculated from performance specifications of the components employed. The error is <5% for 1-350mu;g of water. A high sensitivity and low operating blank recommends application of this method to materials other than thorium dioxide, having very low moisture contents.