Liver biopsy in the post-hepatitis C virus era in Japan.

In Japan, liver biopsies were previously crucial in evaluating the severity of hepatitis caused by the hepatitis C virus (HCV) and diagnosing HCV-related hepatocellular carcinoma (HCC). However, due to the development of effective antiviral treatments and advanced imaging, the necessity for biopsies has significantly decreased. This change has resulted in fewer chances for diagnosing liver disease, causing many general pathologists to feel less confident in making liver biopsy diagnoses. This article provides a comprehensive overview of the challenges and potential solutions related to liver biopsies in Japan. First, it highlights the importance of considering steatotic liver diseases as independent conditions that can coexist with other liver diseases due to their increasing prevalence. Second, it emphasizes the need to avoid hasty assumptions of HCC in nodular lesions, because clinically diagnosable HCCs are not targets for biopsy. Third, the importance of diagnosing hepatic immune-related adverse events caused by immune checkpoint inhibitors is increasing due to the anticipated widespread use of these drugs. In conclusion, pathologists should be attuned to the changing landscape of liver diseases and approach liver biopsies with care and attention to detail.

The Effect of HCV Eradication after Direct-Acting Antiviral Agents on Hepatic Steatosis: A Prospective Observational Study.

BACKGROUND AND AIM: Eradication of hepatitis C virus (HCV) by direct-acting-antiviral- agents (DAAs) was followed by fibrosis regression, but little is available about hepatic steatosis changes after DAAs. The aim of this work was to assess the prevalence of hepatic steatosis among HCV Egyptian patients and the long term changes occuring after viral eradication. METHODS: This prospective cohort study included 150 HCV patients with significant fibrosis. They were examined by Transient elastography to evaluate liver stiffness measurement (LSM) and hepatic steatosis before treatment, at SVR12 and 1 year after the end of therapy. RESULTS: LSM showed a significant positive correlation to pretreatment of hepatic steatosis. LSM significantly decreased and hepatic steatosis significantly increased both at SVR12 and one year after DAAs. Patients with steatosis showed significantly higher median LSM and controlled attenuation parameter (CAP) values at: baseline, SVR12, and one year after therapy. Also, the pretreatment steatosis and body mass index (BMI) had a significant negative correlation with fibrosis regression one year after therapy in all studied groups. CONCLUSION: Hepatic steatosis is common in HCV Egyptian patients and increases after HCV eradication with DAAs. BMI and CAP values are negatively correlated to hepatic fibrosis regression and positively correlated to steatosis progression one year after DAAs. So, HCV patients with hepatic steatosis may need close follow up for atherosclerotic and HCC risk after DAAs, especially if they are overweight.

Comparison of liver biopsies before and after direct-acting antiviral therapy for hepatitis C and correlation with clinical outcome.

Direct-acting antivirals (DAA) have replaced interferon (IFN)-based therapies for hepatitis C virus. In this retrospective clinical study, we examined differences in histopathologic features in paired liver biopsies collected from the same patient before and after DAA and correlated these findings with clinical outcome. Biopsies (n = 19) were evaluated by quantitative imaging analysis to measure steatosis and fibrosis. Most patients had decreased steatosis in their post-treatment, follow-up biopsies. However, one patient had a striking increase in steatosis (from 0.86 to 6.32%) and later developed decompensated cirrhosis and hepatocellular carcinoma (HCC). This patient had a marked increase in fibrosis between biopsies, with a CPA of 6.74 to 32.02. Another patient, who already had bridging fibrosis at the time of her pre-treatment biopsy, developed cholangiocarcinoma after DAA. Even though the overall inflammatory activity in the post-treatment biopsies significantly decreased after treatment, 60% of patients had persistent portal lymphocytic inflammation. In summary, DAAs decreased steatosis and hepatic inflammation in most patients, although some may have persistence of lymphocytic portal inflammation. Patients known to have advanced fibrosis at treatment initiation and who have other risk factors for ongoing liver injury, such as steatosis, should be followed closely for the development of adverse outcomes, such as portal hypertension and primary liver cancers.

Lipid Droplets Accumulation during Hepatitis C Virus Infection in Cell-Culture Varies among Genotype 1-3 Strains and Does Not Correlate with Virus Replication.

Liver steatosis is a common complication of chronic hepatitis C virus (HCV) infection, which can result in accelerated liver fibrosis development, especially in patients infected with genotype 3a. The precise mechanisms of HCV-induced liver steatosis remain unclear, but it is often posited that increased intracellular lipid accumulation is the underlying cause of steatosis. To study experimentally how HCV infection in human liver derived cells by different genotypes and subtypes might affect lipid accumulation, we performed detailed cytofluorimetric and microscopy analyses of intracellular lipid droplets (LDs) in relation to the viral Core and to cell endoplasmic reticulum proteins. Following culture infection with HCV genotype 1a, 2a, 2b, 2c, and 3a strains, we found variable levels of intracellular LDs accumulation, associated to the infecting strain rather than to the specific genotype. Although two genotype 3a strains showed high levels of lipid accumulation, as previously observed, some strains of other genotypes displayed a similar phenotype. Moreover, the analyses of LDs size, number, and shape indicated that the apparent increase in lipid accumulation is due to an increase in the overall number rather than in the size of droplets. Finally, differences in total lipid content across genotypes did not correlate to differences in Core distribution nor Core levels. In conclusion, our study provides a quantitative in-depth analysis of the effect of HCV infection on LDs accumulation in cell-culture.

Association and Interaction Between Serum Interleukin-6 Levels and Metabolic Dysfunction-Associated Fatty Liver Disease in Patients With Severe Coronavirus Disease 2019.

Background and Aim: Circulating levels of interleukin (IL)-6, a well-known inflammatory cytokine, are often elevated in coronavirus disease-2019 (COVID-19). Elevated IL-6 levels are also observed in patients with metabolic dysfunction-associated fatty liver disease (MAFLD). Our study aimed to describe the association between circulating IL-6 levels and MAFLD at hospital admission with risk of severe COVID-19. Methods: A total of 167 patients with laboratory-confirmed COVID-19 from three Chinese hospitals were enrolled. Circulating levels of IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured at admission. All patients were screened for fatty liver by computed tomography. Forty-six patients were diagnosed as MAFLD. Results: Patients with MAFLD (n = 46) had higher serum IL-6 levels (median 7.1 [interquartile range, 4.3-20.0] vs. 4.8 [2.6-11.6] pg/mL, p = 0.030) compared to their counterparts without MAFLD (n = 121). After adjustment for age and sex, patients with MAFLD had a ~2.6-fold higher risk of having severe COVID-19 than those without MAFLD. After adjustment for age, sex and metabolic co-morbidities, increased serum IL-6 levels remained associated with higher risk of severe COVID-19, especially among infected patients with MAFLD (adjusted-odds ratio 1.14, 95% CI 1.05-1.23; p = 0.002). There was a significant interaction effect between serum IL-6 levels and MAFLD for risk of severe COVID-19 (p for interaction = 0.008). Conclusions: Patients with MAFLD and elevated serum IL-6 levels at admission are at higher risk for severe illness from COVID-19.

Fowl Adenovirus Serotype 4 Induces Hepatic Steatosis via Activation of Liver X Receptor-α.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic virus that causes severe hepatic damage characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal necrosis in hepatocytes. Many aspects of FAdV-4 infection and pathogenesis, however, remain unknown. Here, we found that FAdV-4-induced hepatic injury is accompanied by the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in FAdV-4-infected chickens. Significant upregulation of adipose synthesis-related genes, such as liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding protein-1c (SREBP-1c), and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation- and lipid decomposition-related genes were observed in the infected chickens. FAdV-4 infection in cultured leghorn male hepatoma (LMH) cells caused similar signs of steatosis, with alterations in various lipogenesis-related genes. We eliminated the effect of LXR-α activation on FAdV-4-induced steatosis and found that treatment with an LXR-α antagonist (SR9243) and RNA interference (small interfering RNA targeting LXR-α [Si-LXR-α]) decreased the number of oil droplets and the accumulation of lipogenic genes, but treatment with an LXR-α agonist (T0901317) increased the number of oil droplets and the accumulation of lipogenic genes in the cells. Additionally, SR9243 treatment or Si-LXR-α transfection led to significant reductions in viral DNA level, protein expression, and virus production, whereas T0901317 treatment caused significant increases in viral DNA level, protein expression, and virus production. However, inhibition of SREBP-1c activity had no significant effect on virus production. Collectively, these results indicated that FAdV-4-induced steatosis involves activation of the LXR-α signaling pathway, which might be a molecular mechanism underlying the hepatic injury associated with FAdV-4 infection.IMPORTANCE Fowl adenovirus serotype 4 (FAdV-4) is an important hepatotropic adenovirus in chicken, but the underlying mechanism of FAdV-4-induced hepatic injury remains unclear. We report here that infection with FAdV-4 induced the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in the livers of chickens. FAdV-4-induced steatosis might be caused by a disrupted balance of fat metabolism, as evidenced by differential regulation of various lipase genes. The significant upregulation of liver X receptor-α (LXR-α) prompted us to investigate the interplay between LXR-α activation and FAdV-4-induced steatosis. Treatment with an agonist, an antagonist, or RNA interference targeting LXR-α in cultured leghorn male hepatoma (LMH) cells indicated that FAdV-4-induced steatosis was dependent upon LXR-α activation, which contributed to virus replication. These results provide important mechanistic insights, revealing that FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway and highlighting the therapeutic potential of strategies targeting the LXR-α pathway for the treatment of FAdV-4 infection.

The relation of 25-hydroxy vitamin D concentrations to liver histopathology, seasonality and baseline characteristics in chronic hepatitis C virus genotype 2 or 3 infection.

BACKGROUND AND OBJECTIVES: The hydroxylation to 25-hydroxy vitamin D (25(OH)D) occurs in the liver and the impact of liver disease on vitamin D is unclear. This study evaluated the relationship between vitamin D concentrations and hepatic histopathology, seasonality and patient characteristics in well-characterized patients having undergone a liver biopsy. METHOD: 25(OH)D was measured post-hoc in pre-treatment serum from 331 North European patients with chronic HCV genotype 2 or 3 infection (NORDynamIC study). Liver biopsies were scored for fibrosis and inflammation according to the Ishak protocol, and graded for steatosis. Non-invasive markers of hepatic fibrosis as well as baseline viral and host characteristics, including genetic polymorphisms rs2228570, rs7975232, and rs10877012 were also evaluated. RESULTS: Mean 25(OH)D concentration was 59 ±23 nmol/L, with 41% having values <50 nmol/L and 6% were <30 nmol/L. 25(OH)D correlated with fibrosis (r = -0.10, p ≤0.05) in univariate but not in multivariate analyses. No association was observed between 25(OH)D and hepatic inflammation, but with steatosis in HCV genotype 2 infected patients. None of the genetic polymorphisms impacted on 25(OH)D levels or fibrosis. 25(OH)D levels were significantly inversely correlated to BMI (r = -0.19, p = 0.001), and was also associated with season and non-Caucasian ethnicity. CONCLUSION: Fibrosis was not independently associated with 25(OH)D concentration and no association was seen with hepatic inflammation, but HCV genotype 2 infected patients with moderate-to-severe steatosis had lower 25(OH)D levels compared to those without steatosis. A high percentage had potential risk of 25(OH)D deficiency, and BMI, seasonality and ethnicity were independently associated with 25(OH)D as previously reported.

Hepatic Fat-Genetic Risk Score Predicts Hepatocellular Carcinoma in Patients With Cirrhotic HCV Treated With DAAs.

BACKGROUND AND AIMS: Genetic factors and steatosis predispose to hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus; however, their impact in patients with cirrhosis cured by direct-acting antivirals (DAAs) is still undefined. We assessed the association between a genetic risk score (GRS) of hepatic fat accumulation, combining variants in PNPLA3 (patatin-like phospholipase domain containing 3), MBOAT7 (membrane bound O-acyltransferase domain containing 7), TM6SF2 (transmembrane 6 superfamily member 2), GCKR (glucokinase regulator), and HCC in patients treated with DAAs. APPROACH AND RESULTS: We considered 509 consecutive patients with HCV cirrhosis (defined histologically or when liver stiffness ≥12 kPa) treated with DAAs. HCC was diagnosed according to international recommendations. GRS was calculated from the weighted impact of single variants on hepatic fat content quantified by H1 spectrometry in the general population (Dallas Heart Study). During a median follow-up of 43 (3-57) months after DAA start, 36 of 452 (8%) patients developed de novo HCC, 4-year cumulative probability being 9% (95% confidence interval 7%-12%). Male sex (hazard ratio [HR] 2.54, P = 0.02), diabetes (HR 2.39, P = 0.01), albumin (HR 0.35, P = 0.001), and GRS score >0.597 (HR 2.30, P = 0.04) were independent predictors of de novo HCC. In contrast, single genetic risk variants were not useful in stratifying HCC risk. The proportion of patients who developed HCC according to the combination of the independent risk factors ranged from 11% to 67%. HCC recurred in 28 of 57 (49%) patients with previous history; diabetes and ethnicity were the only independent predictors of HCC recurrence. CONCLUSIONS: In a large cohort of DAA-treated patients with cirrhotic HCV, GRS was associated with de novo HCC independently of classical risk factors, including liver disease severity. These data suggest that hepatic fat (i.e., lipotoxicity) promotes HCC in this setting and may represent a target for chemoprevention. Combination of clinical and genetic predictors may improve HCC risk stratification.

Impact of hepatic steatosis on treatment response in nuclesos(t)ide analogue-treated HBeAg-positive chronic hepatitis B: a retrospective study.

BACKGROUND: The impact of hepatic steatosis (HS) on treatment response following nucleos(t)ide analogue (NA) treatment for chronic hepatitis B (CHB) patients has not been clearly elucidated. We aimed to investigate the difference in HBeAg seroclearance between NA-treated HBeAg-positive CHB patients with and without HS. METHODS: We retrospectively recruited HBeAg-positive CHB patients receiving liver biopsy and NA monotherapy. The baseline clinical characteristics and cumulative incidence of HBeAg seroclearance were compared between patients with and without HS and age/gender-matched subgroup analysis was performed. RESULTS: A total of 196 patients were enrolled from 2003 April to 2016 October. The mean age was 39.6 ± 11.2 years, 142 (72.4%) were males and 94 (48%) had histological evidence of HS. Median treatment duration and follow-up period were 24.3 months and 54.9 months, respectively. HBeAg seroclearance was achieved in 56/102 (54.9%) and 54/94 (57.4%) patients with and without HS, respectively (p = 0.830). The 5-year cumulative incidence of HBeAg seroclearance in patients with and without HS was 62.8 and 67.7% in overall population (p = 0.398) and 62.4 and 66.9% in age/gender-matched subgroups (p = 0.395), respectively. The rate of HBeAg seroclearance was comparable between patients with or without HS in different NA monotherapy (all p > 0.05). CONCLUSIONS: HS had no significant impact on HBeAg seroclearance in HBeAg-positive CHB patients with NA monotherapy during long-term follow-up.

Similar prevalence of hepatic steatosis among patients with chronic hepatitis C with and without HIV coinfection.

Hepatic steatosis (HS) is frequently observed in HIV-infected patients. It is not known whether HIV infection is an independent risk factor for HS development. We aimed to analyze whether HIV coinfection was associated with a higher frequency of HS in patients with chronic hepatitis C. This was a retrospective cross-sectional study. 574 subjects with chronic hepatitis C virus (HCV) infection were included, 246 (43%) of them coinfected with HIV. All of them underwent transient elastography with controlled attenuation parameter (CAP) measurement. HS was defined as CAP ≥ 248 dB/m. 147 individuals (45%) showed HS in the HCV-monoinfected group and 100 (40.7%) in the HIV/HCV-coinfected group (p = 0.318). HS was associated with body mass index (BMI) [<25 Kg/m2 vs. ≥25 Kg/m2, 67 (23.5%) vs. 171 (62.9%); p = 0.001], with plasma HDL-cholesterol [<50 mg/dL vs. ≥50 mg/dL, 122 (48.6%) vs. 95 (37.5%), p = 0.012], with plasma triglycerides [<150 mg/dL vs. ≥150 mg/dL, 168 (40.2%) vs. 65 (52.4%); p = 0.016] and with plasma total cholesterol [<200 mg/dL vs. ≥200 mg/dL, 181 (41%) vs. 53 (52.5%); p = 0.035]. In the multivariate analysis, HS was associated with BMI [adjusted OR (AOR) = 1.264 (1.194-1.339); p = 0.001], age [AOR = 1.029 (1.001-1.058); p = 0.047] and HCV genotype 3 infection [AOR = 1.901 (1.081-2.594); p = 0.026]. HIV coinfection was not associated with HS [AOR = 1.166 (0.719-1.892); p = 0.534]. In conclusion, HIV coinfection is not related with an increased frequency of HS in HCV-infected patients.

Relation of Steatosis to Neurocognitive Function in People Living with HIV.

BACKGROUND: In HIV negative population metabolic syndrome and steatosis are related to poorer neurocognitive (NC) performance. We investigated if similar relation exists in people living with HIV (PLWH). METHODS: We included male PLWH aged 20-65, with undetectable viral load for at least 6 months. Data on levels of education, anthropometric measurements, CD4 levels, ART, markers of metabolic syndrome, smoking and concurrent treatment were collected from database. Concentrations of TNF-α and IL-6 were measured. An ultrasound was used to establish the presence of steatosis, visceral fat thickness and carotid intima media thickness. An extensive NC assessment was done by an experienced neuropsychologist. Cognitive domains were defined as executive functions, divergent reasoning, visuo-constructional abilities, delayed recall and working memory and learning and were measured using a battery of 12 tests. RESULTS: 88 PLWH were included (mean age 39,9 years), 51% on PIs, 46% on NNRTI; 20,4% had metabolic syndrome, 42% patients had steatosis. Weak but statistically significant negative correlations were found between the presence of metabolic syndrome, steatosis and VFT and cognitive domains (divergent reasoning, delayed recall and working memory). Poorer perfomrance in the domains of divergent reasoning and in the working memory were found in participants with steatosis (p=0,048 and 0,033 respectively). CONCLUSION: Although the sample size was relatively small, our results show consistent correlations between the observed neurocognitive variables and metabolic parameters. As central obesity is one of the contributors to NCI, it would be one of the modifiable factors to prevent further neurocognitive decline.

Association between PNPLA3[G]/I148M variant, steatosis and fibrosis stage in hepatitis C virus - genetic matters.

There is an established correlation between the PNPLA3 rs738409 C > G single nucleotide polymorphism (SNP) and hepatic steatosis and fibrosis in hepatitis C virus (HCV) infected patients. However not all data is convergent regarding the exact impact of this SNP on the pattern of disease progression in different clinical settings. In this study, we aimed to further bridge the knowledge gap on this topic by investigating the role of the G allele in promoting steatosis, fibrosis and disease progression in relation to other metabolic and anthropometric host factors. Two hundred and fifty consecutive patients, previously diagnosed with chronic hepatitis C (CHC) underwent liver biopsy. Histology was assessed using the Metavir scoring system. Transient elastography was used for follow-up. Ninety-eight patients were genotyped for PNPLA3 rs738409 and followed up for fibrosis progression. PNPLA3 rs738409[G] allele was significantly correlated with severe steatosis (P = 0.04), severe fibrosis at the time of enrollment (P = 0.0005) and fibrosis progression with an OR of 10.31 (95% CI 1.06 - 99.59, P = 0.04), after a mean follow-up time of 62.85 (95%CI: 52.21 - 76.15) months. Severe steatosis at the time of enrollment had an OR of 11.02 (95% CI 1.48 - 82.09, P = 0.01) for the association with fibrosis progression. The HOMA-IR index was also positively correlated with severe fibrosis (P = 0.03) and fibrosis progression on univariate analysis (P = 0.02). PNPLA3 rs738409[G] allele is a reliable predictor for steatosis and fibrosis in CHC. The presence of G allele, along with severe steatosis and insulin resistance are significant predictors for fibrosis progression.

Circulating levels of PRO-C3 reflect liver fibrosis and liver function in HIV positive patients receiving modern cART.

BACKGROUND AND AIMS: Although combined antiretroviral treatment (cART) has improved overall survival of HIV infected patients, liver fibrosis and liver related-mortality still constitute major challenges in HIV positive patients. Collagen accumulates in the liver during fibrogenesis. Recent studies showed that circulating levels of extracellular matrix (ECM) fragments might reflect degree of portal hypertension and fibrosis stage in liver disease. In this study, we analyzed the correlation between liver fibrosis assessed by Fibroscan and levels of the formation and degradation markers of type III and IV collagen in HIV positive patients receiving cART. METHODS: 116 HIV positive patients (82.7% male, median age 47 years) were enrolled into the study. Liver stiffness and liver fat content were determined using a Fibroscan with integrated CAP function. We quantified ECM formation and degradation fragments of collagen III and IV: PRO-C3, PRO-C4, C3M and C4M. These fragments were measured in peripheral serum by using specific ELISAs. RESULTS: Fifteen (12.9%) out of the 116 HIV positive patients had relevant fibrosis with a liver stiffness ≥ 7.1 kPa, and 79 patients had relevant steatosis with a CAP value > 248 dB/m. Circulating PRO-C3 levels significantly correlated with increasing degree of liver fibrosis assessed by Fibroscan (p = 0.0005), as well as with APRI score (p = 0.015). Interestingly, circulating PRO-C3 levels were significantly correlated with bilirubin (p = 0.022), reduced platelet count (p = 0.0008) and low albumin levels (p = 0.001), suggesting the association of type III collagen deposition with impaired liver function. None of the other measured ECM components significantly correlated with fibrosis or steatosis. CONCLUSION: The formation marker of type III collagen, PRO-C3 not only reflects liver fibrosis, but might also mirror liver dysfunction in HIV positive patients receiving cART. Therefore, the circulating levels of PRO-C3 might be suitable to monitor progression of liver fibrosis and deterioration of liver function in HIV positive patients receiving cART.

Nonalcoholic fatty liver disease and steatohepatitis in people living with HIV.

Introduction: The burden of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing worldwide. This phenomenon poses a potentially dangerous risk of rise in mortalities caused by cirrhosis and liver cancer. Owing to a complex combination of factors, NAFLD and NASH arise in a majority of people living with HIV (PLWH), but accurate estimates of prevalence differ, depending on sample selection, type of analysis, and data interpretation. The wide range of diagnostic tools used to assess liver steatosis and lack of control groups in many studies further contributes to current difficulties in properly assessing prevalence of these conditions. Areas covered: Thoroughly scrutinizing the current literature, we compared the prevalence of NAFLD and NASH in PLWH to rates found in the general population. We highlighted strengths and limitations of the studies, in order to determine the effective impact of these medical conditions in PLWH. Expert opinion: The prevalence and progression of NAFLD in human immunodeficiency virus (HIV) infection are reported to be widely variable. HIV infection itself and antiretroviral treatment have been demonstrated to play a role in the development of NAFLD. Larger and more effective studies are needed to evaluate the effects of NASH in PLWH and its progression.

Editorial: Cytokines in liver diseases.

This special issue on 'Cytokines in Liver Diseases' was inspired by many talks and presentations on liver cancer during the 2nd Aegean Conference on Cytokine Signaling in Cancer (ACCSC) held at Heraklion, Crete, Greece on May 30-June 04, 2017 (Cytokine 2018, 108: 225-231). The liver is the biggest blood filtration and detoxification unit, and is a vital metabolic organ. Being constantly exposed to potentially harmful dietary chemicals, drugs, alcohol abuse and pathogens, the liver displays an extraordinary capacity to repair tissue damage and to regenerate. Moreover, only a healthy liver can provide the vast majority of plasma proteins, plus serving as a key organ for body homeostasis and metabolic fitness. Occasionally, the liver may have to deal with chronic damage inflicted by hepatotropic infections such as hepatitis viruses and metabolic derangements caused by obesity and the consequent metabolic syndrome. Overwhelming the natural defenses of the liver can compromise its vital functions and this can lead to more severe liver disease such as fibrosis that may progress towards cirrhosis and eventually to hepatocellular carcinoma (HCC). However, in obesity, a worldwide crisis that has been developing during the last few decades, HCC can develop in the fatty liver bypassing the fibrosis and cirrhosis stages. With the availability of effective therapies and vaccination strategies for hepatitis viruses, over nutrition has become the biggest new threat for a healthy liver. Cytokines and chemokines play a key role in the initiation and perpetuation of acute and chronic injury to the liver, and thus they contribute to most liver pathologies. The topic of cytokines in liver diseases is so vast that it cannot be adequately covered in this special issue. However, we have attempted to provide a glimpse of hot topics through comprehensive reviews and a few accompanying original articles on key research areas.

Development of an in vitro model to study hepatitis C virus effects on hepatocellular lipotoxicity and lipid metabolism.

Hepatic steatosis is common in patients infected with hepatitis C virus (HCV). Particularly in patients infected with non-genotype 3 HCV, hepatic steatosis is closely related to factors of the metabolic syndrome such as hyperlipidemia. However, the molecular mechanisms involved in this "metabolic" steatosis in non-3 genotype HCV infections are not well understood. Here, we aimed to develop an in vitro model to study the effect of genotype 1 HCV infection on hepatic lipotoxicity and lipid metabolism. Cellular lipid accumulation was induced in Huh-7 hepatoma cells transfected with HCV genotype 1b replicon (HCV+) by incubation with increasing doses of palmitic acid (C16:0) or oleic acid (C18:1 n-9) complexed to albumin mimicking hyperlipidemic conditions. Mock transfected hepatoma cells (HCV-) were used as controls. Incubation with oleic acid concentrations as high as 0.5 mM did not induce toxic effects in HCV+ or HCV- cells. In contrast, incubation with palmitic acid caused dose-dependently cytotoxic effects which were more pronounced in HCV+ compared to HCV- cells. Further analysis with subtoxic palmitic and oleic acid concentrations revealed a higher uptake of fatty acids and intracellular triglyceride accumulation in HCV+ compared to HCV- cells. Carnitine palmitoyltransferase I (CPT1) expression, indicative of mitochondrial beta-oxidation, was markedly stimulated by lipid exposure in HCV+ but not in HCV- cells. Furthermore, heme oxygenase 1 (HMOX1) expression levels increased in FA stimulated cells, and this increase was significantly higher in HCV+ compared to HCV- cells. In contrast, expression of the key enzymes of hepatic de novo lipogenesis fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD-1) was significantly reduced upon oleate exposure in HCV- but not in HCV+ cells. In summary, our newly developed cell culture model revealed effects of HCV genotype 1b infection on metabolic susceptibility to lipid accumulation and toxicity particularly to saturated lipids. These results may indicate that HCV (genotype 1b) infected individuals with hyperlipidemia may benefit from dietary or pharmacological intervention.

CD2-Associated Protein Contributes to Hepatitis C, Virus Propagation and Steatosis by Disrupting Insulin Signaling.

Chronic hepatitis C virus (HCV) infection can result in steatosis, a condition displaying aberrant accumulation of neutral lipid vesicles, the component of lipid droplets (LDs), which are essential for HCV assembly. However, the interplay between HCV infection and steatosis remains unclear. Here, we show that HCV-infected cells have higher levels of CD2-associated protein (CD2AP), which plays two distinct, yet tightly linked, roles in HCV pathogenesis: Elevated CD2AP binds to nonstructural protein 5A (NS5A) and participates in the transport of NS5A to LDs to facilitate viral assembly; Up-regulated CD2AP also interacts with casitas B-lineage lymphoma (b) (Cbl/Cbl-b) E3 ligases to degrade insulin receptor substrate 1 (IRS1), which, in turn, disrupts insulin signaling and increases LD accumulation through the IRS1/protein kinase B (Akt)/adenosine monophosphate-activated protein kinase (AMPK)/hormone-sensitive lipase (HSL) signaling axis to accommodate viral assembly. In the HCV-infected mouse model, CD2AP expression is up-regulated during the chronic infection stage and this up-regulation correlates well with liver steatosis. Importantly, CD2AP up-regulation was also detected in HCV-infected human liver biopsies showing steatosis compared to non-HCV-infected controls. Conclusion: CD2AP is indicated as a protein up-regulated by HCV infection, which, in turn, stimulates HCV propagation and steatosis by disrupting insulin signaling; targeting CD2AP may offer an opportunity for alleviating HCV infection and its associated liver pathology. (Hepatology 2018;XX:XXX-XXX.).

Changes in liver stiffness and steatosis among patients with hepatitis C virus infection who received direct-acting antiviral therapy and achieved sustained virological response.

AIM: Whether direct-acting antiviral (DAA) therapy can reduce liver fibrosis and steatosis in patients with chronic hepatitis C virus (HCV) infection remains unclear. We evaluated sequential changes in liver stiffness and steatosis using transient elastography (TE) and the TE-based controlled attenuation parameter (CAP) in patients with HCV who received DAA therapy. PATIENTS AND METHODS: A total of 57 patients with HCV who received DAA therapy and achieved sustained virological response (SVR) were analyzed. Liver stiffness as evaluated with TE, steatosis as evaluated with CAP, and laboratory data were assessed before treatment (baseline), at end of treatment (EOT), 24 weeks after EOT (SVR24), and 48 weeks after EOT (SVR48). RESULTS: Alanine aminotransferase levels, corresponding to the presence of necroinflammatory activity, significantly decreased overall, with significant differences between baseline and EOT, EOT, and SVR24, and baseline and SVR48. However, alanine aminotransferase levels showed no significant changes between SVR24 and SVR48. Median (interquartile range) liver stiffness values at baseline, EOT, SVR24, and SVR48 were 8.3 (5.0-14.8), 7.4 (4.6-14.7), 5.3 (4.1-11.8), and 5.4 (4.0-13.4) kPa, respectively (baseline vs. EOT, P=0.044; EOT vs. SVR24, P=0.011; and SVR24 vs. SVR48, P=0.054). In patients with fatty liver (CAP≥236 dB/m, n=14), CAP values at baseline and SVR48 were 253 (245-278) and 229 (209-249) dB/m, respectively (P=0.020). CONCLUSION: Liver stiffness at SVR24 might reflect liver fibrosis in the patients who received DAA therapy and achieved SVR. In addition, liver steatosis reduces in the same cohort with fatty liver.

Fatty liver in hepatitis C patients post-sustained virological response with direct-acting antivirals.

AIM: To determine steatosis and fibrosis prevalence in hepatitis C patients after a sustained virological response achieved with direct-acting antivirals. METHODS: Transient elastography with controlled attenuation parameter (CAP) was used to assess hepatic steatosis post-sustained virological response (SVR); the CAP technology was not available in the United States at study initiation. Liver stiffness/fibrosis was measured before and 47 wk after treatment completion. Patients with genotype 3 and patients with cirrhosis were excluded. RESULTS: One hundred and one patients were included in the study. Post-SVR there were decreases from baseline in alanine aminotransferase (ALT) (63.1 to 17.8 U/L), aspartate aminotransferase (51.8 to 21.5 U/L) and fibrosis score (7.4 to 6.1 kPa) (P < 0.05). Post-SVR, 48 patients (47.5%) had steatosis on CAP; of these, 6.25% had advanced fibrosis. Patients with steatosis had higher body mass index (29.0 vs 26.1 kg/m2), glucose (107.8 vs 96.6 mg/dL), ALT (20.4 vs 15.3 mg/dL), CAP score (296.3 vs 212.4 dB/m) and fibrosis score (7.0 vs 5.3 kPa); P < 0.05. Interestingly, compared to baseline, both patients with and without steatosis had change in fibrosis score post-SVR (7.7 kPa vs 7.0 kPa and 7.0 kPa vs 5.3 kPa); alternatively, (P < 0.05) and therefore patients with steatosis continued to have clinically significant stiffness (≥ 7 kPa). CONCLUSION: Fatty liver is very common in hepatitis C virus (HCV) patients post-SVR. These patients continue to have elevated mean fibrosis score (≥ 7 kPa) compared to those without fatty liver; some have advanced fibrosis. Long term follow up is needed to assess steatosis and fibrosis in HCV patients post-SVR.

Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

BACKGROUND & AIMS: Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. METHODS: To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. RESULTS: Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. CONCLUSIONS: Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression.