Experimental densities of subcooled deuterium oxide at pressures up to 160 MPa.

In this work, the experimental results of deuterium oxide density at high pressure and in a wide range of temperatures, by means of the pseudo-isochoric method, are presented. A specific stainless steel cell was devised to be used as a pycnometer and filled with variable mass of heavy water. The latter was measured by weighing with an analytical balance and using the substitution method. The volume of the pycnometric cell was measured by the gravimetric method and corrected for the effect of temperature and pressure. Each measurement cycle was performed at constant mass, measuring pressure as a function of temperature at equilibrium. From the mass and volume values, density was calculated according to its definition. Heavy water density was measured for temperatures down to 253 K and for pressures up to 163 MPa, thus both in stable and supercooled metastable states. All terms contributing to the uncertainty in determining the volume and the mass were considered, obtaining an expanded relative uncertainty of deuterium oxide density of 0.04%.

Hepatitis delta virus and hepatocellular carcinoma: an update.

Hepatitis delta virus (HDV) is a defective RNA virus that depends on the presence of hepatitis B virus (HBV) for the creation of new virions and propagation of the infection to hepatocytes. Chronic infection with HDV is usually associated with a worsening of HBV infection, leading more frequently to cirrhosis, increased risk of liver decompensation and hepatocellular carcinoma (HCC) occurrence. In spite of a progressive declining prevalence of both acute and chronic HDV infection observed over several years, mainly due to increased global health policies and mass vaccination against HBV, several European countries have more recently observed stable HDV prevalence mainly due to migrants from non-European countries. Persistent HDV replication has been widely demonstrated as associated with cirrhosis development and, as a consequence, development of liver decompensation and occurrence of HCC. Several treatment options have been attempted with poor results in terms of HDV eradication and improvement of long-term prognosis. A global effort is deemed urgent to enhance the models already existing as well as to learn more about HDV infection and correlated tumourigenesis mechanisms.

Determinants of esophageal varices bleeding in patients with advanced hepatocellular carcinoma treated with sorafenib.

BACKGROUND AND AIMS: Sorafenib is the standard of care for patients with advanced hepatocellular carcinoma (HCC), yet treatment safety may be challenged by portal hypertension. We therefore assessed the prevalence, risk factors and clinical consequences of esophageal varices (EVs) in sorafenib-treated patients with HCC. METHODS: Starting in 2008, all compensated patients with advanced or intermediate HCC not eligible for other therapies were consecutively enrolled in a prospective evaluation of sorafenib therapy, all with pretreatment by upper-gastrointestinal endoscopy (UGE). RESULTS: A total of 150 patients received sorafenib for 4.6 (95% CI, 3.3-5.6) months. At baseline, 61 (41%) patients were EV free (group A), 78 (52%) had EVs (61 small EVs (group B), 17 medium/large EVs (group C)) and 11 (7%) previously endoscopically treated EVs (group D). Propranolol was given to all patients with medium/large EVs and those with previous bleeding. Twelve patients (8%) bled from EVs after 36 (18-260) days of sorafenib. During sorafenib, bleeding occurred in six of 26 group B patients with neoplastic portal vein thrombosis (nPVT), three of nine group C patients with nPVT, two of five group D patients with nPVT and one of six without nPVT (p < 0.0001), nPVT being the strongest independent predictor of bleeding by multivariate analysis (HR = 15.4, 95% CI 1.84-129.6). CONCLUSION: UGE screening is worthwhile in HCC patients allocated to sorafenib since it identifies patients with EVs at risk of bleeding during therapy, particularly those with nPVT.

Density measurements of subcooled water in the temperature range of (243 and 283) K and for pressures up to 400 MPa.

In this work, accurate density measurements of subcooled water (freshly double-distilled water) were performed along eight constant-mass curves in the temperature range of (243 to 283) K and in the pressure range of (140 to 400) MPa, by a pseudo-isochoric method. The experimental apparatus mainly consisted of a high pressure vessel, especially designed for this experiment, of known volume as a function of temperature and pressure, used to perform measurements in the T-p range under study. The density of subcooled water was obtained by measuring the equilibrium pressure at different temperatures, keeping the mass constant. All terms contributing to the uncertainty of subcooled water density measurements were considered; the estimated relative uncertainty, in the investigated temperature and pressure range, is about 0.07%. The experimental results were compared with the literature densities. In particular, the trend of density versus temperature for a constant mass of sample observed experimentally differs from the trend calculated by the equation provided by the International Association for Properties of Water and Steam (IAPWS-95) outside the range of validity, i.e., in the metastable region.

Hepatitis delta virus: Making the point from virus isolation up to 2014.

Chronic infection with hepatitis delta virus (HDV) has lately regained clinical importance because of the recent evidence of increasing prevalence in several European countries, due to immigration from highly endemic areas. HDV requires the mandatory presence of hepatitis B virus (HBV) for propagation to hepatocytes. It is transmitted by the same routes of HBV and it can be acquired either by co-infection (simultaneous transmission of the two viruses) or super-infection (acquisition of HDV by an already chronic carrier of HBV). As a consequence, every HBV carrier is potentially at risk for HDV superinfection. Since the clinical course of super-infection can be severe, early diagnosis of HDV infection is necessary.

High serum levels of HDV RNA are predictors of cirrhosis and liver cancer in patients with chronic hepatitis delta.

Chronic infection with the hepatitis delta virus (HDV) is a risk factor for cirrhosis and hepatocellular carcinoma (HCC), but little is known whether the outcome of hepatitis is predicted by serum markers of HDV and hepatitis B virus (HBV) infection. The aim of the study was to investigate these correlations in 193 patients with chronic HDV infection who had been followed up for a median of 9.5 years (4.8-19.3). HDV-RNA was first measured by qualitative in-house nested RT-PCR and quantified by in-house real-time PCR. HDV RNA levels only appeared significantly associated to HCC (univariate analysis: OR 1.32, 95% CI 1.02-1.71; p = 0.037; multivariate analysis: OR 1.42, 95% CI 1.04-1.95; p = 0.03). In non-cirrhotics at first presentation (n = 105), HDV RNA levels were associated with progression to cirrhosis (univariate analysis: OR = 1.57, 95% CI 1.20-2.05, p<0.001; multivariate analysis: OR = 1.60, 95% CI 1.20-2.12, p = 0.007) and development of HCC (univariate analysis: OR = 1.66, 95% CI 1.04-2.65, p = 0.033; multivariate analysis: OR = 1.88, 95% CI 1.11-3.19, p = 0.019). ROC analysis showed that approximately 600,000 HDV RNA copies/mL was the optimal cut-off value in our cohort of patients for discriminating the development of cirrhosis. High levels of HDV viremia in non-cirrhotic patients are associated with a considerable likelihood of progression to cirrhosis and the development of HCC. Once cirrhosis has developed, the role of HDV replication as a predictor of a negative outcome lessens.

Proteoglycans act as cellular hepatitis delta virus attachment receptors.

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.

[Role of the hepatitis Delta virus on the pathogenesis of hepatic cirrhosis and hepatocellular carcinoma. Recent advances]. / Ruolo del virus dell'epatite delta nella patogenesi della cirrosi epatica e del carcinoma epatocellulare: attuali acquisizioni.

Hepatitis Delta virus is a defective RNA virus that requires HBV as helper virus. The two viruses share the same route of transmission, being prevalently transmitted by contaminated blood and body fluids. HDV can infect simultaneously with HBV (coinfection) or in a patient with already established HBV infection (superinfection). A progressive decline in HDV prevalence both as acute or chronic infection has been observed for several years. More recently, several European countries have observed stable HDV prevalence mainly due to migrants from non European countries. Persistent HDV replication has been demonstrated as correlated to cirrhosis development and hepatocellular carcinoma occurrence. Moreover, persistent HDV replication predicts liver related mortality. Early and prolonged treatment may lead to virus eradication, improving long-term prognosis.

The diagnostic and economic impact of contrast imaging techniques in the diagnosis of small hepatocellular carcinoma in cirrhosis.

BACKGROUND: Contrast-enhanced ultrasound (CE-US), contrast CT scan and gadolinium dynamic MRI are recommended for the characterisation of liver nodules detected during surveillance of patients with cirrhosis with US. AIM: To assess the sensitivity, specificity, diagnostic accuracy and economic impact of all possible sequential combinations of contrast imaging techniques in patients with cirrhosis with 1-2 cm liver nodules undergoing US surveillance. PATIENTS/METHODS: 64 patients with 67 de novo liver nodules (55 with a size of 1-2 cm) were consecutively examined by CE-US, CT, MRI, and a fine-needle biopsy (FNB) as diagnostic standard. Undiagnosed nodules were re-biopsied; non-malignant nodules underwent enhanced imaging follow-up. The typical radiological feature of hepatocellular carcinoma (HCC) was arterial phase hypervascularisation followed by portal/venous phase washout. RESULTS: HCC was diagnosed in 44 (66%) nodules (2, <1 cm; 34, 1-2 cm; 8, >2 cm). The sensitivity of CE-US, CT and MRI for 1-2 cm HCC was 26, 44 and 44%, with 100% specificity, the typical vascular pattern of HCC being identified in 22 (65%) by a single technique versus 12 (35%) by at least two techniques carried out at the same time point (p=0.028). Compared with the cheapest dual examination (CE-US+CT), the cheapest single technique of stepwise imaging diagnosis of HCC was equally expensive (euro 26 440 vs euro 28 667), but led to a 23% reduction of FNB procedures (p=0.031). CONCLUSIONS: In patients with cirrhosis with a 1-2 cm nodule detected during surveillance, a single imaging technique showing a typical contrast pattern confidently permits the diagnosis of HCC, thereby reducing the need for FNB examinations.

A 28-year study of the course of hepatitis Delta infection: a risk factor for cirrhosis and hepatocellular carcinoma.

BACKGROUND & AIMS: Chronic infection with hepatitis Delta virus (HDV) is a risk factor for cirrhosis and hepatocellular carcinoma (HCC); predictors of disease outcome are, however, poorly defined. We tracked the course of HDV infection in 299 patients over a mean period of 233 months. METHODS: We analyzed data from patients who had been HDV positive for at least 6 months (230 males; mean age, 30 years) admitted from 1978 to 2006 to Maggiore Hospital, Milan. HDV infection was defined by the presence of HDV antigen in liver tissue or serum HDV RNA in anti-HDV/hepatitis B surface antigen seropositive patients. At enrollment, 7 patients had acute hepatitis, 101 had mild-moderate chronic hepatitis, 76 had severe chronic hepatitis, and 104 had histologic or clinical cirrhosis. Ninety patients were treated with interferon, 62 with corticosteroids, and 12 with nucleoside analogues; 135 received no therapy. RESULTS: Over a mean period of 233 months, 82 patients developed cirrhosis. Among the 186 total patients with cirrhosis, 46 developed HCC, 43 ascites, 44 jaundice, and 1 encephalopathy. Female sex, alcohol abuse, and HDV replication were associated with liver decompensation; HBV replication and interferon were associated with HCC development. By the end of the study, 186 patients were still alive, 63 had died, and 29 had received liver transplants. The main cause of death was liver failure (n = 37, 59%); HDV replication was the only independent predictor of mortality. CONCLUSIONS: Persistent HDV replication leads to cirrhosis and HCC at annual rates of 4% and 2.8%, respectively, and is the only predictor of liver-related mortality.

Endothelial protein C receptor plasma levels increase in chronic liver disease, while thrombomodulin plasma levels increase only in hepatocellular carcinoma.

INTRODUCTION: Thrombomodulin (TM) and endothelial protein C receptor (EPCR) are two transmembrane endothelial receptors involved in the protein C pathway, that regulates coagulation and inflammation processes. We postulated that soluble thrombomodulin and EPCR are plasmatic markers of progression to hepatocellular carcinoma (HCC) and prognostic indicators in cirrhotic patients. MATERIALS AND METHODS: Plasma levels of TM and EPCR were measured in 104 patients affected by different stages of liver diseases (66 patients with HCC, and 38 without HCC), and in 52 healthy controls. RESULTS: EPCR levels were higher in patients than in controls (239+/-1.8 ng/mL vs. 127+/-1.5 ng/mL, p<0.0001). TM levels were higher in patients with HCC than in those without (42.1+/-2.0 ng/mL vs. 28.3+/-2.1 ng/mL; p=0.039), while EPCR levels were similar in the two groups. No association between TM and clinical outcome was found, while high levels of EPCR were associated with death and thrombosis of the portal vein. CONCLUSIONS: We surmise a possible role for high levels of TM as a marker of HCC development in patients with cirrhosis, whereas high levels of EPCR are a possible marker of worse HCC prognosis, being a sign of endothelial damage of large vessels.

The natural history of compensated cirrhosis due to hepatitis C virus: A 17-year cohort study of 214 patients.

Large databases of consecutive patients followed for sufficiently long periods are needed to establish the rates, chronology, and hierarchy of complications of cirrhosis as well as the importance of other potential causes of liver disease. In accordance with this goal, a cohort of patients with compensated cirrhosis due to hepatitis C virus (HCV) was followed for 17 years. Two hundred and fourteen HCV RNA-seropositive patients with Child-Pugh class A cirrhosis who had no previous clinical decompensation were prospectively recruited and followed up with periodic clinical and abdominal ultrasound examinations. During 114 months (range 1-199), hepatocellular carcinoma (HCC) developed in 68 (32%), ascites in 50 (23%), jaundice in 36 (17%), upper gastrointestinal bleeding in 13 (6%), and encephalopathy in 2 (1%), with annual incidence rates of 3.9%, 2.9%, 2.0%, 0.7%, and 0.1%, respectively. Clinical status remained unchanged in 154 (72%) and progressed to Child-Pugh class B in 45 (21%) and class C in 15 (7%). HCC was the main cause of death (44%) and the first complication to develop in 58 (27%) patients, followed by ascites in 29 (14%), jaundice in 20 (9%), and upper gastrointestinal bleeding in 3 (1%). The annual mortality rate was 4.0% per year and was higher in patients with other potential causes of liver disease than in those without them (5.7% vs. 3.6%; P = .04). In conclusion, hepatitis C-related cirrhosis is a slowly progressive disease that may be accelerated by other potential causes of liver disease. HCC was the first complication to develop and the dominant cause for increased mortality.

No hepatitis recurrence using combination prophylaxis in HBV-positive liver transplant recipients with YMDD mutants.

Recurrence of hepatitis B impairs the outcome of liver transplantation (OLT). In serum hepatitis B virus (HBV)-DNA-positive recipients, prophylaxis using lamivudine and immunoglobulins (HBIg) reduces the risk of recurrence, but it is undefined whether this regimen also protects candidates with YMDD mutants. Seventeen OLT viraemic candidates received pre-emptive lamivudine followed by post-OLT prophylaxis with lamivudine and HBIg. Both sera and liver biopsies were prospectively collected and high-sensitive polymerase chain reaction (PCR) assay was applied for HBV-DNA detection. Finally, the presence of YMDD mutants was explored in all PCR-positive samples. All patients remained hepatitis B recurrence-free after a mean follow up of 32 months. By PCR, serum HBV-DNA was detectable in 64.3% of cases at OLT-baseline, in 64.7% under combined prophylaxis and in 58.8% in patients (70.5% of the total) with a minimum follow up of 24 months. At OLT-baseline, YMDD mutants were found in 44.4% of patients. After OLT, mutants were present in 50% of patients but only in 16.6% of cases in the long period. Although 41% of the native livers and 42.8% of the analysed grafts harboured HBV-DNA, YMDD mutants were detected in 57% of the native positive livers. YMDD mutants were largely detected both at OLT-baseline and post-OLT, but their presence decreased over time. Regardless of the presence of YMDD mutants, no hepatitis B recurrence was observed in our OLT recipients using pre-emptive lamivudine followed by continuous prophylaxis with lamivudine and HBIg.

Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis.

Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.

Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance.

BACKGROUND & AIMS: Significant improvements in management of hepatocellular carcinoma (HCC) have occurred in the last years, but their impact on surveillance outcome is unknown. To clarify this, we compared survival of HCC patients identified along 3 consecutive quinquennia of surveillance. METHODS: A cohort of 417 HCC-free outpatients with compensated cirrhosis was prospectively followed for 148 months (range, 1-213 months) with periodic ultrasound examinations. RESULTS: HCC developed in 112 patients, at a 3.4% rate per year, and was the prime cause of death (n = 54). Forty-six (41%) patients had a single tumor, with a mean size of 3.7 cm, 3.0 cm, and 2.2 cm in the 3 quinquennia (first vs. second: ns; first vs. third: P = 0.017; second vs. third: P = 0.02), and 38 (44%) underwent radical therapy. Mortality rates in HCC patients fell from 45% in the first quinquennium to 37% in the second and 10% in the third (first vs. second: ns; first vs. third: P = 0.0009; second vs. third: P = 0.018) in parallel with a reduction in yearly mortality of treated patients (34%, 28%, and 5%, respectively; first vs. second: ns; second vs. third: P = 0.036; first vs. third: P = 0.0024). After stratification for quinquennium, tumor staging, according to Cancer of the Liver Italian Program (CLIP), was the only independent predictor of survival (P = 0.015). CONCLUSIONS: Cirrhotic patients developing a HCC during the last 5 years of surveillance survived longer than previously, as a consequence of improved management of the tumor and complications of cirrhosis.

Long-term suppression of hepatitis B e antigen-negative chronic hepatitis B by 24-month interferon therapy.

To assess whether extended treatment with interferon improves the outcome of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 101 consecutive patients were treated with 6 MU of interferon alfa 2b 3 times weekly for 24 months. During the 68-month study, 30 patients (30%) had a sustained response (i.e., normal serum transaminase levels and undetectable hepatitis B virus DNA by non-polymerase chain reaction [PCR] assays), and 15 cleared serum surface antigen. Twenty-five nonresponders, 16 relapsers, and 30 who discontinued treatment were considered treatment failures. Multivariate analysis predicted a sustained response for young age (odds ratio, 0.94; 95% confidence interval, 0.89-0.99; P =.041) and high pretreatment serum levels of immunoglobulin M (IgM) anti-hepatitis B core antigen (HBc) (odds ratio, 4.52; 95% confidence interval, 1.63-12.5; P =.004). Liver disease progressed in none of the sustained responders but in 16 with treatment failure (0% vs. 22%, P =.002); hepatocellular carcinoma (HCC) developed with similar frequency in both groups (7%). Overall, estimated 8-year complication-free survival was longer for the 30 sustained responders than the 71 patients with treatment failure (90% vs. 60%, P <.001), but 8-year patient survival was similar in the 2 groups (100% and 90%). Short complication-free survival was predicted by failure to respond to interferon (hazard ratio, 7.8; 95% confidence interval, 1.8-34.0; P =.006) and high scores for liver fibrosis (hazard ratio, 1.71; 95% confidence interval, 1.17-2.50; P =.005). In conclusion, 24 months of treatment with interferon alfa 2b led to sustained disease suppression in a significant proportion of patients with HBeAg-negative chronic hepatitis B.

The natural history of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a multistage disease whose occurrence is linked to environmental, dietary and life-style factors. Risk factors for HCC have been grouped into two clusters, physiologic and pathologic factors. Much evidence indicates that these risk factors may interact with each other either as a group or as a single factor to modulate development of liver cancer.