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Background & Aims Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. Methods To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. Results We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. Conclusions These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach., Graphical abstract

Background and aims Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. Approach and results Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. Conclusions Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

This is an editorial about non-invasive fibrosis markers in patients with NASH and type 2 diabetes mellitus.

The cardiac glycoside digoxin was identified as a potent suppressor of pyruvate kinase isoform 2-hypoxia-inducible factor-α (PKM2-HIF-1α) pathway activation in liver injury mouse models via intraperitoneal injection. We have assessed the therapeutic effects of digoxin to reduce nonalcoholic steatohepatitis (NASH) by the clinically relevant oral route in mice and analyzed the cellular basis for this effect with differential involvement of liver cell subsets. C57BL/6J male mice were placed on a high-fat diet (HFD) for 10 wk and started concurrently with the gavage of digoxin (2.5, 0.5, 0.125 mg/kg twice a week) for 5 wk. Digoxin significantly reduced HFD-induced hepatic damage, steatosis, and liver inflammation across a wide dosage range. The lowest dose of digoxin (0.125 mg/kg) showed significant protective effects against liver injury and sterile inflammation. Consistently, digoxin attenuated HIF-1α sustained NLRP3 inflammasome activation in macrophages. We have reported for the first time that PKM2 is upregulated in hepatocytes with hepatic steatosis, and digoxin directly improved hepatocyte mitochondrial dysfunction and steatosis. Mechanistically, digoxin directly bound to PKM2 and inhibited PKM2 targeting HIF-1α transactivation without affecting PKM2 enzyme activation. Thus, oral digoxin showed potential to therapeutically inhibit liver injury in NASH through the regulation of PKM2-HIF-1α pathway activation with involvement of multiple cell types. Because of the large clinical experience with oral digoxin, this may have significant clinical applicability in human NASH. NEW & NOTEWORTHY This study is the first to assess the therapeutic efficacy of oral digoxin on nonalcoholic steatohepatitis (NASH) in a high-fat diet (HFD) mouse model and to determine the divergent of cell type-specific effects. Oral digoxin reduced liver damage, steatosis, and inflammation in HFD mice. Digoxin attenuated hypoxia-inducible factor (HIF)-1α axis-sustained inflammasome activity in macrophages and hepatic oxidative stress response in hepatocytes via the regulation of PKM2-HIF-1α axis pathway activation. Oral digoxin may have significant clinical applicability in human NASH.

Shc expression rises in human nonalcoholic steatohepatitis (NASH) livers, and Shc-deficient mice are protected from NASH–thus Shc inhibition could be a novel therapeutic strategy for NASH. Idebenone was recently identified as the first small-molecule Shc inhibitor drug. We tested idebenone in the fibrotic methionine-choline deficient (MCD) diet and the metabolic fast food diet (FFD) mouse models of NASH. In the fibrotic MCD NASH model, idebenone reduced Shc expression and phosphorylation in peripheral blood mononuclear cells and Shc expression in the liver; decreased serum alanine aminotransferase and aspartate aminotransferase; and attenuated liver fibrosis as observed by quantitative polymerase chain reaction (qPCR) and hydroxyproline quantification. In the metabolic FFD model, idebenone administration improved insulin resistance, and reduced inflammation and fibrosis shown with qPCR, hydroxyproline measurement, and histology. Thus, idebenone ameliorates NASH in two mouse models. As an approved drug with a benign safety profile, Idebenone could be a reasonable human NASH therapy.

Type 2 diabetes is clinically associated with progressive necroinflammation and fibrosis in nonalcoholic steatohepatitis (NASH). Advanced glycation end-products (AGEs) accumulate during prolonged hyperglycemia, but the mechanistic pathways that lead to accelerated liver fibrosis have not been well defined. In this study, we show that the AGEs clearance receptor AGER1 was downregulated in patients with NASH and diabetes and in our NASH models, whereas the proinflammatory receptor RAGE was induced. These findings were associated with necroinflammatory, fibrogenic, and pro-oxidant activity via the NADPH oxidase 4. Inhibition of AGEs or RAGE deletion in hepatocytes in vivo reversed these effects. We demonstrate that dysregulation of NRF2 by neddylation of cullin 3 was linked to AGER1 downregulation and that induction of NRF2 using an adeno-associated virus–mediated approach in hepatocytes in vivo reversed AGER1 downregulation, lowered the level of AGEs, and improved proinflammatory and fibrogenic responses in mice on a high AGEs diet. In patients with NASH and diabetes or insulin resistance, low AGER1 levels were associated with hepatocyte ballooning degeneration and ductular reaction. Collectively, prolonged exposure to AGEs in the liver promotes an AGER1/RAGE imbalance and consequent redox, inflammatory, and fibrogenic activity in NASH.

A variety of signaling pathways contribute to initiating the pathologies associated with fibrotic disease. Galectins are a group of beta-galactoside-binding proteins that are involved in a host of cellular processes, some of which contribute to fibrosis in different organs. Accumulating evidence indicates that of these, Galectin-3 (Gal-3) is a pathogenic mediator in fibrotic diseases in many different organs. The atypical Gal-3 contains a single carbohydrate recognition domain (CRD) attached to an N-terminal peptide sequence that putatively nucleates the formation of oligomers that can form lattice networks when bound to multiple cellular glycans. Pharmacological or genetic knockdown of Gal-3 has been shown to inhibit fibrosis in several organs, and thus has emerged as a valid therapeutic target. This chapter will review the structure and function of Gal-3 and attempt to validate the important role it plays in fibrosis. In addition, the current state of pharmaceutical discovery of Gal-3 inhibitors will be outlined and discussed in the context of fibrotic disease of the heart, liver, lungs and kidneys. A discussion of the challenges facing future Gal-3 inhibitor development for targeting fibrosis will also be included.

Author(s): Tepper, Clifford G; Dang, Julie HT; Stewart, Susan L; Fang, Dao M; Wong, Kimberly A; Liu, Stephenie Y; Davis, Ryan R; Dao, Doan Y; Gregg, Jeffrey P; Torok, Natalie J; Chen, Moon S | Abstract: BACKGROUND:An exploratory study was performed to determine the prevalence of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs78409 [G] allele among the Hmong as a risk factor for nonalcoholic fatty liver disease (NAFLD). NAFLD/nonalcoholic steatohepatitis is the world's most common chronic liver disease and is expected to replace viral hepatitis as the leading cause of cirrhosis and potential precursor to hepatocellular carcinoma (HCC). Of all populations in California, the Hmong experience the highest risk of death from HCC and the highest prevalence of metabolic syndrome risk factors among Asians that predispose them to NAFLD. Here a genetic explanation was sought for the high rates of chronic liver disease among the Hmong. The literature pointed to the PNPLA3 rs738409 [G] allele as a potential genetic culprit. METHODS:Cell-free DNA was isolated from 26 serum samples previously collected in community settings. Quantitative polymerase chain reaction-based single-nucleotide polymorphism (SNP) genotyping was performed with a validated TaqMan SNP genotyping assay, and results were analyzed with TaqMan Genotyper software. RESULTS:The PNPLA3 rs738409 [CgG] variant occurred at a frequency of 0.46 (12 of 26; 95% confidence interval, 0.27-0.67). This carrier rate would rank the Hmong as the third highest population in the 1000 Genomes Project. CONCLUSIONS:Although this small sample size limits the generalizability, the high frequency rates of this allele along with the presence of metabolic syndrome risk factors warrant further studies into the etiology of NAFLD among the Hmong. Cancer 2018;124:1583-9. © 2018 American Cancer Society.

Apoptosis, or programmed cell death is a regulated event, which plays a major role in maintaining homeostasis in the biliary epithelium. The disruption of normal apoptotic pathways can result in different disease states: (1) increases in apoptosis result in decreasing number of biliary epithelial cells, as observed in different cholestatic conditions; and (2) a decrease and/or dysregulation of apoptosis can lead to development of cholangiocarcinoma. This chapter reviews briefly the current knowledge on different apoptotic pathways in the biliary epithelium, the alteration of these pathways during different disease processes, and finally summarizes current concepts on dysregulation of apoptosis and biliary carcinogenesis.

Background and aims Cardiovascular disease (CVD) is a leading cause of mortality in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship of leptin-to-adiponectin (L/A) ratio with hepatic steatosis and arterial stiffness in NAFLD. Methods The subjects were 871 Japanese adults who participated in a health survey. Dietary intake, body composition, lipid profile, serum interleukin-6 (IL-6), leptin, and adiponectin were analyzed. NAFLD was defined as fatty liver on ultrasonography in the absence of other causes of steatosis. Arterial stiffness was evaluated by the brachial-ankle pulse wave velocity (baPWV). Results The subjects with NAFLD had a greater body mass index (BMI) and body fat percentage (BFP); a higher intake of daily energy (kcal) and carbohydrates; and a higher prevalence of hypertension, diabetes, and hyperlipidemia. The subjects with NAFLD had higher serum leptin and lower serum adiponectin concentrations and a higher L/A ratio than subjects without NAFLD. The L/A ratio increased with increasing severity of steatosis. The L/A ratio showed positive correlations with BMI and BFP, and a negative correlation with age. Women had higher L/A ratio and BFP levels than men regardless of the presence or absence of NAFLD. There was a weak positive correlation between baPWV and severity of steatosis. BaPWV was strongly correlated with age, while no relation was found between baPWV and L/A ratio. IL-6 level was correlated with baPVW and age, while the correlation between Il and 6 level and L/A ratio was very weak. The L/A ratio was correlated with triglycerides and the ratio of total cholesterol to high-density lipoprotein-cholesterol. Conclusion L/A ratio and arterial stiffness were associated with the severity of steatosis, whereas there was no correlation between L/A ratio and arterial stiffness in NAFLD. These findings suggest that not only leptin and adiponectin but also other factors might be involved in the pathogenesis for atherosclerosis in NAFLD.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-β) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4−/−) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4−/− and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4−/− mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-β pathway. In fact, levels of active TGF-β1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-β1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects., Graphical abstract Image 1, Highlights • General or hepatocyte-specific Nox4 deletion accelerates mice liver regeneration. • Increased hepatocyte proliferation is observed, coincident with higher Myc expression. • RNA-seq analysis reveals a role for Myc in a node of regulation of gene expression. • Transcriptional and functional attenuation of the TGF-β1 pathway is observed in vivo. • In vitro, Nox4 deleted hepatocytes maintain the growth inhibitory response to TGF-β1.

Alcoholic liver disease is one of the most prevalent liver diseases worldwide, and a major cause of morbidity and mortality. Alcoholic hepatitis is a severe form of liver injury in patients with alcohol abuse, can present as an acute on chronic liver failure associated with a rapid decline in liver synthetic function, and consequent increase in mortality. Despite therapy, about 30%–50% of patients with severe alcoholic hepatitis eventually die. The pathogenic pathways that lead to the development of alcoholic hepatitis are complex and involve oxidative stress, gut dysbiosis, and dysregulation of the innate and adaptive immune system with injury to the parenchymal cells and activation of hepatic stellate cells. As accepted treatment approaches are currently limited, a better understanding of the pathophysiology would be required to generate new approaches that improve outcomes. This review focuses on recent advances in the diagnosis, pathogenesis of alcoholic hepatitis and novel treatment strategies.

There is an urgent need to develop antifibrotic therapies for chronic liver disease, and clarify which endpoints in antifibrotic trials will be acceptable to regulatory agencies. The American Association for the Study of Liver Diseases sponsored an endpoints conference to help accelerate the efficient testing of antifibrotic agents and develop recommendations on clinical trial design for liver fibrosis. In this review, we summarize the salient and novel elements of this conference and provide directions for future clinical trial design. The article follows the structure of the conference and is organized into five areas: (1) antifibrotic trial design; (2) preclinical proof-of-concept studies; (3) pharmacological targets, including rationale and lessons to learn; (4) rational drug design and development; and (5) consensus and recommendations on design of clinical trials in liver fibrosis. Expert overviews and collaborative discussions helped to summarize the key unmet needs and directions for the future, including: (1) greater clarification of at-risk populations and study groups; (2) standardization of all elements of drug discovery and testing; (3) standardization of clinical trial approaches; (4) accelerated development of improved noninvasive markers; and (5) need for exploration of potential off-target toxicities of future antifibrotic drugs.

Sterile inflammation after tissue damage is a ubiquitous response, and yet has the highest amplitude in the liver. This has major clinical consequences with alcoholic and non-alcoholic steatohepatitis (ASH and NASH) accounting for the majority of liver disease in industrialized countries, and both lacking therapy. Requirements for sustained sterile inflammation include increased oxidative stress, and activation of the HIF-1α signaling pathway. We demonstrate the ability of digoxin, a cardiac glycoside, to protect from liver inflammation and damage in ASH and NASH. Digoxin was effective in maintaining cellular redox homeostasis, and suppressing HIF-1α pathway activation. A proteomic screen revealed that digoxin binds pyruvate kinase M2 (PKM2), and independent of PKM2 kinase activity results in chromatin remodeling and down-regulation of HIF-1α transactivation. These data identify PKM2 as a mediator and therapeutic target for regulating liver sterile inflammation, and demonstrate a novel role for digoxin which can effectively protect the liver from ASH and NASH.

The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.

Background & Aims Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. Methods A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. Results HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B–induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. Conclusions The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI’s Gene Expression Omnibus (GEO accession number: GSE98153)., Graphical abstract

Recruitment of inflammatory cells is a major feature of alcoholic liver injury however; the signals and cellular sources regulating this are not well defined. C-C chemokine receptor type 2 (CCR2) is expressed by active hepatic stellate cells (HSC) and is a key monocyte recruitment signal. Activated HSC are also important sources of hydrogen peroxide resulting from the activation of NADPH oxidase 4 (NOX4). As the role of this NOX in early alcoholic liver injury has not been addressed, we studied NOX4-mediated regulation of CCR2/CCL2 mRNA stability. NOX4 mRNA was significantly induced in patients with alcoholic liver injury, and was co-localized with αSMA-expressing activated HSC. We generated HSC-specific NOX4 KO mice and these were pair-fed on alcohol diet. Lipid peroxidation have not changed significantly however, the expression of CCR2, CCL2, Ly6C, TNFα, and IL-6 was significantly reduced in NOX4HSCKO compared to fl/fl mice. NOX4 promoter was induced in HSC by acetaldehyde treatment, and NOX4 has significantly increased mRNA half-life of CCR2 and CCL2 in conjunction with Ser221 phosphorylation and cytoplasmic shuttling of HuR. In conclusion, NOX4 is induced in early alcoholic liver injury and regulates CCR2/CCL2 mRNA stability thereby promoting recruitment of inflammatory cells and production of proinflammatory cytokines.

ObjectivesTo investigate risk factor patterns and the simultaneous occurrence of multiple risk factors in the viral, metabolic and lifestyle domains among Asian Americans, who have had the highest mortality rates from hepatocellular carcinoma (HCC).SettingSacramento County, California, USA.ParticipantsEligible participants were county residents ages 18 and older who had not been screened for chronic hepatitis B virus (HBV) and were born in a CDC-defined endemic area or whose parent was born in that area. Of 1004 enrolled, 917 were foreign-born Chinese (130 women, 94 men), Hmong (133 women, 75 men), Korean (178 women, 90 men) or Vietnamese (136 women, 81 men) with complete risk factor data.Primary and secondary outcome measuresWe tested participants for HBV and chronic hepatitis C virus (HCV); measured haemoglobin A1c and waist circumference; and recorded self-reported history of diabetes, hypertension, alcohol use and smoking status. We identified risk factor patterns using cluster analysis and estimated gender-specific age-standardised prevalence rates.ResultsWe identified four patterns: (1) viral (chronic HBV or HCV); (2) lifestyle (current smoker or alcohol user, no viral); (3) metabolic (≥2 metabolic, no lifestyle or viral); and (4) lower risk (≤1 metabolic, no lifestyle or viral). Vietnamese men (16.3%, 95% CI 7.4% to 25.3%) and Hmong women (15.1%, 95% CI 7.8% to 22.5%) had the highest viral pattern prevalence. Hmong women had the highest metabolic (37.8%, 95% CI 29.8% to 45.9%), and Vietnamese men the highest lifestyle (70.4%, 95% CI 59.1% to 81.7%) pattern prevalence. In multiple domains, Hmong men and women were most likely to have viral+metabolic risk factors (men: 14.4%, 95% CI 6.0% to 22.7%; women: 11.9%, 95% CI 5.6% to 18.3%); Vietnamese men were most likely to have lifestyle+viral (10.7%, 95% CI 2.7% to 18.8%), and lifestyle+metabolic but not viral (46.4%, 95% CI 34.4% to 58.5%) risk factors.ConclusionsEfforts to reduce HCC must comprehensively address multiple risk factors.Trial registration numberNCT02596438.

Fungal endophthalmitis is a rare but serious infection. In March 2012, several cases of probable and laboratory-confirmed fungal endophthalmitis occurring after invasive ocular procedures were reported nationwide. We identified 47 cases in 9 states: 21 patients had been exposed to the intraocular dye Brilliant Blue G (BBG) during retinal surgery, and the other 26 had received an intravitreal injection containing triamcinolone acetonide. Both drugs were produced by Franck’s Compounding Lab (Ocala, FL, USA). Fusarium incarnatum-equiseti species complex mold was identified in specimens from BBG-exposed case-patients and an unopened BBG vial. Bipolaris hawaiiensis mold was identified in specimens from triamcinolone-exposed case-patients. Exposure to either product was the only factor associated with case status. Of 40 case-patients for whom data were available, 39 (98%) lost vision. These concurrent outbreaks, associated with 1 compounding pharmacy, resulted in a product recall. Ensuring safety and integrity of compounded medications is critical for preventing further outbreaks associated with compounded products. Download MP3 Length: 1:42

Advanced glycation endproducts (AGEs) accumulate in patients with diabetes, yet the link between AGEs and inflammatory and fibrogenic activity in nonalcoholic steatohepatitis (NASH) has not been explored. Tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) is at the center of inflammatory processes. Because the main natural regulator of TACE activity is the tissue inhibitor of metalloproteinase 3 (Timp3), we hypothesized that AGEs induce TACE through nicotinamide adenine dinucleotide phosphate reduced oxidase 2 (NOX2); and the down-regulation of Sirtuin 1 (Sirt1)/Timp3 pathways mediate fibrogenic activity in NASH. The role of NOX2, Sirt1, Timp3, and TACE was evaluated in choline-deficient L-amino acid defined (CDAA) or Western diet (WD)-fed wild-type (WT) and NOX2−/− mice. To restore Timp3, mice were injected with adenovirus (Ad)-Timp3. Sirt1 and Timp3 expressions were studied in livers from NASH patients, and we found that their levels were significantly lower than in healthy controls. In WT mice on the CDAA or WD, Sirt1 and Timp3 expressions were lower, whereas production of reactive oxidative species and TACE activity significantly increased with an increase in active TNF-α production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) expression. NOX2−/− mice on the CDAA or WD had no significant change in Sirt1, Timp3, and TACE activity or the fibrosis markers assessed. In vitro, AGE exposure decreased Sirt1 and Timp3 in hepatic stellate cells by a NOX2-dependent pathway, and TACE was induced after exposure to AGEs. Conclusion: TACE activation is central to the pathogenesis of NASH and is mediated by AGEs through NOX2 induction and down-regulation of Sirt1/Timp3 pathways. (Hepatology 2013;58:1339–1348)

UnlabelledHepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels.ConclusionReduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.

Objective. To examine our experience with cytology and histology biopsy of the liver and to define methods for improvement of diagnosis of primary liver tumors.Methods. This include retrospective study of 189 biopsies of 185 liver masses for cytological or histological analysis. Patients were subdivided into two groups. Group 1 consisted of 124 suspected metastasis. Group 2 consisted of 61 suspected primary neoplasms. Biopsies were considered positive or equivocal. In equivocal cases, special stains were performed. In Group 2, cases were classified by contrast CT or MRI as to (I) classic HCC, (II) infiltrated HCC, or (Ill) equivocal.Results. Definitive diagnosis was obtained in 117/124 masses (94%) in Group 1, 48/61 masses (79%) in Group 2, and (Ill) equivocal 13 cases in Group II. In two equivocal cases in which special stains were performed, they were reclassified as HCC. In 8/13 cases, CT findings were consistent with HCC.Conclusion. Liver biopsies are useful in obtaining a definitive diagnosis of suspected metastatic liver disease. Biopsy results are less reliable in patients with suspected primary liver tumors. In these situations, strategies can include basing treatment on imaging criteria or use of newer special pathological stains.Advances in Knowledge. Use of newer special immunological stains improves accuracy in definitive diagnosis of primary liver tumors.

Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin-3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL-17 signaling. In liver tissues from patients with PBC and dnTGF-βRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis-associated speck-like protein was induced, with the downstream activation of caspase-1 and IL-1β. In wild-type hepatic macrophages, deoxycholic acid induced the association of Gal3 and NLRP3 with direct activation of the inflammasome, resulting in an increase in IL-1β. Downstream retinoid-related orphan receptor C mRNA, IL-17A, and IL-17F were induced. In Gal3-/- macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the pathogenesis of cholestatic liver injury, we generated dnTGF-βRII/galectin-3-/- (dn/Gal3-/-) mice, which showed impaired inflammasome activation along with significantly improved inflammation and fibrosis. Taken together, our data point to a novel role of Gal3 as an initiator of inflammatory signaling in autoimmune cholangitis, mediating the activation of NLRP3 inflammasome and inducing IL-17 proinflammatory cascades. These studies provide a rationale to target Gal3 in autoimmune cholangitis and potentially other cholestatic diseases.-Tian, J., Yang, G., Chen, H.-Y., Hsu, D. K., Tomilov, A., Olson, K. A., Dehnad, A., Fish, S. R., Cortopassi, G., Zhao, B., Liu, F.-T., Gershwin, M. E., Torok, N. J., Jiang, J. X. Galectin-3 regulates inflammasome activation in cholestatic liver injury.

Background The relationship between gut microbial community composition at the higher-taxonomic order level and local and systemic immunologic abnormalities in HIV disease may provide insight into how bacterial translocation impacts HIV disease. Methods Antiretroviral-naive patients with HIV underwent upper endoscopy before and 9 months after starting antiretroviral treatment. Duodenal tissue was paraffin-embedded for immunohistochemical analysis and digested for fluorescence activated cell sorting for T-cell subsets and immune activation (CD38+/HLA-DR+) enumeration. Stool samples were provided from patients and control subjects for comparison. Metagenomic microbial DNA was extracted from feces for optimized 16S ribosomal RNA gene (rDNA) real-time quantitative polymerase chain reaction assays designed to quantify panbacterial loads and the relative abundances of proinflammatory Enterobacteriales order and the dominant Bacteroidales and Clostridiales orders. Results Samples from 10 HIV subjects before initiating and from six subjects receiving antiretroviral treatment were available for analysis. There was a trend for a greater proportion of Enterobacteriales in HIV-positive subjects compared with control subjects (P = 0.099). There were significant negative correlations between total bacterial load and duodenal CD4 and CD8 T-cell activation levels (r = -0.74, P = 0.004 and r = -0.67, P = 0.013, respectively). The proportions of Enterobacteriales and Bacteroidales were significantly correlated with duodenal CD4 T-cell depletion and peripheral CD8 T-cell activation, respectively. Conclusions These data represent the first report of quantitative molecular and cellular correlations between total/universal and order-level gut bacterial populations and gastrointestinal-associated lymphoid tissue levels of immune activation in HIV-infected subjects. The correlations between lower overall 16S rDNA levels and tissue immune activation suggest that the gut microbiome may contribute to immune activation and influence HIV progression.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of young adults that is associated with significant morbidity and mortality. No effective medical therapy is available. Minocycline has been found to exert biological effects independent of its antimicrobial properties, including anti-inflammatory activities such as inhibition of inducible nitric oxide synthase, upregulation of interleukin 10, and direct suppressive effect on B- and T-cell function. Minocycline may also inhibit cell death pathways by reducing both proapoptotic and proinflammatory enzyme activation. We sought to investigate the safety and efficacy of minocycline among patients with PSC.We evaluated the efficacy of minocycline in patients with PSC in a pilot study. Sixteen patients with PSC were enrolled. Minocycline, 100 mg orally twice daily, was given for 1 year.A statistically significant improvement in serum alkaline phosphatase activity (330 U/l vs. 265 U/l, P=0.04) and Mayo risk score (0.55 vs. 0.02, P=0.05) occurred with treatment. Serum bilirubin and albumin remained essentially unchanged while on treatment.The results of this pilot study indicate that minocycline is reasonably well tolerated and potentially effective in patients with PSC. These findings might be explained by the anti-inflammatory and antiapoptotic properties of minocycline. Though the data presented are too preliminary to support the clinical use of minocycline in the treatment of PSC at this time, its use should be further investigated.

Leptin, a profibrogenic cytokine, plays an important role in the development of non-alcoholic steatohepatitis. Leptin also regulates immune responses, including macrophage phagocytic activity. Stellate cells are key elements in liver fibrogenesis, and previously we have demonstrated that phagocytosis of apoptotic bodies by stellate cells is profibrogenic. To study the effects of leptin on the phagocytic activity of hepatic stellate cells, we exposed both LX-2 cells and primary stellate cells to leptin, and we have observed increased phagocytic activity. In stellate cells isolated from Zucker (fa/fa) rats, the rate of phagocytosis was significantly decreased. To investigate the mechanism by which leptin induces phagocytosis, we focused on the role of Rho-guanosine triphosphate (GTP)-ases. We found that leptin induced the PI3K-dependent activation of Rac1, and that nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase activation was also implicated in this process. Leptin also induced RhoA activation and translocation to the phagosomes. Expression of the constitutive active Rac1 and RhoA both increased the phagocytic rate, whereas inhibition of the Rho-dependent kinase decreased the phagocytic activity. Conclusion: We describe a novel role of leptin in the fibrogenic process, the induction of phagocytosis of apoptotic bodies by hepatic stellate cells. The data provide strong evidence of a Rho-GTPase–mediated regulation of the cytoskeleton during stellate cell phagocytosis. Leptin-mediated phagocytic activity of stellate cells therefore could be an important mechanism responsible for progression of fibrosis in non-alcoholic steatohepatitis. (HEPATOLOGY 2008.)

Nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease in adults and children. It is one of the consequences of the current obesity epidemic, and can progress to nonalcoholic steatohepatitis (NASH), characterized by steatosis, inflammation, and progressive fibrosis, ultimately leading to cirrhosis and end-stage liver disease. The factors implicated in this progression are poorly understood. NASH is closely associated with obesity and the metabolic syndrome. Recent studies emphasize the role of insulin resistance, oxidative stress, lipid peroxidation, and cytokine release in the development of NASH. This review summarizes the current knowledge on the etiology and pathomechanism of NASH and the role of the metabolic syndrome in NASH development.

Reactive oxygen species are implicated in physiological signaling and cell fate decisions. In chronic liver diseases persistent and increased production of oxidative radicals drives a fibrogenic response that is a common feature of disease progression. Despite our understanding the biology of the main prooxidant enzymes, their targets, and antioxidant mechanisms in the liver, there is still lack of knowledge concerning their precise role in the pathogenesis of fibrosis. This review will examine the role of physiological redox signaling in the liver, provide an overview on recent advances in prooxidant and antioxidant pathways that are dysregulated during fibrosis, and highlight possible novel treatment targets.

Oxidative stress is a common feature observed in a wide spectrum of chronic liver diseases including viral hepatitis, alcoholic, and nonalcoholic steatohepatitis. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are emerging as major sources of reactive oxygen species (ROS). Several major isoforms are expressed in the liver, including NOX1, NOX2, and NOX4. While the phagocytic NOX2 has been known to play an important role in Kupffer cell and neutrophil phagocytic activity and inflammation, the nonphagocytic NOX homologues are increasingly recognized as key enzymes in oxidative injury and wound healing. In this review, we will summarize the current advances in knowledge on the regulatory pathways of NOX activation, their cellular distribution, and their role in the modulation of redox signaling in liver diseases.

Background & Aims Reactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis. Methods Liver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4 hepKO ) and NOX4 floxp+/+ C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase−mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C. Results Levels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase−mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity. Conclusions NOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

The basic patterns of thyroid hormones [thyroxine (T4) and 3,3',5-triiodothyronine (T3)] and the T4 and T3 responses induced by thyrotropin releasing hormone (TRH) are reported in captive female barn owls (Tyto alba) during the non-breeding period. The main findings of the study, conducted on a total of 10 owls, are as follow: (1) The thyroid gland of barn owl can be stimulated by the classical TRH stimulation test. (2) T3 response was much more pronounced both under cold (around 10°C) and warm (around 20°C) conditions, whereas T4 response ranged so widely that we could not point out any significant change in it. (3) Basal T3 plasma level was significantly (p = 0.036) higher in birds exposed to cold temperature, and they responded to TRH treatment with a lower plasma T3 elevation than the birds kept in a warm chamber. This pattern, however, cannot be explained by increased food intake, but is in agreement with the fact that enhanced T3 level may account for higher avUCP mRNA expression, which results in higher heat production on the cell level. From the results it is concluded that altering T3 plasma level plays a significant role in cold-induced thermoregulation.

Objectives. This study examined whether differences in heat alone, as opposed to public health interventions or other factors, accounted for the reduction in heat-related deaths and paramedic emergency medical service (EMS) runs between 1995 and 1999 during 2 heat waves occurring in Milwaukee, Wis. Methods. Two previously described prediction models were adapted to compare expected and observed heat-related morbidity and mortality in 1999 based on the city's 1995 experience. Results. Both models showed that heat-related deaths and EMS runs in 1999 were at least 49% lower than levels predicted by the 1995 relation between heat and heat-related deaths or EMS runs. Conclusions. Reductions in heat-related morbidity and mortality in 1999 were not attributable to differences in heat levels alone. Changes in public health preparedness and response may also have contributed to these reductions.

BACKGROUND: Porcine clotting factor has been used for more than 15 years to treat severe bleeding episodes in persons with hemophilia who have antibodies to human clotting factor. In 1996, QC procedures revealed for the first time the presence of porcine parvovirus (PPV) in the product. This report describes an investigation to determine the extent of product contamination and to evaluate past recipients of porcine clotting factor (Hyate:C, Speywood Biopharm) for evidence of PPV infection. STUDY DESIGN AND METHODS: Stored specimens from 22 lots of previously released Hyate:C were tested for the presence of PPV DNA by PCR and nested PCR assays. Serum specimens from 98 recipients of Hyate:C and 24 controls who did not receive Hyate:C were tested for PPV antibodies by an immunofluorescence assay. RESULTS: PPV DNA was detected in product from 21 of the 22 lots of Hyate:C, primarily by nested PCR testing. In contrast, none of the serum specimens from the 98 Hyate:C recipients tested positive for PPV IgG antibodies. CONCLUSION: The risk of human disease from percutaneous exposure to low levels of PPV seems to be low. Nevertheless, the theoretical risk of human infection with PPV has led to manufacturing changes, including PCR screening of all porcine plasma, which are designed to eliminate this risk.

To better define the contribution of human parainfluenza viruses (HPIVs) to lower respiratory tract infection in adults, we tested acute- and convalescent-phase serum specimens from hospitalized adults participating in a population-based prospective study of lower respiratory tract infection during 1991-1992. We tested all available specimens from the epidemic seasons for each virus and approximately 300 randomly selected specimens from the corresponding off-seasons for antibodies to HPIV-1, HPIV-2, or HPIV-3. During the respective epidemic season, HPIV-1 infection was detected in 18 (2.5%) of 721 and HPIV-3 infection in 22 (3.1%) of 705 patients with lower respiratory tract infection. Only 2 (0.2%) of 1,057 patients tested positive for HPIV-2 infection. No HPIV-1 infections and only 2 (0.7% of 281 patients tested) HPIV-3 infections were detected during the off-seasons. HPIV-1 and HPIV-3 were among the four most frequently identified infections associated with lower respiratory tract infection during their respective outbreak seasons.

A 38-year-old woman with alcoholic cirrhosis was admitted with a recurrent lower extremity rash. She had presented 1 year prior with a history of alcohol abuse, abdominal pain, fatigue, and a violaceous rash over the abdomen and proximal lower extremities. A skin biopsy showed multiple small vessels with calcification of the outer wall suggesting calciphylaxis. The patient was treated with sodium thiosulfate 25 mg intravenously 3 times weekly and serial wound debridement. She had symptom improvement within 3 weeks and resolution of the rash. One year later, the patient presented with recurrence of the painful violaceous rash (Figure 1). She denied any alcohol use over the last year. She had not been receiving albumin infusions or blood transfusions, and her body mass index (BMI) was 22 kg/m2. Evidence of a healed skin graft was noted on her left thigh, with surrounding tender punctate erythema. Her calcium, phosphorus, parathyroid hormone, creatinine, and protein C and S levels were normal. An abdominal CT was significant for findings consistent with cirrhosis. The patient was started on sodium thiosulfate therapy and local wound care, and responded well without recurrence to date.

To look for genetic changes in human parvovirus B19 that might be associated with chronic infection, we sequenced B19 DNA obtained from serum specimens collected over an approximately 1-year period from a patient with systemic vasculitis. A comparison of the nucleotide sequences of the VP1/VP2 gene from four specimens revealed an abrupt change in the B19 genotype that coincided with initiation of intravenous immune globulin (IVIG) therapy. We suspect that one or more of the lots of IVIG administered to the patient were contaminated with B19. If true, this finding suggests that investigators must be careful in linking B19 infection to disease based on detection of B19 DNA in persons who have received multiple unit blood products. J. Med. Virol. 53:233–236, 1997. Published 1997 Wiley-Liss, Inc.1

Background. Rotavirus is the leading cause of severe pediatric gastroenteritis worldwide. A vaccine may soon be licensed for use in the United States to prevent this disease. To characterize US geographic and temporal trends in rotavirus activity, we made contour maps showing the timing of peak rotavirus activity. Methods. From July, 1991, through June, 1996, 79 laboratories participating in the National Respiratory and Enteric Virus Surveillance System reported on a weekly basis the number of stool specimens that tested positive for rotavirus. The peak weeks in rotavirus detections from each laboratory were mapped using kriging, a modeling technique originally developed for geostatis-tics. Results. During the 5-year period 118 716 fecal specimens were examined, of which 27 616 (23%) were positive for rotavirus. Timing of rotavirus activity varied by geographic location in a characteristic pattern in which peak activity occurred first in the Southwest from October through December and last in the Northeast in April or May. The Northwest exhibited considerable year-to-year variability (range, December to May) in the timing of peak activity, whereas the temporal pattern in the remainder of the contiguous 48 states was relatively constant. Conclusion. Kriging is a useful method for visualizing geographic and temporal trends in rotavirus activity in the United States. This analysis confirmed trends reported in previous years, and it also identified unexpected variability in the timing of peak rotavirus activity in the Northwest. The causes of the seasonal differences in rotavirus activity by region are unknown. Tracking of laboratory detections of rotavirus may provide an effective surveillance tool to assess the impact of a rotavirus vaccination campaign in the United States.

We describe an outbreak of 22 sternal surgical site infections following cardiac surgery, including 4 Gordonia infections. Possible operation room environmental contamination and suboptimal infection control practices regarding scrub attire may have contributed to the outbreak.

Liver fibrosis is a wound healing process, the end result of chronic liver injury elicited by different noxious stimuli. Activated hepatic stellate cells or myofibroblasts and portal myofibroblasts are considered as the main producers of the extracellular matrix in the liver. Upon liver injury the quiescent stellate cells transdifferentiate into myofibroblasts a process highlighted by the loss of vitamin A stores, upregulation of interstitial type collagens, smooth muscle α actin, matrix metalloproteinases, proteoglycans, and the induction of cell survival pathways. Activation of hepatic stellate cells is a result of a complex interplay between the parenchymal cells, immune cells, extracellular matrix mechanics and extrahepatic milieu such as the gut microbiome. In this review we will focus on the pathomechanism of stellate cell activation following chronic liver injury; with the aim of identifying possible treatment targets for anti-fibrogenic agents.

A suspected nosocomial outbreak of parvovirus B19 infection in a maternity ward was investigated in February 1994. Questionnaires were administered and sera collected from maternity ward staff (n = 91), other ward staff in the same hospital (n = 101), and maternity ward staff at a nearby hospital (n = 81). Blood donors (n = 265) were used as community controls. Recent infection (parvovirus B19 IgM positivity) in susceptible persons (parvovirus B19 IgG-negative or IgM-positive) was common among all 4 groups (23%-30%). This high rate of recent infection occurred during a large community outbreak of fifth disease. Environmental samples collected from a room where a stillborn parvovirus B19-infected fetus was delivered were positive for parvovirus B19 DNA. Thus, this suspected nosocomial outbreak actually reflected transmission outside the hospital, but contaminated environmental surfaces were identified as one potential source for transmission of parvovirus B19.

Reactive oxygen species (ROS) play a key role in chronic liver injury and fibrosis. Homologues of NADPH oxidases (NOXs) are major sources of ROS, but the exact role of the individual homologues in liver disease is unknown. Our goal was to determine the role of NOX4 in liver fibrosis induced by bile duct ligation (BDL) with the aid of the pharmacological inhibitor GKT137831, and genetic deletion of NOX4 in mice. GKT136731 was either applied for the full term of BDL (preventive arm), or starting at 10 days post-operatively (therapeutic arm). Primary hepatic stellate cells (HSC) from control mice with and without BDL were analyzed and the effect of NOX4 inhibition on HSC activation was also studied. FasL or TNFα/actinomycin D induced apoptosis was studied in wild type and NOX4−/− hepatocytes. Results: NOX4 was upregulated by a TGF-β/Smad3-dependent mechanism in HSC. Downregulation of NOX4 decreased ROS production and the activation of NOX4−/− HSC was attenuated. NOX4−/− hepatocytes were more resistant to FasL or TNFα/actinomycin D induced apoptosis. Similarly, after pharmacological NOX4 inhibition, ROS production, the expression of fibrogenic markers and hepatocyte apoptosis were reduced. NOX4 was expressed in human livers with stage 2–3 autoimmune hepatitis. Fibrosis was attenuated by the genetic deletion of NOX4. BDL mice gavaged with GKT137831 both in the preventive or the therapeutic arm displayed less ROS production; significantly attenuated fibrosis and decreased hepatocyte apoptosis. In conclusion, NOX4 plays a key role in liver fibrosis. GKT137831 is a potent inhibitor of fibrosis and hepatocyte apoptosis; therefore it is a promising therapeutic agent for future translational studies.

Non-alcoholic steatohepatitis (NASH) is one of the most common chronic liver diseases worldwide (1). Despite the large number of studies published in the field, the molecular signals triggering the progression of NASH from simple steatosis to necroinflammation are still poorly understood. One of the most important and early features of progressive NASH is lipoapoptosis of hepatocytes that creates a proinflammatory and fibrogenic environment (2, 3). The activation of c-jun N-terminal kinase 1(JNK1) in both hepatocytes and macrophages has been described as a key event in NASH (4, 5), thus the signals governing its induction need to be better elucidated. The article published by Ibrahim et al. (6) has focused on the role of the mixed-lineage kinase 3 (MLK3) as an important proximal JNK activator that has a major role in progressive liver injury in diet-induced NASH. Several studies demonstrated that MLK3/JNK activation plays an essential role in saturated fatty acid-induced insulin resistance and hepatocyte lipotoxicity (7–9). Exposure to free fatty acid induced MLK3/JNK in mouse embryonic fibroblasts, and the MLK3-/- cells displayed increased insulin sensitivity (8); the same trend was found in the MLK3-/- mice on high-fat diet (8, 10). In hepatocytes, palmitate induced the recruitment of cdc42/Rac1 and the activation of MLK3/JNK, leading to downstream ER stress signalling (7). In a different study, the palmitate-induced M1 macrophage polarization was diminished in the MLK3-/- cells (10). All of these suggest that MLK3/JNK signalling plays an important role in the progression of NASH. MLKs are MAPK-kinase kinases (MKKKs) that activate JNK and p38 signalling cascades via the MAPK kinases (MKKs) by either forming a homodimer or associating with Rho GTPases (11–13). The selective activation of MKKs and the downstream induction of JNK1/2 or p38 by MLKs are mediated by the MAPK scaffold proteins such as JNK-interacting proteins (JIPs) (13, 14). How these JIPs direct selective activation of their targets is still under investigation. JIP3 was shown to bind to MLK3/MKK7 inducing JNK1 activation in neurons (14–16). It is however, not clear which JIPs are predominant in the liver and whether they selectively mediate JNK1 and 2 activation. c-Jun N-terminal kinase activation plays a key role in saturated fatty acid-induced hepatocyte apoptosis (5), both in humans and in animal models (17–19). In HSC, phosphorylated-JNK1 directly mediates transdifferentiation contributing to fibrosis (20). MLK3/JNK/P38 activation in LX-2 cells has been observed when cells were stimulated with a PPARβ/δ ligand, leading to cell proliferation (21). JNK1 and JNK2 have differential effects in NASH: JNK1 mediates steatohepatitis and lipotoxicity, whereas JNK2 activation is more protective (22, 23). It is not yet known whether MLK3 has differential effects on JNK1 or JNK2 in the liver, and if so, how the differential induction is mediated. This study is in agreement with previous findings demonstrating that the MLK3-/- mice developed less hepatic steatosis, decreased liver injury, inflammation and fibrosis. The diet used in this study mimics the fast food diet consumed by humans inducing insulin resistance, steatohepatitis and fibrosis (24). It has been recently reported that compared to the WT mice, the global MLK3-deficient mice on high-fat diet displayed significantly less weight gain, and reduced macrophage infiltration in the adipose tissue and also decreased systemic inflammation (10). Interestingly, these mice had increased energy expenditure that could have accounted for the slower weight gain. The improved inflammation in the liver could be explained by the decreased macrophage recruitment and JNK inactivation in the MLK3-/- mice (10). Using the MLK2/3 double knockout mice on the high-fat diet model, Davis and colleagues showed that the obesity-resistant phenotype of these animals was related to the upregulation of the sympatho-adrenal system, as using a selective antagonist to the β3-adrenergic receptor prevented the increase in body temperature and decreased the expression of the adrenergic target genes (25). Distinct from the studies described above, the authors in this study chose high-fat diet combined with high fructose consumption. The mice in this study gained similar amount of weight in both treatment arms, and this could be attributed to the high fructose intake. As fibrosis was diminished in the MLK3-/- mice, it is likely that MLK3 in stellate cells was involved in their transdifferentiation, which has been observed in lung fibroblasts by Lin et al. (26). Macrophage polarization could also be affected by MLK3 (10); and there is evidence showing that MLK3 interacts with TLR signalling by directly binding to Myd88 (27). The dominant cell type and mechanisms for the pro-inflammatory and fibrogenic activity of MLK3 in the liver would require further investigation, and could be addressed in the future by the generation of conditional knockout mice.