Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Measurement of the whole body clearance of infused glycerol as a test of liver function after major hepatectomy.

Major liver resection can be used in the treatment of liver cancer. The functional capacity of liver parenchyma needs to be evaluated preoperatively because it conditions the outcome. We assessed whether the whole body clearance of glycerol, a substrate essentially metabolized in liver cells, may be suitable as a simple test of liver function. Seven patients after major hepatectomy, six patients after colectomy and 12 healthy subjects were studied. Patients were investigated on the first day after surgery. All participants were studied during a 150-min basal period followed by a 120-min infusion of 16 mumol kg-1 min-1 13C-labelled glycerol. Whole body glycerol clearance was calculated from the change in plasma glycerol concentration. Whole body glucose production was measured with 6,6 2H2 glucose infused as a tracer in the basal state and during glycerol infusion. In addition, 13C glucose synthesis was monitored to quantitate gluconeogenesis from glycerol. Patients after liver resection had higher plasma glycerol concentrations and lower whole body glycerol clearance than healthy subjects and patients after colectomy. They also had higher plasma glucagon concentrations. Their fasting glucose production was mildly elevated in the fasting state and did not change after glycerol infusion, indicating a normal hepatic autoregulation of glucose production. These results indicate that whole body glycerol clearance can be simply determined from plasma glycerol concentrations during exogenous glycerol infusion. It is significantly reduced in patients after major hepatectomy, suggesting that it constitutes a sensitive test of hepatic function. Its use as a preoperative testing procedure remains to be evaluated.

IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis c than the IL28b (IFN lambda 3) genotype

Background: Therapy of chronic hepatitis C (CHC) with pegIFNa/ribavirin achieves sustained virologic response (SVR) in ~55%. Pre-activation of the endogenous interferon system in the liver is associated non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. Methods: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. Results: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. Conclusions: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Atrial natriuretic peptides: reproducibility of renal effects and response of liver blood flow.

To assess the variability of the response to exogenous atrial natriuretic peptide (ANP), it was infused at the rate of 1 microgram/min for 2 h in 6 salt-loaded normal volunteers under controlled conditions on 2 occasions at an interval of 1 week. The effect on solute excretion and the haemodynamic and endocrine actions were highly reproducible. The constant ANP infusion caused a delayed and prolonged excretion of sodium, chloride and calcium, no change in potassium or phosphate excretion or in glomerular filtration rate but a marked decrease in renal plasma flow. Blood pressure, heart rate and the plasma levels of angiotensin II, aldosterone, arginine vasopressin and plasma renin activity were unaltered. The effect of a 2-h infusion of ANP 0.5 microgram/min or its vehicle on apparent hepatic blood flow (HBF) was also studied in 14 normal volunteers by measuring the indocyanine green clearance. A 21% decrease in HBF was observed in subjects who received the ANP infusion (p less than 0.01 vs vehicle). Thus, ANP infused at a dose that did not lower blood pressure decreased both renal and liver blood flow in normotensive volunteers. The renal and endocrine responses to ANP were reproducible over a 1-week interval.

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

A stable phage lysin (Cpl-1) dimer with increased antipneumococcal activity and decreased plasma clearance.

Bacteriophages (phages) produce endolysins (lysins) as part of their lytic cycle in order to degrade the peptidoglycan layer of the infected bacteria for subsequent release of phage progeny. Because these enzymes maintain their lytic and lethal activity against Gram-positive bacteria when added extrinsically to the cells, they have been actively exploited as novel anti-infectives, sometimes termed enzybiotics. As with other relatively small peptides, one issue in their clinical development is their rapid inactivation through proteolytic degradation, immunological blockage and renal clearance. The antipneumococcal lysin Cpl-1 was shown to escape both proteolysis and immunological blockage. However, its short plasma half-life (20.5 min in mice) may represent a shortcoming for clinical usefulness. Here we report the construction of a Cpl-1 dimer with a view to increasing both the antipneumococcal specific activity and plasma half-life of Cpl-1. Dimerisation was achieved by introducing specific cysteine residues at the C-terminal end of the enzyme, thus favouring disulphide bonding. Compared with the native monomer, the constructed dimer demonstrated a two-fold increase in specific antipneumococcal activity and a ca. ten-fold decrease in plasma clearance. As several lysins are suspected to dimerise on contact with their cell wall substrate to be fully active, stable pre-dimerised enzymes may represent a more efficient alternative to the native monomer.

Actualités diagnostiques et thérapeutiques dans le domaine de l'encéphalopathie hépatique [Hepatic encephalopathy: recent developments].

Hepatic encephalopathy is a neurological syndrome occurring in patients with liver failure or in those with a large porto-systemic shunt. In cirrhotic patients, the current classification comprises covert and overt encephalopathy. Diagnosis of covert encephalopathy requires sensitive tests. Lactulose and rifaximin are the two leading therapeutic options. Rifaximin is efficacious for maintaining remission from hepatic encephalopathy. Liver transplantation should be discussed in cirrhotic patients with encephalopathy.

Fractional hepatic de novo lipogenesis in healthy subjects during near-continuous oral nutrition and bed rest: a comparison with published data in artificially fed, critically ill patients.

BACKGROUND AND AIMS: In critically ill patients, fractional hepatic de novo lipogenesis increases in proportion to carbohydrate administration during isoenergetic nutrition. In this study, we sought to determine whether this increase may be the consequence of continuous enteral nutrition and bed rest. We, therefore, measured fractional hepatic de novo lipogenesis in a group of 12 healthy subjects during near-continuous oral feeding (hourly isoenergetic meals with a liquid formula containing 55% carbohydrate). In eight subjects, near-continuous enteral nutrition and bed rest were applied over a 10 h period. In the other four subjects, it was extended to 34 h. Fractional hepatic de novo lipogenesis was measured by infusing(13) C-labeled acetate and monitoring VLDL-(13)C palmitate enrichment with mass isotopomer distribution analysis. Fractional hepatic de novo lipogenesis was 3.2% (range 1.5-7.5%) in the eight subjects after 10 h of near continuous nutrition and 1.6% (range 1.3-2.0%) in the four subjects after 34 h of near-continuous nutrition and bed rest. This indicates that continuous nutrition and physical inactivity do not increase hepatic de novo lipogenesis. Fractional hepatic de novo lipogenesis previously reported in critically ill patients under similar nutritional conditions (9.3%) (range 5.3-15.8%) was markedly higher than in healthy subjects (P&lt;0.001). These data from healthy subjects indicate that fractional hepatic de novo lipogenesis is increased in critically ill patients.

Variable viral clearance despite adequate ganciclovir plasma levels during valganciclovir treatment for cytomegalovirus disease in D+/R- transplant recipients.

ABSTRACT: BACKGROUND: Valganciclovir, the oral prodrug of ganciclovir, has been demonstrated equivalent to iv ganciclovir for CMV disease treatment in solid organ transplant recipients. Variability in ganciclovir exposure achieved with valganciclovir could be implicated as a contributing factor for explaining variations in the therapeutic response. This prospective observational study aimed to correlate clinical and cytomegalovirus (CMV) viral load response (DNAemia) with ganciclovir plasma concentrations in patients treated with valganciclovir for CMV infection/disease. METHODS: Seven CMV D+/R- transplant recipients (4 kidney, 2 liver and 1 heart) were treated with valganciclovir (initial dose was 900-1800 mg/day for 3-6.5 weeks, followed by 450-900 mg/day for 2-9 weeks). DNAemia was monitored by real time quantitative PCR and ganciclovir plasma concentration was measured at trough (Ctrough) and 3 h after drug administration (C3h) by HPLC. RESULTS: Four patients presented with CMV syndrome, two had CMV tissue-invasive disease after prophylaxis discontinuation, and one liver recipient was treated pre-emptively for asymptomatic rising CMV viral load 5 weeks post-transplantation in the absence of prophylaxis. CMV DNAemia decreased during the first week of treatment in all recipients except in one patient (median decrease: -1.2 log copies/mL, range: -1.8 to 0) despite satisfactory ganciclovir exposure (AUC0-12 = 48 mg.h/L, range for the 7 patients: 40-118 mg.h/L). Viral clearance was obtained in five patients after a median of time of 34 days (range: 28-82 days). Two patients had recurrent CMV disease despite adequate ganciclovir exposure (65 mg.h/L, range: 44-118 mg.h/L). CONCLUSIONS: Valganciclovir treatment for CMV infection/disease in D+/R- transplant recipients can thus result in variable viral clearance despite adequate ganciclovir plasma concentrations, probably correlating inversely with anti-CMV immune responses after primary infection.

The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms.

Background: The modulation of energetic homeostasis by pollutants has recently emerged as a potential contributor to the onset of metabolic disorders. Diethylhexyl phthalate (DEHP) is a widely used industrial plasticizer to which humans are widely exposed. Phthalates can activate the three peroxisome proliferatoractivated receptor (PPAR) isotypes on cellular models and induce peroxisome proliferation in rodents.Objectives: In this study, we aimed to evaluate the systemic and metabolic consequences of DEHP exposure that have remained so far unexplored and to characterize the underlying molecular mechanisms of action.Methods: As a proof of concept and mechanism, genetically engineered mouse models of PPARs were exposed to high doses of DEHP, followed by metabolic and molecular analyses.Results: DEHP-treated mice were protected from diet-induced obesity via PPARalpha-dependent activation of hepatic fatty acid catabolism, whereas the activity of neither PPARbeta nor PPARgamma was affected. However, the lean phenotype observed in response to DEHP in wild-type mice was surprisingly abolished in PPARalpha-humanized mice. These species differences are associated with a different pattern of coregulator recruitment.Conclusion: These results demonstrate that DEHP exerts species-specific metabolic actions that rely to a large extent on PPARalpha signaling and highlight the metabolic importance of the species-specific activation of PPARalpha by xenobiotic compounds. Editor's SummaryDiethylhexyl phthalate (DEHP) is an industrial plasticizer used in cosmetics, medical devices, food packaging, and other applications. Evidence that DEHP metabolites can activate peroxisome proliferatoractivated receptors (PPARs) involved in fatty acid oxidation (PPARalpha and PPARbeta) and adiposite function and insulin resistance (PPARgamma) has raised concerns about potential effects of DEHP on metabolic homeostasis. In rodents, PPARalpha activation also induces hepatic peroxisome proliferation, but this response to PPARalpha activation is not observed in humans. Feige et al. (p. 234) evaluated systemic and metabolic consequences of high-dose oral DEHP in combination with a high-fat diet in wild-type mice and genetically engineered mouse PPAR models. The authors report that mice exposed to DEHP gained less weight than controls, without modifying their feeding behavior; they also exhibited lower triglyceride levels, smaller adipocytes, and improved glucose tolerance compared with controls. These effects, which were observed in mice fed both high-fat and standard diets, appeared to be mediated by PPARalpha-dependent activation of hepatic fatty acid catabolism without apparent involvement of PPARbeta or PPARgamma. However, mouse models that expressed human (versus mouse) PPARalpha tended to gain more weight on a high-fat diet than their DHEP-unexposed counterparts. The authors conclude that findings support species-specific metabolic effects of DEHP mediated by PPARalpha activation.

Prognostic value of postoperative carcinoembryonic antigen concentration and extent of invasion of resection margins after hepatic resection for colorectal metastases.

OBJECTIVE: To evaluate the prognostic value of postoperative concentration of carcinoembryonic antigen (CEA) and extent of surgical margins after resection of liver metastases from colorectal cancer. DESIGN: Retrospective study. SETTING: Teaching hospital, Switzerland. SUBJECTS: 49 patients with hepatic metastases after primary colorectal cancer. INTERVENTIONS: Resection of hepatic metastases MAIN OUTCOME MEASURES: Assessment of prognostic value of variables by univariate and multivariate analysis. RESULTS: Median survival was 24 months (range 5-86 months). Resection margins were clear (> 1-cm) in 10, close (< 1-cm) in 25 and invaded in 9 patients. On univariate analysis, a postoperative concentration of CEA of <4ng/ml was correlated with prolonged survival (p < 0.001), but the width of the resection margin was not of prognostic importance. There was no correlation between width of resection margins and postoperative concentration of CEA (p = 0.5). On multivariate analysis, postoperative concentrations of CEA of 4 ng/ml or more were associated with increased risk of death (relative risk 7.3; 95% confidence interval (CI) 2.8-18.7, p < 0.001). CONCLUSION: Postoperative CEA offers better prognostic discrimination than the width of resection margins after resection of liver metastases from colorectal tumours. Some patients with invaded resection margins did survive for 3 years, but no patient did whose CEA concentration was 4 ng/ml or more. The definition of a potentially curative hepatic resection should include a postoperative CEA concentration of <4 ng/ml (within the reference range).

Mechanisms of postprandial hyperglycemia in liver transplant recipients: comparison of liver transplant patients with kidney transplant patients and healthy controls.

Impaired glucose tolerance or diabetes mellitus are frequent complications after organ transplantation, and are usually attributed to glucocorticoid and immunosuppressive treatments. Liver transplantation results in total hepatic denervation which may also affect glucoregulation. We therefore evaluated postprandial glucose metabolism in a group of patients with liver cirrhosis before and after orthotopic liver transplantation. Seven patients with liver cirrhosis of various etiologies, 6 patients having received a kidney transplant, and 6 healthy subjects were studied. Their glucose metabolism was evaluated in the basal state and over 4 hours after ingestion of a glucose load with 6.6 (2) H glucose dilution analysis. The patients with liver cirrhosis were studied before, and again 4 weeks (range 2-6) and 38 weeks (range 20-76, n=6) after orthotopic liver transplantation. Basal glucose metabolism was similar in liver and kidney transplant recipients. Impaired glucose tolerance was present in both groups, but postprandial hyperglycemia was exaggerated and lasted longer in liver transplant patients. Postprandial insulinemia was lower in liver transplant recipients, while C-peptide concentrations were comparable to those of kidney transplant recipients, indicating increased insulin clearance. Glucose turnover was not altered in both groups of patients during the initial 3 hours after glucose ingestion, but was higher in liver transplant early after transplantation during the fourth hour. Postprandial hyperglycemia remained unchanged in liver transplant recipients 38 weeks after liver transplantation, despite substantial reduction of immunosuppressive and glucocorticoid doses. We conclude that liver transplant recipients have severe postprandial hyperglycemia which can be attributed to insulinopenia (secondary, at least in part, to increased insulin clearance) and a late increased glucose turnover. These changes may be secondary to hepatic denervation.