Yaron Rotman

The association of genetic variability in patatin-like phospholipase domain-containing protein 3 (PNPLA3) with histological severity of nonalcoholic fatty liver disease.

Genome‐wide association studies identified single‐nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). To investigate whether these SNPs are associated with histological severity of NAFLD, 1117 (894 adults/223 children) individuals enrolled in the Nonalcoholic Steatohepatitis (NASH) Clinical Research Network and National Institutes of Health Clinical Center studies with histologically confirmed NAFLD were genotyped for six SNPs that are associated with hepatic fat or liver enzymes in genome‐wide association studies. In adults, three SNPs on chromosome 22 showed associations with histological parameters of NASH. After adjustment for age, sex, diabetes, and alcohol consumption, the minor allele of rs738409 C/G, a nonsynonymous coding SNP in the patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) (adiponutrin) gene encoding an Ile148Met change, was associated with steatosis (P = 0.03), portal inflammation (P = 2.5 × 10−4), lobular inflammation (P = 0.005), Mallory‐Denk bodies (P = 0.015), NAFLD activity score (NAS, P = 0.004), and fibrosis (P = 7.7 × 10−6). Two other SNPs in the same region demonstrated similar associations. Three SNPs on chromosome 10 near the CHUK (conserved helix‐loop‐helix ubiquitous kinase) gene were independently associated with fibrosis (P = 0.010). In children, no SNP was associated with histological severity. However, the rs738409 G allele was associated with younger age at the time of biopsy in multivariate analysis (P = 0.045). Conclusion: In this large cohort of histologically proven NAFLD, we confirm the association of the rs738409 G allele with steatosis and describe its association with histological severity. In pediatric patients, the high‐risk rs738409 G allele is associated with an earlier presentation of disease. We also describe a hitherto unknown association between SNPs at a chromosome 10 locus and the severity of NASH fibrosis. (Hepatology 2010)

Natural Killer Cells Are Polarized Toward Cytotoxicity in Chronic Hepatitis C in an Interferon-Alfa-Dependent Manner

BACKGROUND & AIMS Patients with chronic hepatitis C virus (HCV) infection display great variability in disease activity and progression. Although virus-specific adaptive immune responses have been characterized extensively and found to be impaired in chronic hepatitis C, the role of innate immune responses in disease activity and progression of chronic hepatitis C is not well understood. METHODS We studied 42 HCV-infected patients and 12 healthy uninfected controls. RESULTS We found an increased frequency of natural killer (NK) cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), NKp44, NKG2C, and CD122 in chronic hepatitis C as compared with healthy controls (P < .05 for all markers) and stronger activation of NK cells in the liver than in the blood (P < .05). This NK cell phenotype was associated with polarization of NK cell function toward CD107a expression as a marker of degranulation, but with not increased interferon (IFN)-gamma production of CD56(dim) NK cells. The polarized NK cell phenotype correlated with alanine aminotransferase levels (r(2) = 0.312, P = .03). To investigate whether in vivo exposure of NK cells to HCV-induced type I IFN was causing this NK cell phenotype, peripheral blood mononuclear cells from 10 healthy controls and 8 HCV-infected patients were stimulated in the presence of IFN-alfa, which resulted in increased NK cell expression of TRAIL and CD107a (P < .001), but not IFN-gamma. CONCLUSIONS Collectively, these results describe a polarized NK cell phenotype induced by chronic exposure to HCV-induced IFN-alfa. This phenotype may contribute to liver injury through TRAIL expression and cytotoxicity, whereas the lacking increase in IFN-gamma production may facilitate the inability to clear HCV.

HCV Infection Induces a Unique Hepatic Innate Immune Response Associated With Robust Production of Type III Interferons

BACKGROUND & AIMS Polymorphisms in the IL28B gene have been associated with clearance of hepatitis C virus (HCV), indicating a role for type III interferons (IFNs) in HCV infection. Little is known about the function of type III IFNs in intrinsic antiviral innate immunity. METHODS We used in vivo and in vitro models to characterize the role of the type III IFNs in HCV infection and analyzed gene expression in liver biopsy samples from HCV-infected chimpanzees and patients. Messenger RNA and protein expression were studied in HCV-infected hepatoma cell lines and primary human hepatocytes. RESULTS HCV infection of primary human hepatocytes induced production of chemokines and type III IFNs, including interleukin (IL)-28, and led to expression of IFN-stimulated genes (ISGs). Chimpanzees infected with HCV showed rapid induction of hepatic type III IFN, associated with up-regulation of ISGs and minimal induction of type I IFNs. In liver biopsy specimens from HCV-infected patients, hepatic expression of IL-28 correlated with levels of ISGs but not of type I IFNs. HCV infection produced extensive changes with gene expression in addition to ISGs in primary human hepatocytes. The induction of type III IFNs is regulated by IFN regulatory factor 3 and nuclear factor κB. Type III IFNs up-regulate ISGs with a different kinetic profile than type 1 IFNs and induce a distinct set of genes, which might account for their functional differences. CONCLUSIONS HCV infection results predominantly in induction of type III IFNs in livers of humans and chimpanzees; the level of induction correlates with hepatic levels of ISGs. These findings might account for the association among IL-28, level of ISGs, and recovery from HCV infection and provide a therapeutic strategy for patients who do not respond to IFN therapy.

Emerging Trends in Hepatocellular Carcinoma Incidence and Mortality

The rise in incidence of hepatocellular carcinoma (HCC) in the United States has been well documented. The purpose of this analysis was to examine temporal trends in HCC incidence, mortality, and survival within the U.S. population. The Surveillance, Epidemiology, and End Results data were used to examine incidence and incidence‐based (IB) mortality in HCC from 1973 to 2011. Secular trends in age‐adjusted incidence and IB mortality by sex and cancer stage were characterized using the Joinpoint Regression program. In 1973, HCC incidence was 1.51 cases per 100,000, whereas in 2011, HCC incidence was 6.20 cases per 100,000. Although HCC incidence continues to increase, a slowing of the rate of increase occurs around 2006. In a sensitivity analysis, there was no significant increase in incidence and IB mortality from 2009 to 2011. There was a significant increase in overall median survival from the 1970s to 2000s (2 vs. 8 months; P < 0.001). On multivariable Coxs regression analysis, age, sex, race, tumor grade, stage at diagnosis, lymph/vascular invasion, number of primary tumors, tumor size, and liver transplant were independently associated with mortality. Conclusion: Our results indicate a deceleration in the incidence of HCC around 2006. Since 2009 and for the first time in four decades, there is no increase in IB mortality and incidence rates for HCC in the U.S. population. The nonsignificant increase in incidence and IB mortality in recent years suggest that the peak of the HCC epidemic may be near. A significant survival improvement in HCC was also noted from 1973 to 2010, which seems to be driven by earlier detection of HCC at a curative stage and greater utilization of curative modalities (especially transplant). (Hepatology 2015;61:191–199)

Early Changes in Natural Killer Cell Function Indicate Virologic Response to Interferon Therapy for Hepatitis C

BACKGROUND & AIMS Mathematical modeling of hepatitis C virus (HCV) kinetics indicated that cellular immune responses contribute to interferon (IFN)-induced clearance of HCV. We investigated a potential role of natural killer (NK) cells in this process. METHODS Phenotype and function of blood and liver NK cells were studied during the first 12 weeks of treatment with pegylated IFN-alfa and ribavirin, the time period used to define the early virological response. RESULTS Within hours of treatment initiation, NK cells of patients that had an early virological response increased expression of activating receptors NKG2D, NKp30, and CD16 and decreased expression of NKG2C and 2B4, along with inhibitory receptors SIGLEC7 and NKG2A, resulting in NK cell activation. NK cell cytotoxicity, measured by degranulation and tumor necrosis factor-related apoptosis-inducing ligand production, peaked after 24 hours (P<.01), concomitant with an increase in alanine aminotransferase levels (P<.05), whereas IFN-γ production decreased within 6 hours and did not recover for more than 4 weeks (P<.05). NK cells from liver biopsies taken 6 hours after treatment initiation had increased numbers of cytotoxic CD16+NK cells (P<.05) and a trend toward increased production of tumor necrosis factor-related apoptosis-inducing ligand. Degranulation of peripheral blood NK cells correlated with treatment-induced, first-phase decreases in viral load (P<.05) and remained higher in early virological responders than in nonresponders for weeks. CONCLUSIONS IFN activates NK cells early after treatment is initiated. Their cytotoxic function, in particular, is strongly induced, which correlates to virologic response. Therefore, NK cell activation indicates responsiveness to IFN-α-based treatment and suggests the involvement of the innate immune cells in viral clearance.

Ribavirin Improves Early Responses to Peginterferon Through Improved Interferon Signaling

BACKGROUND & AIMS The therapeutic mechanisms of ribavirin for hepatitis C are unclear. Microarray analyses have shown that ribavirin increases induction of interferon-stimulated genes. We evaluated viral kinetics, serum cytokine expression, and viral mutagenesis during early stages of peginterferon therapy with and without ribavirin. METHODS Fifty patients with chronic hepatitis C virus (HCV) infection genotype 1 were randomly assigned to groups that were given peginterferon alpha-2a, with or without ribavirin, for 4 weeks; all patients then received an additional 44 weeks of combination therapy. First- and second-phase viral kinetics were evaluated. Serum levels of interferon-gamma-inducible protein-10 (IP10), monokine induced by interferon-gamma, and monocyte chemoattractant protein 1 were quantified as measures of the interferon-stimulated genes response. NS5A and NS5B were partially sequenced, and mutation rates were calculated. RESULTS The first-phase decrease in HCV RNA was similar between groups. Patients who received ribavirin had a more rapid second-phase decrease, compared with patients who did not receive ribavirin-particularly those with an adequate first-phase decrease (0.61 vs 0.35 log10 IU/mL/week; P = .018). At 12 hours, fold induction of serum IP10 was higher in patients given the combination therapy than those given peginterferon only (7.6- vs 3.8-fold; P = .01); however, the difference was greatest in patients with an adequate first-phase decrease in HCV RNA. IP10-induction correlated with first- and second-phase kinetics and with ribavirin serum concentrations on day 3. HCV mutation rates were similar between groups. CONCLUSIONS Ribavirin improves the kinetics of the early response to therapy in patients with an adequate initial response to peginterferon. Induction of interferon-stimulated cytokines correlates with viral kinetics following ribavirin therapy, suggesting that ribavirin promotes interferon signaling.

Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease

Given the high prevalence and rising incidence of non-alcoholic fatty liver disease (NAFLD), the absence of approved therapies is striking. Although the mainstay of treatment of NAFLD is weight loss, it is hard to maintain, prompting the need for pharmacotherapy as well. A greater understanding of disease pathogenesis in recent years was followed by development of new classes of medications, as well as potential repurposing of currently available agents. NAFLD therapies target four main pathways. The dominant approach is targeting hepatic fat accumulation and the resultant metabolic stress. Medications in this group include peroxisome proliferator-activator receptor agonists (eg, pioglitazone, elafibranor, saroglitazar), medications targeting the bile acid-farnesoid X receptor axis (obeticholic acid), inhibitors of de novo lipogenesis (aramchol, NDI-010976), incretins (liraglutide) and fibroblast growth factor (FGF)-21 or FGF-19 analogues. A second approach is targeting the oxidative stress, inflammation and injury that follow the metabolic stress. Medications from this group include antioxidants (vitamin E), medications with a target in the tumour necrosis factor α pathway (emricasan, pentoxifylline) and immune modulators (amlexanox, cenicriviroc). A third group has a target in the gut, including antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin). Finally, as the ongoing injury leads to fibrosis, the harbinger of liver-related morbidity and mortality, antifibrotics (simtuzumab and GR-MD-02) will be an important element of therapy. It is very likely that in the next few years several medications will be available to clinicians treating patients with NAFLD across the entire spectrum of disease.

Coinfection with Hepatitis C Virus and Human Immunodeficiency Virus: Virological, Immunological, and Clinical Outcomes

Infection with hepatitis C virus (HCV) is estimated to affect 2% of the world population ([90][1]) and is a leading cause of liver-related morbidity and mortality. Human immunodeficiency virus (HIV) is also a very important global public health problem, infecting about 33 million people worldwide ([

Early changes in interferon signaling define natural killer cell response and refractoriness to interferon‐based therapy of hepatitis C patients

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN‐γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)‐induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN‐α‐based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN‐α‐based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor–apoptosis‐inducing ligand [TRAIL] expression and degranulation) and decreased IFN‐γ production. NK cells from patients with a greater than 2 log10 first‐phase HCV RNA decline to IFN‐α‐based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first‐phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN‐α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN‐α during the second‐phase virological response. Conclusion: These data show that IFN‐α‐induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN‐γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN‐α‐based therapy. (Hepatology 2012)

Effect of ribavirin on viral kinetics and liver gene expression in chronic hepatitis C

Objective Ribavirin improves treatment response to pegylated-interferon (PEG-IFN) in chronic hepatitis C but the mechanism remains controversial. We studied correlates of response and mechanism of action of ribavirin in treatment of hepatitis C. Design 70 treatment-naive patients were randomised to 4 weeks of ribavirin (1000–1200 mg/d) or none, followed by PEG-IFNα-2a and ribavirin at standard doses and durations. Patients were also randomised to a liver biopsy 24 h before or 6 h after starting PEG-IFN. Hepatic gene expression was assessed by microarray and interferon-stimulated gene (ISG) expression quantified by nCounter platform. Temporal changes in ISG expression were assessed by qPCR in peripheral-blood mononuclear cells (PBMC) and by serum levels of IP-10. Results After 4 weeks of ribavirin monotherapy, hepatitis C virus (HCV) levels decreased by 0.5±0.5 log10 (p=0.009 vs controls) and ALT by 33% (p<0.001). Ribavirin pretreatment, while modestly augmenting ISG induction by PEG-IFN, did not modify the virological response to subsequent PEG-IFN and ribavirin treatment. However, biochemical, but not virological, response to ribavirin monotherapy predicted response to subsequent combination treatment (rapid virological response, 71% in biochemical responders vs 22% non-responders, p=0.01; early virological response, 100% vs 68%, p=0.03; sustained virological response 83% vs 41%, p=0.053). Ribavirin monotherapy lowered serum IP-10 levels but had no effect on ISG expression in PBMC. Conclusions Ribavirin is a weak antiviral but its clinical effect seems to be mediated by a separate, indirect mechanism, which may act to reset IFN-responsiveness in HCV-infected liver.