Virus Genotype-Dependent Transcriptional Alterations in Lipid Metabolism and Inflammation Pathways in the Hepatitis C Virus-infected Liver.

Despite advances in antiviral therapy, molecular drivers of Hepatitis C Virus (HCV)-related liver disease remain poorly characterised. Chronic infection with HCV genotypes (1 and 3) differ in presentation of liver steatosis and virological response to therapies, both to interferon and direct acting antivirals. To understand what drives these clinically important differences, liver expression profiles of patients with HCV Genotype 1 or 3 infection (n = 26 and 33), alcoholic liver disease (n = 8), and no liver disease (n = 10) were analysed using transcriptome-wide microarrays. In progressive liver disease, HCV genotype was the major contributor to altered liver gene expression with 2151 genes differentially expressed >1.5-fold between HCV Genotype 1 and 3. In contrast, only 6 genes were altered between the HCV genotypes in advanced liver disease. Induction of lipogenic, lipolytic, and interferon stimulated gene pathways were enriched in Genotype 1 injury whilst a broad range of immune-associated pathways were associated with Genotype 3 injury. The results are consistent with greater lipid turnover in HCV Genotype 1 patients. Moreover, the lower activity in inflammatory pathways associated with HCV genotype 1 is consistent with relative resistance to interferon-based therapy. This data provides a molecular framework to explain the clinical manifestations of HCV-associated liver disease.

Assessment of adult patients with chronic liver failure for liver transplantation in 2015: who and when?

In 2015, there are a few absolute contraindications to liver transplantation. In adult patients, survival post-liver transplant is excellent, with 1-year survival rate >90% and 5-year survival rates >80% and predicted median allograft survival beyond 20 years. Patients with a Child-Turcotte Pugh score ≥9 or a model for end-stage liver disease (MELD) score >15 should be referred for liver transplantation, with patients who have a MELD score >17 showing a 1-year survival benefit with liver transplantation. A careful selection of hepatocellular cancer patients results in excellent outcomes, while consideration of extra-hepatic disease (reversible vs irreversible) and social support structures are crucial to patient assessment. Alcoholic liver disease remains a challenge, and the potential to cure hepatitis C virus infection together with the emerging issue of non-alcoholic fatty liver disease-associated chronic liver failure will change the landscape of the who in the years ahead. The when will continue to be determined largely by the severity of liver disease based on the MELD score for the foreseeable future.

Longitudinal assessment of hepatitis C fibrosis progression by collagen and smooth muscle actin morphometry in comparison to serum markers.

BACKGROUND: Assessment of fibrosis progression in chronic liver disease relies upon non-invasive tools and changes in semi-quantitative histopathology scores that may not be reliable. AIM: To assess the diagnostic performance of the FibroSURE (FS) index and collagen/alpha smooth muscle actin (α-SMA) morphometry in relation to longitudinal changes in fibrosis on paired biopsies. METHODS: The study cohort included 201 chronic hepatitis C (CHC) nonresponders enrolled in a prior phase II anti-fibrotic study. Serum FS and paired biopsies, with both collagen and α-SMA morphometry, were evaluated at baseline and week 52. RESULTS: Study patients were mostly male (67%) and Caucasian (77%), with Ishak stages 2 (n = 79), 3 (n = 88) and 4 (n = 30), excluded (n = 4 stage 1 or 5). Mean biopsy length was 22.9 mm. For baseline Ishak 2/3 vs. 4, there were no significant differences in AUROCs for collagen (0.71), SMA (0.66) or FS (0.70). At week 52, 62% of patients had no change in Ishak stage, but collagen/α-SMA increased by 34-51% (P < 0.0001), and FS decreased by 5% (P = 0.008). Among the 33% of patients with +/-1 Ishak stage change, FS changes were not significant, but α-SMA increased 29-72%, and collagen increased by 12-38% (P = 0.01 for +1 only). CONCLUSIONS: Longitudinal changes in collagen and α-SMA morphometry are apparent prior to change in histological stage or FibroSURE in CHC nonresponders with intermediate fibrosis. This likely reflects quantitative morphological differences that are not detected by routine histological staging or serum markers such as FibroSURE.

Lower serum fibroblast activation protein shows promise in the exclusion of clinically significant liver fibrosis due to non-alcoholic fatty liver disease in diabetes and obesity.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) is common in diabetes and obesity but few have clinically significant liver fibrosis. Improved risk-assessment is needed as the commonly used clinical-risk algorithm, the NAFLD fibrosis score (NFS), is often inconclusive. AIMS: To determine whether circulating fibroblast activation protein (cFAP), which is elevated in cirrhosis, has value in excluding significant fibrosis, particularly combined with NFS. METHODS: cFAP was measured in 106 with type 2 diabetes who had transient elastography (Cohort 1) and 146 with morbid obesity who had liver biopsy (Cohort 2). RESULTS: In Cohort 1, cFAP (per SD) independently associated with median liver stiffness (LSM) ≥ 10.3 kPa with OR of 2.0 (95% CI 1.2-3.4), p=0.006. There was 0.12 OR (95% CI 0.03-0.61) of LSM ≥ 10.3 kPa for those in the lowest compared with the highest FAP tertile (p=0.010). FAP levels below 730 pmol AMC/min/mL had 95% NPV for LSM ≥ 10.3 kPa and reclassified 41% of 64 subjects from NFS 'indeterminate-risk' to 'low-risk'. In Cohort 2, cFAP (per SD), associated with 1.7 fold (95% CI 1.1-2.8) increased odds of significant fibrosis (F ≥ 2), p=0.021, and low cFAP reclassified 49% of 73 subjects from 'indeterminate-risk' to 'low-risk'. CONCLUSIONS: Lower cFAP, when combined with NFS, may have clinical utility in excluding significant fibrosis in diabetes and obesity.

Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection.

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

Terlipressin therapy for moderate-to-severe hyponatraemia in patients with liver failure.

BACKGROUND: Hyponatraemia in liver failure is associated with increased morbidity and mortality. Improving serum sodium in liver failure has been observed in patients receiving terlipressin. METHODS: We assessed the response of hyponatraemia in patients with liver failure to terlipressin using comparative retrospective analysis. RESULTS: Twenty-three patients received terlipressin for hyponatraemia after failed conservative management (median age 52 years (27-67), model for end-stage liver disease score 28 (16-38)). The median therapy was 7 days (1-27), with an average total dose of 25 mg (4-90) and a mean follow up of 51 days (5-1248). These patients were compared with 11 hyponatraemic patients managed conservatively during the same period with comparable age, baseline serum sodium and follow up. After 1 week of terlipressin therapy, serum sodium increased from a median of 120 (115-128) to 129 mmol/L (121-144) (P < 0.001), and at the end of terlipressin therapy, the serum sodium had increased significantly to 131 mmol/L (120-148) (P < 0.001). In comparison, in the conservatively managed group, the serum sodium did not increase significantly from the baseline of 123 (117-127) mmol/L. Adverse events occurred in 26% of patients receiving terlipressin, which predominantly pulmonary oedema. Importantly, more hyponatraemic patients treated with terlipressin (48%) were alive compared with the conservative group (18%), despite the latter having a significantly lower baseline median MELD score of 21 (16-30) (P = 0.008). Moreover, the transplant-free survival was higher in the terlipressin (30%) compared with the conservative group (0%). CONCLUSIONS: Terlipressin is effective in treating hyponatraemia in liver failure. Importantly, terlipressin use results in better transplant-free survival but also more adverse events.

Diabetes and nonalcoholic Fatty liver disease: a pathogenic duo.

Recent data increasingly support a complex interplay between the metabolic condition diabetes mellitus and the pathologically defined nonalcoholic fatty liver disease (NAFLD). NAFLD predicts the development of type 2 diabetes and vice versa, and each condition may serve as a progression factor for the other. Although the association of diabetes and NAFLD is likely to be partly the result of a "common soil," it is also probable that diabetes interacts with NAFLD through specific pathogenic mechanisms. In particular, through interrelated metabolic pathways currently only partly understood, diabetes appears to accelerate the progression of NAFLD to nonalcoholic steatohepatitis, defined by the presence of necroinflammation, with varying degrees of liver fibrosis. In the research setting, obstacles that have made the identification of clinically significant NAFLD, and particularly nonalcoholic steatohepatitis, difficult are being addressed with the use of new imaging techniques combined with risk algorithms derived from peripheral blood profiling. These techniques are likely to be used in the diabetes population in the near future. This review examines the pathogenic links between NAFLD and diabetes by exploring the epidemiological evidence in humans and also through newer animal models. Emerging technology to help screen noninvasively for differing pathological forms of NAFLD and the potential role of preventive and therapeutic approaches for NAFLD in the setting of diabetes are also examined.

Increasing liver transplantation waiting list mortality: a report from the Australian National Liver Transplantation Unit, Sydney.

BACKGROUND: We aimed to describe the demand for liver transplantation (LTx) and patient outcomes on the waiting list at the Australian National Liver Transplantation Unit, Sydney over the last 20 years. METHODS: We performed a retrospective analysis with the data divided into three eras: 1985-1993, 1994-2000 and 2001-2008. RESULTS: The number of patients accepted for LTx increased from 320 to 372 and 548 (P < 0.001) with the number of LTx being performed increasing from 262 to 312 and 452 respectively (P < 0.001). The median adult recipient age increased from 45 to 48 and 52 years (P < 0.001) while it decreased in children from 4 to 2 and 1 years respectively (P = 0.001). In parallel, the deceased donor offers decreased from 1003 to 720 and 717 (P < 0.001). Methods to improve access to donor livers have been used with the use of split livers, extended criteria and non-heart beating donors, resulting in increased acceptance of deceased donor offers by 65% and 115% in the second and third eras when compared with the first era (P < 0.001). However, the adult median waiting time has increased from 23 to 41 and 120 days respectively (P < 0.001). This was associated with increased adult mortality on the waiting list from 23 to 40 and 122 respectively (P < 0.001). CONCLUSIONS: Despite the increasing proportion of donor offers being used, the waiting list mortality is increasing. A solution to this problem is an increase in organ donation to keep pace with the escalating demand for LTx.

Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications.

CD147 is a highly glycosylated transmembrane protein that is known to play a role in regulation of many protein families. It has the unique ability to maintain functional activity in both the membrane bound state and in the soluble form. CD147 is known to play a role in regulation of matrix metalloproteinase (MMP) expression, but whether its expression is affected by the diabetic milieu is not known, and its role in regulation of monocyte MMPs in this environment has not been investigated. Therefore, in this study we investigated the effect of advanced glycation end products (AGEs) and high glucose (HG; 25 mM), on monocyte CD147 expression. Culture of THP-1 monocytes in the presence of AGEs or HG significantly increased CD147 at the gene and protein level. THP-1 cell results were confirmed using freshly isolated monocytes from human volunteers. The effect of AGEs and HG on CD147 expression was also mimicked by addition of proinflammatory cytokines. Addition of AGEs or HG also increased expression of monocyte MMP-1 and MMP-9 but not MMP-2. This increase in MMPs was significantly attenuated by inhibition of CD147 using either a small interfering RNA or an anti-CD147 antibody. Inhibition of NF-κB or addition of antibodies to either TNF-α or the receptor for AGE (RAGE) each significantly prevented in a dose-dependent manner the induction of CD147 gene and protein by AGE and also decreased MMP-1 and MMP-9. This novel result shows that AGEs can induce monocyte CD147 expression, an effect mediated by inflammatory pathways and RAGE. Because MMPs play a role in monocyte migration, inhibition of their regulator CD147 may assist in the prevention of diabetic complications, particularly those where monocyte infiltration is an early initiating event.

Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

BACKGROUND: Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood. AIM: To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes. METHODS: Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real-time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (> or =1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low-level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance.

The hallmarks of chronic liver diseases are chronic inflammation, cellular damage, regeneration and fibrosis. An appreciation of intrahepatic molecular expression patterns in normal and diseased liver provides clues for understanding pathogenic pathways whilst studies of the structure and function of molecules implicated in liver disease provide insights into their potential as therapeutic targets. We have examined the expression, function, molecular structure and structure-function relationships of type IV dipeptidyl aminopeptidases. In particular, the roles of CD26/DPPIV in T-cell proliferation and chemotaxis and of fibroblast activation protein in human cirrhosis are discussed. We have investigated the pathogenesis of liver disease by characterising patterns of cytokine and growth factor expression in experimental and human cirrhosis. We have quite recently expanded this approach to use differential gene expression analyses to elucidate overall pathways of gene activation and suppression in human cirrhosis. In addition, our detailed molecular and cellular studies of the mechanisms of spontaneous liver transplant tolerance have generated novel insights into this process. This review touches on these diverse aspects of liver function and disease.

Video push enteroscopy in the investigation of small bowel disease: defining clinical indications and outcomes.

BACKGROUND: Push enteroscopy is a new technique for investigation of the small intestine. The clinical indications are still being defined. It also offers the potential for therapeutic intervention in suitable cases. AIMS: To evaluate further the role of push enteroscopy in the diagnosis and treatment of patients with suspected or known small bowel disease. METHODS: A prospective record was kept of all patients having enteroscopy at Royal Prince Alfred Hospital between March 1995 and July 1997. The procedure was performed 73 times in 68 patients. Indications and diagnoses were noted. The outcome in patients with obscure gastrointestinal bleeding or anaemia in whom a vascular lesion was treated with a heater probe was also determined. RESULTS: Enteroscopy was performed in 23 patients for gastrointestinal bleeding of obscure origin. An active or possible bleeding source was found in 13 (57%). The commonest of these was jejunal angiodysplasia. In the 21 patients with chronic iron deficiency anaemia a lesion was found in ten (48%). The majority of these were in the stomach, as described by others. The diagnostic yield in the 16 patients having enteroscopy for known or suspected small bowel disease was 56%. One patient underwent balloon dilatation of a postoperative jejunal stricture. Eleven patients with obscure bleeding or anaemia had ablation of a vascular lesion with a heater probe. Transfusion requirements fell after this procedure, particularly in those with active bleeding at the time of the examination. In five of the 11 no further transfusions were required in over six months of follow-up. CONCLUSIONS: The most common indications for enteroscopy are obscure gastrointestinal bleeding, chronic anaemia and known or suspected small bowel disease. A positive result can be expected in over 50% of patients. The treatment of vascular lesions via the enteroscope has a significant impact of subsequent transfusion requirements.

Copper-salicylate gel for pain relief in osteoarthritis: a randomised controlled trial.

OBJECTIVE: To assess the efficacy and safety of a copper-salicylate gel in osteoarthritis of the hip and knee. DESIGN: Randomised, double-blind, placebo-controlled study. SETTING: Rheumatology Clinic of St Vincent's Hospital, Sydney, New South Wales (a tertiary referral hospital), June 1993 to October 1994. PATIENTS: 116 patients with pain associated with osteoarthritis of the hip and/or knee (diagnosed by criteria of the European League against Rheumatism), drawn from patients attending the Clinic or self-referred after newspaper advertisements. INTERVENTION: Copper-salicylate or placebo gel (1.5 g) applied twice daily to the forearm for four weeks. OUTCOME MEASURES: Self-assessment of pain before the trial and after two and four weeks of treatment; patient and investigator assessments of efficacy; additional analgesia required; adverse reactions; and withdrawal rates. RESULTS: Pain scores at rest and on movement decreased in both the copper-salicylate and placebo groups by 13%-20%. There was no significant difference between the two groups for decrease in pain score, patient and investigator efficacy ratings, number of patients requiring paracetamol for extra analgesia (active, 77%; placebo, 71%) and average dose of paracetamol (active, 555 mg/day; placebo, 600 mg/day). Significantly more patients in the copper-salicylate group reported adverse reactions (83% versus 52% of the placebo group), most commonly skin reactions, and withdrew from the trial because of these reactions (17% versus 1.7% of the placebo group). CONCLUSION: Copper-salicylate gel applied to the forearm was no better than placebo gel as pain relief for patients with osteoarthritis of the hip or knee, but produced significantly more skin rashes.

Hepatic glutathione degradation within the sinusoids of guinea pig livers shows a periportal localization of gamma-glutamyl transferase activity.

In utilizing intact perfused guinea pig livers a probable periportal localization of gamma-glutamyl transferase (GGT) activity was apparent. Untreated livers perfused from the portal to hepatic vein had a significantly greater glutathione efflux than the same livers perfused from the hepatic to portal vein. Switching the perfusion direction also saw a significant rise in the cysteinylglycine efflux. It was apparent from the results that the efflux of glutathione degradation products from the guinea pig livers was significantly greater than those of the intact tripeptide. However, with maximal GGT inhibition glutathione accounted for 90% of the glutathione-related thiols exported from the liver with cysteine accounting for the remaining 10%. Therefore, glutathione exported from hepatocytes may act as a means of transporting its constituent amino acids. The periportal localization of GGT activity ensures that the more susceptible perivenous region faces the greatest concentration of glutathione degradation products.