Woodchuck hepatitis virus core gene deletions and proliferative responses of peripheral blood mononuclear cells stimulated by an immunodominant epitope: a viral immune escape in the woodchuck model of chronic hepatitis B?

Marmota monax and its natural infection by woodchuck hepatitis virus (WHV) could be used as a predictive model for evaluating mechanisms of viral persistence during chronic hepatitis B virus (HBV) infection. The aim of this study was to investigate the presence of viral variants in the core gene of chronically WHV-infected woodchucks that showed two different patterns of peripheral blood mononuclear cells' (PBMCs') responses after stimulation with a specific WHV core peptide. Sequences' analysis of the WHV core region from eight WHV chronically infected woodchucks have been performed after in vitro stimulation with an immunodominant epitope of the WHV core protein (amino acids [aa] 96-110). Following this stimulation, positive PBMC responses at each point of follow-up were observed for four animals (group A), and weak immune responses at one or a few points of follow-up were observed for the remaining four animals (group B). The WHV core gene sequences contained amino acid deletions (aa 84-126, aa 84-113) in three of four group A animals and in none of group B animals. In the group A animals, the same deletions were observed in liver specimens and in two of four tumor specimens. Hepatocellular carcinoma (HCC) was diagnosed in all group A animals and in one group B animal. In conclusion, internal deletions in the core region correlated with a sustained PBMC response to the immunogenic peptide (96-110) of the core protein. A possible role of this relationship in hepatocarcinogenesis could be hypothesized; however, this needs to be investigated in patients with chronic HBV infection. The evaluation of virus-specific T-cell responses and T-cell epitopes that are possibly related to the mechanisms of viral evasion should be further investigated in order to design combined antiviral and immune approaches to control chronic HBV infection.

Ribavirin priming improves the virological response to antiviral treatment in transplanted patients with recurrent hepatitis C: a pilot study.

INTRODUCTION: Patients with hepatitis C recurrence after liver transplantation represent a clinical challenge. Antiviral treatment in transplant patients has usually poor tolerability and limited efficacy, with a mean sustained virological response (SVR) of 30%. Our pilot study was aimed at evaluating whether 8-week ribavirin pre-treatment could increase either adherence or antiviral effect of a 48-week combination therapy. METHODS: Ribavirin pre-treatment (8 weeks) was started with 600 mg daily and increased to 10.4 mg/kg/day. After pre-treatment, 1.5 µg/kg/week pegylated interferon-α2b was added for 48 additional weeks of combination therapy. Blood count, liver function tests and plasma HCV-RNA were examined monthly. Ribavirin plasma concentrations were determined by HPLC. RESULTS: Thirteen patients (mean age 53±2 years, 11 males) were treated: eight were HCV genotype 1/4; five were genotype 2/3. The median baseline HCV RNA level was 6.5 log(10) (range 5.84-7.42 log(10)). During ribavirin pre-treatment the median HCV RNA levels decreased significantly (5.7 log(10) ; P=0.023). During combination therapy 6/13 (46%) patients exhibited a rapid virological response (RVR) and 10/13 (77%) patients a complete early virological response, two were non-responders. A decline of 0.5 log(10) HCV RNA during pre-treatment predicted RVR. SVR occurred in six patients (46%): four were genotype 2/3. Stable ribavirin dose reduction was required in only two patients (15%) in whom transient interferon reduction was also required. CONCLUSION: This proof-of-concept study indicates that ribavirin pre-treatment increased the tolerability of the antiviral treatment, and improved its efficacy in liver transplant patients. Moreover, the degree of HCV RNA decline during pre-treatment allowed one to predict on-treatment response.

Natural history of gallstone disease: Expectant management or active treatment? Results from a population-based cohort study.

BACKGROUND AND AIMS: The knowledge of natural history is essential for disease management. We evaluated the natural history (e.g. frequency and characteristics of symptoms and clinical outcome) of gallstones (GS) in a population-based cohort study. METHODS: A total of 11 229 subjects (6610 men, 4619 women, age-range: 29-69 years, mean age: 48 years) were studied. At ultrasonography, GS were present in 856 subjects (338 men, 455 women) (7.1%). GS were followed by means of a questionnaire inquiring about the characteristics of specific biliary symptoms. RESULTS: At enrollment, 580 (73.1%) patients were asymptomatic, 94 (11.8%) had mild symptoms and 119 (15.1%) had severe symptoms. GS patients were followed up for a mean period of 8.7 years; 63 subjects (7.3%) were lost to follow up. At the end of the follow up, of the asymptomatic subjects, 453 (78.1%) remained asymptomatic; 61 (10.5%) developed mild symptoms and 66 (11.4%) developed severe symptoms. In subjects with mild symptoms, the symptoms disappeared in 55 (58.5%), became severe in 23 (24.5%), remained stable in 16 (17%); in subjects with severe symptoms, the symptoms disappeared in 62 (52.1%), became mild in 20 (16.8%) and remained stable in 37 (31.1%). A total of 189 cholecystectomies were performed: 41.3% on asymptomatic patients, 17.4% on patients with mild symptoms and 41.3% on patients with severe symptoms. CONCLUSIONS: This study indicates that: (i) asymptomatic and symptomatic GS patients have a benign natural history; (ii) the majority of GS patients with severe or mild symptoms will no longer experience biliary pain; and (iii) a significant proportion of cholecystectomies are performed in asymptomatic patients. Expectant management still represents a valid therapeutic approach in the majority of patients.

Atorvastatin-induced prolonged cholestasis with bile duct damage.

We report a case of acute-onset, long-lasting cholestasis induced by atorvastatin. This antihyperlipidaemic drug was taken for 40 days by a 72-year-old male as a treatment for his mixed dyslipidaemia. At that point, the patient presented with asthenia, nausea, painless icterus, acholic stools and hyperchromic urine with biochemical analyses showing a dramatic increase in bilirubin (total bilirubin 22 mg/dL; direct bilirubin 21 mg/dL) and alkaline phosphatase (up to 4-fold over the normal level) with less marked increases in transaminases. Liver histology showed a pattern of cholestasis with evident signs of cholangiolitis and damage of the interlobular bile ducts. Serum transaminase and bilirubin levels returned to normal within 5 months after atorvastatin withdrawal while alkaline phosphatase normalized after only 8 months. Scores on both the Maria and Victorino clinical scale for the diagnosis of drug-induced hepatitis and the Naranjo Adverse Drug Reaction Probability Scale indicated that atorvastatin was the probable cause of prolonged cholestasis in this patient. This is a rare case of cholestasis probably caused by atorvastatin and unusually characterized by bile duct damage.

Systemic thrombolysis of portal vein thrombosis in cirrhotic patients: a pilot study.

BACKGROUND: Portal vein thrombosis is a frequent complication in liver cirrhosis. Encouraging reports of systemic thrombolysis in non-cirrhotic patients suffering from acute portal vein thrombosis led us to start a pilot study on the efficacy and safety of systemic low dose recombinant tissue plasminogen activator (Actilyse, Boheringer Ingelheim, Florence, Italy). PATIENTS: Nine cirrhotic patients (6 males and 3 females) with recent portal vein thrombosis were enrolled. Exclusion criteria were portal cavernomatosis, recent (30 days) surgery, active bleeding, hepatocellular carcinoma and cancer in other sites. METHODS: All cases were treated for a maximum of 7 days by continuous i.v. infusion of 0.25mg/kg/die of r-tPA plus subcutaneous low molecular weight heparin. Efficacy was evaluated by colour doppler sonography monitoring and confirmed by contrast enhanced computerized tomography. RESULTS: The combined r-tPA/LMWH treatment was well tolerated without clinically significant side effects. Complete resolution of thrombosis occurred in 4 cases, partial regression in 4 and none in 1. Retreatment of a complete recurrence in 1 patient was successful. Variceal pressure dropped from 30.7+/-4.5 mmHg to 21.2+/-6.6 mmHg (p=0.012). CONCLUSIONS: Our preliminary data demonstrate that thrombolytic treatment of recent portal vein thrombosis with i.v. r-tPA and LMWH in patients with cirrhosis appears to be safe and effective and can significantly reduce pressure in oesophageal varices.

Incidence of gallstone disease in Italy: results from a multicenter, population-based Italian study (the MICOL project).

AIM: To evaluate gallstone incidence and risk factors in a large population-based study. METHODS: Gallstone incidence and risk factors, were evaluated by structured questionnaire and physical examination, respectively, in 9611 of 11 109 (86.5%) subjects who were gallstone-free at the cross-sectional study. RESULTS: Six centers throughout Italy enrolled 9611 subjects (5477 males, 4134 females, aged 30-79 years), 9517 of whom were included into analysis: 424 subjects (4.4%) had gallstones and 61 (0.6%) had been cholecystectomized yielding a cumulative incidence of 0.67% per year (0.66% in males, 0.81% in females). Increasing age, a high body mass index (BMI), a history of diabetes, peptic ulcer and angina, and low cholesterol and high triglyceride levels were identified as risk factors in men while, in females, the only risk factors were increasing age and a high BMI. Increasing age and pain in the right hypocondrium in men and increasing age in females were identified as predictors of gallstones. Pain in the epigastrium/right hypocondrium was the only symptom related to gallstones; furthermore, some characteristics of pain (forcing to rest, not relieved by bowel movements) were significantly associated with gallstones. No correlation was found between gallstone characteristics and clinical manifestations, while increasing age in men and increasing age and BMI in females were predictors of pain. CONCLUSION: Increasing age and BMI represent true risk factors for gallstone disease (GD); pain in the right hypocondrium and/or epigastrium is confirmed as the only symptom related to gallstones.

Serum and biliary insulin-like growth factor I and vascular endothelial growth factor in determining the cause of obstructive cholestasis.

BACKGROUND: Cholangiocarcinoma cells express and secrete insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF). OBJECTIVE: To measure IGF-I and VEGF in bile and serum of patients with extrahepatic cholangiocarcinoma and to evaluate their performance as diagnostic markers. DESIGN: Cross-sectional study. SETTING: Inpatients at the Division of Gastroenterology, University Hospital, Ancona, Italy. PATIENTS: 73 patients who consecutively had endoscopic retrograde cholangiopancreatography (ERCP), including patients with extrahepatic cholangiocarcinoma (n = 29), pancreatic cancer (n = 19), and benign biliary abnormalities (n = 25; bile duct stones, primary sclerosing cholangitis, and cholangitis). MEASUREMENTS: Diagnosis was based on conventional radiology, ERCP, and follow-up. Insulin-like growth factor I and VEGF were measured by using enzyme-linked immunosorbent assay. RESULTS: The biliary IGF-I concentration was 15- to 20-fold higher (P < 0.001) in extrahepatic cholangiocarcinoma (mean, 84.6 nmol/L [95% CI, 74.0 to 95.2 nmol/L]) than in pancreatic cancer (5.8 nmol/L [CI, 4.0 to 7.5 nmol/L]) or benign biliary abnormalities (4.1 nmol/L [CI, 3.1 to 5.2 nmol/L]). The area under the receiver-operating characteristic curve was 1 when biliary IGF-I values in the extrahepatic cholangiocarcinoma were compared with benign biliary abnormalities or pancreatic cancer. In contrast, biliary VEGF concentration was similar in the 3 groups. Serum IGF-I levels were similar among the groups, whereas serum VEGF levels were higher in the cholangiocarcinoma (0.97 ng/mL [CI, 0.59 to 1.35 ng/mL]; P = 0.0016) and pancreatic cancer groups (0.66 ng/mL [CI, 0.43 to 0.88 ng/mL]; P < 0.001) compared with patients with benign biliary abnormalities (0.28 ng/mL [CI, 0.17 to 0.37 ng/mL]). LIMITATIONS: Data were obtained in a small sample, the study was performed in a single center, and few patients had a tissue diagnosis. CONCLUSIONS: Biliary IGF-I levels in patients undergoing ERCP for biliary obstruction may differentiate extrahepatic cholangiocarcinoma from either pancreatic cancer or benign biliary abnormalities.

Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.

Estrogens and insulin-like growth factor 1 modulate neoplastic cell growth in human cholangiocarcinoma.

We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-alpha, ER-beta, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-alpha was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-beta, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17beta-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17beta-estradiol and IGF-1 were associated with enhanced protein expression of ER-alpha, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-beta. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma.

High prevalence of spontaneous portal-systemic shunts in persistent hepatic encephalopathy: a case-control study.

Large spontaneous portal-systemic shunts have been occasionally described in patients with cirrhosis. This study was undertaken to assess the prevalence of portal-systemic shunts in patients with cirrhosis with recurrent or persistent hepatic encephalopathy (HE) as compared with patients with cirrhosis without HE. Fourteen patients with cirrhosis with recurrent or persistent HE (cases) and 14 patients with cirrhosis without previous or present signs of overt HE matching for age and degree of liver failure (controls) were studied. Each patient underwent neurological assessment and cerebral magnetic resonance (MR) imaging to exclude organic neurological pathological conditions. HE evaluation included psychometric performance (Trail-Making Test A), electroencephalogram (EEG), mental status examination and grading, arterial, venous, and partial pressure of ammonia determination. The presence of portal-systemic shunts was assessed by portal venous phase multidetector-row spiral computed tomography (CT). Large spontaneous portal-systemic shunts were detected in 10 patients with HE and in only 2 patients without HE (71% vs. 14%; chi square = 9.16; df = 1.0; P = .002). The patients with HE presented ascites (P = .002) and medium/large esophageal varices (P = .02) less frequently than the control group. In conclusion, our study suggests that large spontaneous shunts may often sustain the chronicity of HE; the presence of large shunts should be sought in patients with cirrhosis with recurrent or persistent HE.

Pharmacological prophylaxis of hepatic encephalopathy after transjugular intrahepatic portosystemic shunt: a randomized controlled study.

BACKGROUND/AIMS: Hepatic encephalopathy is a frequent event after transjugular-intrahepatic-portosystemic-shunt (TIPS), especially during the first months. Aim of this study was to compare two different treatments (lactitol 60 g/day, rifaximin 1200 mg/day) with no-treatment in the prevention of post-TIPS hepatic encephalopathy. METHODS: Seventy-five consecutive cirrhotics submitted to TIPS were randomized to receive either one of the above treatments or no-treatment. The main end-point was the occurrence of an episode of overt hepatic encephalopathy during the first month post-TIPS. Before the procedure and weekly thereafter the patients were evaluated by examining their mental status, asterixis, ammonia and trail-making-test Part-A (TMT-A). RESULTS: The three groups were comparable for age, sex, etiology, Child-Pugh-score, post-TIPS porto-systemic gradient, previous hepatic encephalopathy, basal values of ammonia and psychometric performance. Twenty-five patients developed hepatic encephalopathy (33%, CI 95%=22-45%). One-month incidence was similar in the three groups (P=0.97). Previous hepatic encephalopathy (Relative Hazard=3.79;1.27-11.31) and basal-TMT-A Z-score>1.5 (RH=3.55;1.24-10.2) were predictors of post-TIPS encephalopathy at multivariate analysis. A <5 mmHg porto-systemic gradient was also significantly related to the occurrence of encephalopathy. CONCLUSIONS: Our data show that treatment with lactitol or rifaximin is not effective in the prophylaxis of hepatic encephalopathy during the first month after a TIPS.

Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.

BACKGROUND & AIMS: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. METHODS: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. RESULTS: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. CONCLUSIONS: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Effect of ovariectomy on the proliferative capacity of intrahepatic rat cholangiocytes.

BACKGROUND & AIMS: We evaluated the effects of ovariectomy (OVX) and estrogen replacement treatment on cholangiocyte proliferation induced by bile duct ligation (BDL). METHODS: BDL (2 weeks) was performed in ovariectomized rats and the proliferative and apoptotic activity compared with normal, with BDL control rats, and with BDL +/- OVX rats treated with 17-beta estradiol. RESULTS: OVX induced a significant (P < 0.01) reduction of bile duct mass in BDL rats. The reduction of bile duct mass induced by OVX was associated with a decreased expression of estrogen receptor (ER)-alpha (2.5-fold) and, mainly, ER-beta (35-fold). Proliferating cellular nuclear antigen (PCNA) expression in cholangiocytes was impaired by OVX, indicating depression of proliferation, whereas terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and Fas positivity were markedly enhanced, indicating activation of Fas-mediated apoptosis. Administration of 17-beta estradiol during BDL in OVX rats induced a normalization of bile duct mass, ER expression, cholangiocyte proliferation, and apoptosis (Fas and TUNEL) in comparison with untreated BDL rats. CONCLUSIONS: Our findings support the role of endogenous estrogens in sustaining the enhanced proliferative and secretory activities of cholangiocytes in cholestasis. On the basis of these data, the hypothesis of an estrogenic functional deficiency in chronic cholestatic liver diseases should merit careful attention.

Corticosteroids modulate the secretory processes of the rat intrahepatic biliary epithelium.

BACKGROUND & AIMS: We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion. METHODS: GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H+/HCO3- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes. RESULTS: GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P < 0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P < 0.05) activity of the Na+/H+ exchanger (NHE1 isoform) and Cl-/HCO3- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P < 0.05) in isolated cholangiocytes by dexamethasone treatment. CONCLUSIONS: The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.