Anuradha Krishnan

Broadly neutralizing antibodies abrogate established hepatitis C virus infection

HCV-specific neutralizing antibodies protect humanized mice from challenge and suppress established infections. Neutralizing Antibodies Take Down the HCV Establishment In most individuals infected with hepatitis C virus (HCV), the HCV sets up shop—establishing a long-term, chronic infection that damages the liver and can lead to cirrhosis or liver cancer. de Jong et al. now report that a trio of neutralizing antibodies not only can prevent infection but also can treat and maybe even cure already established infection in multiple animal models. The broadly neutralizing antibodies, which could block multiple genotypes of HCV, were delivered into the muscle by a virus—an adeno-associated vector that does not cause disease—resulting in prolonged expression of the antibodies. If these data hold true in people, this approach may provide a new tool for treating HCV infection. In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. We demonstrate that three broadly nAbs—AR3A, AR3B, and AR4A—delivered with adeno-associated viral vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.

Differential expression of lumican and fatty acid binding protein-1: New insights into the histologic spectrum of nonalcoholic fatty liver disease†

The basis of hepatocellular injury and progressive fibrosis in a subset of patients with nonalcoholic fatty liver disease (NAFLD) is poorly understood. We sought to identify hepatic proteins that are differentially abundant across the histologic spectrum of NAFLD. Hepatic protein abundance was measured in liver samples from four groups (n = 10 each) of obese (body mass index >30 kg/m2) patients: (1) obese normal group (normal liver histology), (2) simple steatosis (SS), (3) nonalcoholic steatohepatitis (NASH)‐mild (steatohepatitis with fibrosis stage 0‐1), and (4) NASH‐progressive (steatohepatitis with fibrosis stage 2‐4). Hepatic peptides were analyzed on an API Qstar XL quadrupole time‐of‐flight mass spectrometer using Analyst QS software. Linear trends tests were performed and used to screen for differential abundance. Nine known proteins were expressed with differential abundance between study groups. For seven proteins differential abundance is likely to have been on the basis increased hepatic lipid content and/or inflammation. Lumican, a 40‐kDa keratin sulfate proteoglycan that regulates collagen fibril assembly and activates transforming growth factor‐beta and smooth muscle actin, was expressed similarly in obese normal and SS but was overexpressed in a progressive manner in NASH‐mild versus SS (124%, P < 0.001), NASH‐progressive versus NASH‐mild (156%, P < 0.001) and NASH‐progressive versus obese normal (178%, P < 0.001). Fatty acid binding protein‐1 (FABP‐1), which is protective against the detergent effects of excess free fatty acids, facilitates intracellular free fatty acid transport and is an important ligand for peroxisome proliferator‐activated receptor–mediated transcription, was overexpressed in SS when compared to the obese normal group (128%, P < 0.001), but was paradoxically underexpressed in NASH‐mild versus SS (73%, P < 0.001), NASH‐progressive versus NASH‐mild (81%, P < 0.001), and NASH‐progressive versus obese normal (59%, P < 0.001). Conclusion: Histologically progressive NAFLD is associated with overexpression of lumican, an important mediator of fibrosis in nonhepatic tissues, whereas FABP‐1 is paradoxically underexpressed in NASH, suggesting a new potential mechanism of lipotoxicity in NAFLD. Further studies are needed to determine the biologic basis of lumican and/or FABP‐1 dysregulation in NAFLD. (HEPATOLOGY 2009;49:1375–1384.)

Low Circulating Levels of Dehydroepiandrosterone in Histologically Advanced Nonalcoholic Fatty Liver Disease

The biological basis of variability in histological progression of nonalcoholic fatty liver disease (NAFLD) is unknown. Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone and has been shown to influence sensitivity to oxidative stress, insulin sensitivity, and expression of peroxisome proliferator‐activated receptor alpha and procollagen messenger RNA. Our aim was to determine whether more histologically advanced NAFLD is associated with low circulating levels of DHEA. Serum samples were obtained prospectively at the time of liver biopsy in 439 patients with NAFLD (78 in an initial and 361 in validation cohorts) and in controls with cholestatic liver disease (n = 44). NAFLD was characterized as mild [simple steatosis or nonalcoholic steatohepatitis (NASH) with fibrosis stage 0‐2] or advanced (NASH with fibrosis stage 3‐4). Serum levels of sulfated DHEA (DHEA‐S) were measured by enzyme‐linked immunosorbent assay. Patients with advanced NAFLD had lower plasma levels of DHEA‐S than patients with mild NAFLD in both the initial (0.25 ± 0.07 versus 1.1 ± 0.09 μg/mL, P < 0.001) and validation cohorts (0.47 ± 0.06 versus 0.99 ± 0.04 μg/mL, P < 0.001). A “dose effect” of decreasing DHEA‐S and incremental fibrosis stage was observed with a mean DHEA‐S of 1.03 ± 0.05, 0.96 ± 0.07, 0.83 ± 0.11, 0.66 ± 0.11, and 0.35 ± 0.06 μg/mL for fibrosis stages 0, 1, 2, 3, and 4, respectively. All patients in both cohorts in the advanced NAFLD group had low DHEA‐S levels, with the majority in the hypoadrenal range. The association between DHEA‐S and severity of NAFLD persisted after adjusting for age. A relationship between disease/fibrosis severity and DHEA‐S levels was not seen in patients with cholestatic liver diseases. Conclusion: More advanced NAFLD, as indicated by the presence of NASH with advanced fibrosis stage, is strongly associated with low circulating DHEA‐S. These data provide novel evidence for relative DHEA‐S deficiency in patients with histologically advanced NASH. (HEPATOLOGY 2008;47:484–492.)

Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes

BACKGROUND & AIMS Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. RESULTS Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.

TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess

BACKGROUND & AIMS Low-grade chronic inflammation is a cardinal feature of the metabolic syndrome, yet its pathogenesis is not well defined. The purpose of this study was to examine the role of TRAIL receptor (TR) signaling in the pathogenesis of obesity-associated inflammation using mice with the genetic deletion of TR. METHODS TR knockout (TR(-/-)) mice and their littermate wild-type (WT) mice were fed a diet high in saturated fat, cholesterol and fructose (FFC) or chow. Metabolic phenotyping, liver injury, and liver and adipose tissue inflammation were assessed. Chemotaxis and activation of mouse bone marrow-derived macrophages (BMDMϕ) was measured. RESULTS Genetic deletion of TR completely repressed weight gain, adiposity and insulin resistance in FFC-fed mice. Moreover, TR(-/-) mice suppressed steatohepatitis, with essentially normal serum ALT, hepatocyte apoptosis and liver triglyceride accumulation. Gene array data implicated inhibition of macrophage-associated hepatic inflammation in the absence of the TR. In keeping with this, there was diminished accumulation and activation of inflammatory macrophages in liver and adipose tissue. TR(-/-) BMDMϕ manifest reduced chemotaxis and diminished activation of nuclear factor-κ B signaling upon activation by palmitate and lipopolysaccharide. CONCLUSIONS These data advance the concept that macrophage-associated hepatic and adipose tissue inflammation of nutrient excess requires TR signaling.

Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis

Lumican, an extracellular matrix proteoglycan was previously shown to be upregulated with increasing severity of nonalcoholic steatohepatitis (NASH). Although lumican is involved in collagen fibrillogenesis in extra-hepatic tissues, little is known about the role of lumican in hepatic disease. We therefore determined lumican expression in etiologies other than clinical NASH. Our results indicated that lumican is upregulated in clinical samples of hepatitis C virus infection, in experimental rodent models of chronic and acute liver injury and could additionally be induced in vitro in response to the pro-fibrotic cytokine transforming growth factor β1 (TGFβ1) and to lipotoxic palmitic acid. Together, these results suggested a role for lumican in hepatic fibrosis. To investigate the functional role of lumican in hepatic fibrosis, lumican null (Null) and wild-type (WT) littermates were administered carbon tetrachloride intra-peritoneally. Serum and liver tissue were analyzed for indices of liver injury, fibrosis, matrix turnover, and proliferation. Hepatic fibrosis was greatly reduced in null animals (P<0.05). Paradoxically, gene expression of fibrosis-related genes such as TGFβ1 and collagen 1 was numerically higher in null animals though statistically insignificant from WT animals. On the other hand, α smooth muscle actin expression (α-SMA), a marker for activated fibroblasts, the main contributors of collagen production was significantly higher (P<0.05) in null animals as compared with WT littermates. Among the matrix metalloproteases (MMP), MMP13 was significantly increased (P<0.05) in null animals. Ultra-structural imaging indicated differences in the organization and spatial distribution of hepatic collagen fibrils of null and WT mice. Cell proliferation was significantly increased (P<0.05) in null animals. We conclude that lumican is a prerequisite for hepatic fibrosis. The protective effect of lumican deficiency in hepatic fibrosis appears to be downstream of collagen production and mediated through the combined effects of impaired collagen fibrillogenesis, increased matrix turnover, and an enhanced proliferative response.

Investigation of PNPLA3 and IL28B Genotypes on Diabetes and Obesity After Liver Transplantation: Insight Into Mechanisms of Disease

To identify genetic risks for obesity and diabetes postliver transplantation (LT), LT recipients underwent genotyping for IL28B rs12979860 (n = 295) and PNPLA3 rs738409 (n = 205) polymorphism in both donors and recipients. The development of obesity and diabetes/impaired fasting glucose (IFG) was determined 1–5 years post‐LT. Recipient PNPLA‐3 genotype was independently associated with obesity (BMI > 30) at 3 years posttransplant (genotype CC 33.7%, CG 48.3% and GG 82.4%, p = 0.002), with an odds ratio (OR 2.54, CI 1.38–4.66, p = 0.003), associated with the G allele. Diabetes/IFG diagnosed within 5 years posttransplant associated with PNPLA‐3 non‐CC genotype (HR 1.59, 1.12–2.26, p = 0.010), but not IL28B TT genotype (HR 1.46, 0.94–2.27, p = 0.092). No genotype variable was independently predictive of diabetes/IFG. The combination of PNPLA‐3 non‐CC and IL28B TT genotype was associated with increased risk of diabetes/IFG compared to PNPLA‐3 CC, IL28B non‐TT (HR 2.64, CI 1.30–5.39, p = 0.008). Donor genotypes were not associated with any of the outcomes analyzed. In conclusion, PNPLA‐3 non‐CC genotype is associated with posttransplant obesity but not independently with diabetes/IFG. The lack of donor related risk suggests a peripheral rather than central mechanism of insulin resistance in liver transplant recipients.

Growth hormone, dehydroepiandrosterone and adiponectin levels in non-alcoholic steatohepatitis: an endocrine signature for advanced fibrosis in obese patients.

Liver‐related clinical consequences of non‐alcoholic fatty liver disease (NAFLD) are seen only in the minority of patients with advanced fibrosis. The aim of our study was to generate insight into a potential endocrine basis of steatohepatitis with advanced fibrosis in NAFLD.

Noninvasive diagnosis of acute cellular rejection in liver transplant recipients: A proteomic signature validated by enzyme‐linked immunosorbent assay

The diagnosis of acute cellular rejection (ACR) requires liver biopsy with its attendant expense and risk. Our first aim was to prospectively determine in an exploratory analysis whether there is a serum proteome signature associated with histologically confirmed ACR. Our second aim was to use simpler and faster enzyme‐linked immunosorbent assay (ELISA)‐based assays for proteins identified as differentially abundant in the proteomic analysis to identify patients with ACR in a separate validation cohort. We used sequential high‐abundance protein depletion and isobaric tag for relative and absolute quantitation liquid chromatography–tandem mass spectrometry to characterize the serum proteome in serum samples of patients with or without ACR. Seven of the 41 proteins identified as differentially abundant [serum amyloid A, complement component 4 (C4), fibrinogen, complement component 1q (C1q), complement component 3, heat shock protein 60 (HSP60), and HSP70] could be measured with ELISA‐based assays in a validation cohort consisting of patients with ACR (n = 25) and patients without ACR (n = 21). The mean alanine aminotransferase (ALT) levels in patients with ACR and in patients without ACR were 198 ± 27 and 153 ± 34 U/L, respectively. Among the 7 proteins for which ELISA assays were available, C4 and C1q were both independent predictors of ACR. C4 had the greatest predictivity for differentiating patients with or without ACR. A C4 level ≤ 0.31 g/L had a sensitivity of 97%, a specificity of 62%, a positive predictive value of 74%, and a negative predictive value of 94%. A C4 level ≤ 0.31 g/L and an ALT level ≥ 70 IU/mL together had a sensitivity of 96%, a specificity of 81%, a positive predictive value of 86%, and a negative predictive value of 94%. In summary, in this exploratory analysis, serum C4 and ALT levels were highly predictive of ACR in liver transplant recipients. Confirmation in a prospective, larger, and diverse population is needed. Liver Transpl 17:723‐732, 2011.

A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.