A STAT4 variant increases liver fibrosis risk in Caucasian patients with chronic hepatitis B.

BACKGROUND: Host genetic modifiers of the natural history of chronic hepatitis B (CHB) remain poorly understood. Recently, a genome-wide association study (GWAS)-identified polymorphism in the STAT4 gene that contributes to the risk for hepatocellular carcinoma (HCC) was shown to be associated with the full spectrum of hepatitis B virus (HBV) outcomes in Asian patients. However, the functional mechanisms for this effect are unknown and the role of the variant in modulating HBV disease in Caucasians has not been investigated. AIMS: To determine whether STAT4 genetic variation is associated with liver injury in Caucasian patients with CHB and to investigate potential mechanisms mediating this effect. METHODS: STAT4 rs7574865 was genotyped in 1085 subjects (830 with CHB and 255 healthy controls). STAT4 expression in liver, PBMCs and NK cells, STAT4 phosphorylation and secretion of interferon-gamma (IFN-γ) according to STAT4 genetic variation was examined. RESULTS: STAT4 rs7574865 genotype was independently associated with hepatic inflammation (OR: 1.42, 95% CI: 1.07-2.06, P = 0.02) and advanced fibrosis (OR: 1.83, 95% CI: 1.19-2.83, P = 0.006). The minor allele frequency of rs7574865 was significantly lower than that in healthy controls. rs7574865 GG risk carriers expressed lower levels of STAT4 in liver, PBMCs and in NK cells, while NK cells from patients with the risk genotype had impaired STAT4 phosphorylation following stimulation with IL-12/IL-18 and a reduction in secretion of IFN-γ. CONCLUSION: Genetic susceptibility to HBV persistence, hepatic inflammation and fibrosis in Caucasians associates with STAT4 rs7574865 variant. Downstream effects on NK cell function through STAT4 phosphorylation-dependent IFN-γ production likely contribute to these effects.

IFNL3/4 genotype is associated with altered immune cell populations in peripheral blood in chronic hepatitis C infection.

Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO-; CD8CD45RO+ to CD8CD45RO-, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.

Prospective data collection of off-label use of rituximab in Australian public hospitals.

BACKGROUND: Rituximab is a chimeric, anti-CD20 monoclonal antibody registered for the treatment of B-cell malignancies and refractory rheumatoid arthritis in Australia. In addition to these approved indications, there has been growing interest in the use of off-label rituximab in the management of a variety of diseases. AIMS: To determine the current usage of off-label rituximab in Australia, we collected nationwide data. METHODS: Information regarding patients receiving rituximab for off-label indications was prospectively collected for a 6-month period from Australian public hospitals. Data recorded included clinical indication, dosing schedule, previous therapy and efficacy assessment. The level of evidence for the use of rituximab was determined for each off-label indication. RESULTS: During the 6-month period, a total of 364 instances of off-label rituximab use was recorded in the national database. A total of 63 underlying diagnoses was identified. These were subclassified into haematological disorders (19%), autoimmune connective tissue diseases (12%), vasculitis (12%), neurological disorders (12%), transplant-related uses (12%), haematological malignancies (11%), muscle disorders (8%), renal diseases (6%), dermatological conditions (5%), other conditions (2%) and ocular diseases (1%). Forty percent of these requests were supported only by level 4 evidence of benefit. Data highlighted the non-standardised approaches to drug approval mechanisms, dosing schedules and monitoring for efficacy. CONCLUSIONS: Off-label rituximab is prescribed for a diverse range of clinical conditions. Determining a safe and effective means of regulating this use within an evidence-based framework remains an ongoing challenge.

Inflammation and altered drug clearance in cancer: transcriptional repression of a human CYP3A4 transgene in tumor-bearing mice.

A tumor-associated inflammatory response has recently been found to contribute to the considerable interindividual variability in cytotoxic drug clearance seen in cancer patients. Circulating inflammatory markers, such as C-reactive protein (CRP) and interleukin-6 (IL-6), correlate with excessive drug toxicity caused by reduced CYP3A4-mediated metabolism. This article outlines the use of a transgenic mouse model of human CYP3A4 regulation to demonstrate that extrahepatic tumors elicit an inflammatory response, leading to transcriptional repression of the CYP3A4 gene as well as of other drug clearance pathways.

Downregulation of drug transport and metabolism in mice bearing extra-hepatic malignancies.

There is increasing evidence of a systemic inflammatory response associated with malignancy, which may have an impact on both drug disposition and resistance to cytotoxic therapy. The impact of inflammation on drug disposition was studied in mice bearing a number of common tumour xenografts. C57BL/6 mice were inoculated with tumour xenografts. Hepatic expressions of Cyp3a and drug transporters were analysed at the mRNA, protein and functional levels (Cyp3a only). Circulating serum cytokines and the hepatic expression of acute phase proteins (APPs) were measured. Intratumoral levels of multidrug resistance genes were determined. Tumour xenografts elicited an inflammatory response that coincided with repression in hepatic Cyp3a11 activity and the expression of a number of hepatic drug transporters. With tumour growth, a progressive reduction in hepatic Cyp3a11 mRNA expression was seen. Conversely, an increase in the hepatic APP expression and circulating interleukin (IL)-6 levels was observed. Furthermore, a correlation was seen between increased intratumoral expression of the multidrug resistance gene, Mdr1a, and levels of circulating IL-6. Malignancy results in reduced hepatic drug disposition that correlates with an associated inflammatory response. Reduction of inflammation may improve the clinical outcome for patients receiving chemotherapeutic agents that undergo hepatic metabolism.

Evaluation of the off-label usage of rituximab in a large teaching hospital in New South Wales.

A retrospective review of patients receiving rituximab off label in a large teaching hospital was conducted between July 2002 and January 2006. The indication, dosing regimen, efficacy and cost of rituximab were evaluated. Rituximab was prescribed for three clinical indications; acute organ transplant rejection, post-transplant lymphoproliferative disease and autoimmune disease. On average, 600 mg of rituximab was prescribed weekly for 4 weeks, costing the hospital $108,739.37. We suggest an initial approval for a limited number of doses with subsequent approval dependent on improvement in predefined clinical or biochemical end-points. Furthermore, we suggest an Australia-wide central database be established to enable delineation of the optimal dosing schedule, as well as monitoring of clinical outcome.

Shielding evaluation of a medical linear accelerator vault in preparation for installing a high-dose rate 252Cf remote afterloader.

In support of the effort to begin high-dose rate 252Cf brachytherapy treatments at Tufts-New England Medical Center, the shielding capabilities of a clinical accelerator vault against the neutron and photon emissions from a 1.124 mg 252Cf source were examined. Outside the clinical accelerator vault, the fast neutron dose equivalent rate was below the lower limit of detection of a CR-39 etched track detector and below 0.14 +/- 0.02 muSv h(-1) with a proportional counter, which is consistent, within the uncertainties, with natural background. The photon dose equivalent rate was also measured to be below background levels (0.1 muSv h(-1)) using an ionisation chamber and an optically stimulated luminescence dosemeter. A Monte Carlo simulation of neutron transport through the accelerator vault was performed to validate measured values and determine the thermal-energy to low-energy neutron component. Monte Carlo results showed that the dose equivalent rate from fast neutrons was reduced by a factor of 100,000 after attenuation through the vault wall, and the thermal-energy neutron dose equivalent rate would be an additional factor of 1000 below that of the fast neutrons. Based on these findings, the shielding installed in this facility is sufficient for the use of at least 5.0 mg of 252Cf.

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576.

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.

Which patients with hepatitis C develop liver complications?

To identify variables that are independent predictors of adverse outcomes in chronic hepatitis C, we analyzed a cohort of 455 patients followed for a median of 4.7 years. Associations were sought between demographic and behavioral factors, hepatitis C virus (HCV) genotype, liver histology and liver tests at entry, and development of liver complications, hepatocellular carcinoma (HCC), hepatic transplantation and liver-related death. Independent predictors were identified by multivariate analysis. The following were associated with a significantly higher rate of liver complications: age; birth in Asia, Europe, Mediterranean region, or Egypt; transmission by blood transfusion or sporadic cases; HCV genotypes 1b and 4 (compared with 1/1a); fibrosis stage 3 or 4 (cirrhosis); serum albumin; bilirubin; prothrombin time; and alpha-fetoprotein. However, the only independent predictors of liver-related complications were sporadic transmission (P <.001), advanced fibrosis (P =.004), and low albumin (P <.001). The corresponding independent risk factors for HCC were male gender (P =. 07), sporadic transmission (P <.001), and albumin (P <.001); bilirubin (P =.02) was an additional predictor of transplantation or liver-related death. It is concluded that only patients with advanced hepatic fibrosis or cirrhosis, are at risk of developing hepatic complications of chronic hepatitis C during 5-year follow-up. Among such patients, abnormalities in serum albumin, bilirubin, or prothrombin time indicate a high probability of complications. Patients without definite risk factors for HCV (sporadic cases) are at higher risk of complications, possibly because of interaction between older age, duration of infection, country of birth, and HCV genotypes 1b and 4.

The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module.

Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases -7836 to -7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to approximately 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region and cis-acting elements in the proximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction of CYP3A4 by activators of hPXR.

Risk factors for development of hepatocellular carcinoma among Australians with hepatitis C: a case-control study.

BACKGROUND: Older patients with cirrhosis due to hepatitis C are at risk of developing hepatocellular carcinoma (HCC), but additional risk factors may vary between countries. AIM: In the present study, we sought to identify additional risk factors for HCC among a cohort of Australian patients with chronic hepatitis C. METHODS: Case-control study of patients with advanced fibrosis stage hepatitis C who developed HCC during five-year follow up at a referral liver clinic. Cases were compared to twice the number of age-matched patients with chronic hepatitis C of similar fibrotic severity who did not develop HCC over a similar interval, using conditional logistic regression analysis (CLRA) and multivariate analysis. The main outcome measures were demographic and disease-related variables at first presentation in relation to the development of HCC. RESULTS: HCC developed in 17 cases, an annual incidence among those considered to be at risk of 2%. The duration of follow up since first assessment was comparable among the cases and 34 selected age-matched controls (4.1 and 5.2 years respectively, p=0.5). Cases were more often male (p=0.03), born in Asia (p=0.05), and had poorer liver function as indicated by serum albumin concentration (p=0.02). Anti-hepatitis B core-antibody (anti-HBc) was detected in 59% (ten/17) of cases, compared to 21% (seven/34) of the controls (p=0.01). No patient with a sustained response to interferon developed HCC during follow up. There were no significant differences in the mode of HCV transmission, HCV genotype, alcohol exposure, serum bilirubin level or prothrombin time between the cases and the controls. Although the data set was small, multivariate CLR analysis identified serum albumin < or = 35 g/L and anti-HBc positivity to be independent risk factors for development of HCC. CONCLUSIONS: Among older Australian patients (over the age of 40 years) with advanced fibrosis stage hepatitis C, the annual incidence of HCC is about 2%. Those who have low serum albumin and evidence of previous exposure to hepatitis B virus (anti-HBc positivity) appear to have the highest risk of developing HCC during follow up, but males and those born in Asia could also be at increased risk.

Hepatocellular proliferation and development of hepatocellular carcinoma: a case-control study in chronic hepatitis C.

Patients with hepatitis C have an increased risk of developing hepatocellular carcinoma (HCC). This is related to the stage of chronic liver disease, as characterized histologically by hepatic fibrosis and architectural distortion, but it is unclear whether histological markers can define the risk of developing HCC. We conducted a case-control immunohistochemical study of Ki-67, a marker for hepatocellular proliferation, in livers of 18 patients who had developed HCC more than 2 years after the biopsy specimen had been taken. Using conditional logistic regression analysis, the results were compared with 18 selected controls, who were age-matched patients with hepatitis C of similar histological stage who had not developed HCC. We also examined livers for cellular dysplasia, p53 mutations, and bcl-2 overexpression, and assessed whether the results could be correlated with demographic and disease-related variables, such as gender, region of birth, alcohol consumption, severity of liver disease, HCV genotype, and markers of hepatitis B virus (HBV) infection. Livers from patients who developed HCC were more often positive for Ki-67 (13 of 18 [72%] v 9 of 18 [50%]; P = .06) and tended to have higher mean Ki-67 scores (6 +/- 7.5 v 3 +/- 4.4; P = .10) compared with control cases. In the HCC-predisposed group, three livers showed large cell dysplasia, two were positive for p53 mutations, and two for bcl-2 overexpression. In contrast, in the non-HCC group, only one case had dysplasia, and none were positive for immunostaining for p53 or bcl-2 mutations. With the exception of one case, all livers with large cell dysplasia or p53 mutations and bcl-2 overexpression were also positive for Ki-67. Twelve (55%) of the 22 Ki-67-positive cases were anti-HBc-positive in the serum, in contrast to 2 of 14 (14%) patients in the Ki-67-negative group (P = .01). Patients with evidence of past infection with HBV were more often Ki-67 positive than those who had no evidence of past infection (85% [11 of 13] v 45% [10 of 22]; P = .02). There were no other associations between demographic or disease-related variables and Ki-67 expression. Increased hepatocellular proliferative activity, as assessed by Ki-67 expression, may be one factor indicative of an increased risk of developing HCC among patients with chronic hepatitis C. Furthermore, past infection with HBV appears to be an important correlate of increased hepatocellular proliferation in hepatitis C.

Effects of bile duct ligation on hepatic expression of female-specific CYP2C12 in male and female rats.

Gender differences in hepatic sex steroid and drug metabolism result from hormonal regulation of specific cytochrome P450 genes (CYP). In male rats, bile duct ligation (BDL) is associated with down-regulation of the male-specific genes, CYP2C11 and CYP3A2, together with a decrease in serum testosterone levels and a two- to three-fold increase in serum estradiol concentrations. We anticipated that if estrogen is responsible for down-regulation of male-specific CYPs in BDL male rats, the female-specific CYP2C12, which is not normally present in adult male rat liver, should be up-regulated. We examined this proposal by determining the profile of hepatic cytochrome P450 enzymes in female rats subjected to BDL, and by seeking evidence for expression of CYP2C12 in male rats that do not normally express this gene. In female rats killed 5 days after BDL, total cytochrome P450 content and NADPH-cytochrome P450-reductase (P450-reductase) were decreased to 74% and 58% of control, respectively. Microsomal enzyme activities attributable to CYP2A1, CYP2C6, and CYP2E1 were 50% to 60% of control, but ethylmorphine N-demethylase, which in female liver is catalyzed by CYP2C12 and to a lesser extent CYP2C6, was significantly less affected (81% of control). Likewise, levels of CYP2C6 and P450-reductase proteins were decreased in proportion to the corresponding enzyme activities (50% to 60%), while CYP2C12 protein (and mRNA levels) were not altered in BDL female rat liver. In sham-operated male rats, transcripts for CYP2C12 were rarely detected, but mRNA levels rose to appreciable levels within 24 hours of BDL, and CYP2C12 protein was expressed in hepatic microsomes of BDL male rats. Administration of estradiol to male rats produced a similar elevation of CYP2C12 mRNA, to values approximately 40% of female rats. It is concluded that CYP2C12 is up-regulated in male rats with cholestasis caused by BDL, while CYP2C12 protein is preserved in female rats when other microsomal proteins are decreased. These changes may be related to the increase in serum estradiol levels that result from altered hepatic steroid metabolism. The results demonstrate that activities of individual drug-metabolizing enzymes in liver disease can be determined by dysregulation of the constitutive expression of hepatic CYP genes.

How much does alcohol contribute to the variability of hepatic fibrosis in chronic hepatitis C?

In order to determine the contribution of alcohol intake to the severity of hepatic fibrosis in patients with chronic hepatitis C, we studied associations between various levels of alcohol intake, other demographic variables and semiquantitative liver histology in 434 cases of chronic hepatitis C. Clinical, demographic and disease-related data were entered into a relational database. Liver histology was scored according to Scheuer. The average daily alcohol intake for the year preceding liver biopsy (recent exposure) and for earlier periods (past exposure) was categorized into five levels of intake. One-third of patients gave a history of alcohol intake that had exceeded 40 g/day for at least 5 years. By univariate analysis, age, but not recent or past alcohol intake or other baseline variables, was associated with portal score (r = 0.14, P = 0.004), fibrosis score (r = 0.46, P < 0.001), total Scheuer score (r = 0.35, P < 0.001). However, by multivariate analysis, age (P < 0.001), past (but not present) alcohol intake (P < 0.001) and birth in Egypt (P = 0.006) were independently associated with fibrosis score. Age, past alcohol and birth place in Egypt contributed 27% to total variance of the hepatic fibrosis score, while age alone accounted for 23%. Age also independently predicted portal activity (P = 0.02) and total Scheuer score (P < 0.001), whereas past alcohol intake correlated with total Scheuer score (P = 0.002) but not with other histological indices. A separate multivariate analysis was performed on a more homogeneous subgroup of 196 patients who acquired hepatitis C by injection drug use. In this subgroup, age (P < 0.05) and past alcohol (P < 0.05) were independently associated with fibrosis score. In both the overall and subgroup analyses, there was a threshold level of past alcohol intake (>80 g/day) beyond which the risk of fibrosis increased significantly. It is concluded that toxic levels of alcohol exposure for at least 5 years accentuate hepatic fibrosis in hepatitis C but the influence of alcohol appears to be minor compared with age and other variables and is exerted only at toxic levels of intake.

Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) has multiple etiologic associations, but the pathogenesis is poorly understood. Cytochrome P450 (CYP) 2E1 is induced in the liver of patients who drink alcohol to excess and is important in the pathogenesis of alcoholic liver disease (ALD). We have previously shown that hepatic CYP2E1 is also increased in a rat dietary model of steatohepatitis. The aim of the present study was to test the hypothesis that hepatic CYP2E1 is induced in the liver of patients with NASH, defined on the basis of compatible liver histology and the exclusion of excessive alcohol intake. Sections of paraffin-embedded liver biopsy material from 31 subjects with NASH were evaluated and compared with sections from 10 histologically normal livers and 6 patients with ALD. Hepatic CYP2E1 and CYP3A were detected in liver sections by immunohistochemistry using specific anti-human CYP2E1 and CYP3A antibodies. As expected, normal livers showed CYP2E1 immunostaining confined to a rim, two to three cells thick, around terminal hepatic venule, while livers from alcoholic hepatitis patients showed increased CYP2E1 staining. CYP2E1 immunostaining was also increased in livers from patients with NASH, irrespective of the etiologic association. Further, the pattern of CYP2E1 distribution was similar to ALD, with increased perivenular intensity and more extensive acinar distribution of staining. As in the rat model, the hepatic distribution of CYP2E1 corresponded to that of steatosis. In contrast to CYP2E1, CYP3A immunostaining was decreased in patients with NASH. We conclude that hepatic CYP2E1 is increased in patients with NASH compared with normal livers. Thus, despite many possible etiologic factors for NASH, the pathogenetic mechanisms may be similar and, like alcoholic steatohepatitis, may involve induction of CYP2E1.

Antipyrine clearance and response to interferon treatment in patients with chronic active hepatitis C.

To determine whether hepatic metabolic function affects the response to interferon treatment, we measured antipyrine clearance (APC) in 85 patients with chronic active hepatitis C and compared the results with treatment outcome. Among 55 patients who responded to interferon by normalization of alanine transaminase (ALT), median APC before treatment was 0.47 (range, 0.12 to 0.98; normal range, 0.34 to 1.02 mL/min/kg body wt), a value that was significantly greater than in 30 nonresponders (0.23; 0.08 to 0.67 mL/min/kg body wt, P < .001). APC was closely associated with response to interferon. The response rate among cases with values > 0.25 mL/min/kg body weight was 79%, the same as in cases without cirrhosis. Cases without cirrhosis and with APC of > 0.25 mL/min/kg body weight had an 85% chance of responding to interferon; this was unlikely a simple reflection of histological activity, because the correlation with Scheuer score was poor in this subgroup (r = -.31, P < .05). A second, independent group of 43 patients was used to test the predictive value of APC (using 0.25 mL/min/kg body wt as a cut-off) for response to interferon treatment. In this group, APC correctly predicted positive response to interferon in 75% of cases. APC was also used to measure the effects of treatment on hepatic metabolic function. Regardless of outcome, there was no change in APC at the end of a 6-month course of interferon treatment. Six months later, however, improvement in APC (14%; P < .05) was evident among responders but not in those who had failed to respond to interferon.(ABSTRACT TRUNCATED AT 250 WORDS)

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: importance to reduced hepatic content of cytochrome P450 in cholestasis.

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased to 32% to 52% of control, but the activities of two male sex-specific CYPs, 2C11 and 3A2, were reduced to a significantly greater extent (P < .05). The microsomal contents of CYP proteins 2C6 and 2E1 were decreased after BDL to 61% and 63% of control, whereas 2C11 and 3A2 proteins were 21% and 45% of control. Corresponding reductions of the messenger RNA (mRNA) species for CYP 2C11 (9% of control) and 3A2 (37%) were detected, whereas there was no reduction of 2C6 mRNA. These findings are consistent with downregulation of the CYP 2C11 and 3A2 genes. Nuclear run-on studies performed 3 days after BDL showed that there was a generalized impairment of gene transcription after BDL, but a disproportionate reduction in transcription of CYPs 2C11 and 3A2. A possible explanation for downregulation of CYP 2C11 and 3A2 was provided by the observation that serum estradiol concentrations were threefold greater in BDL male rats, while serum testosterone was reduced; estradiol is known to downregulate CYPs 2C11 and 3A2. It is concluded that male sex-specific CYP enzymes are decreased to a greater extent than other microsomal proteins in BDL male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Coinfection with hepatitis B and C or B, C and delta viruses results in severe chronic liver disease and responds poorly to interferon-alpha treatment.

Chronic coinfection with the hepatitis B (HBV) and hepatitis delta (HDV) viruses is known to cause severe liver disease, but the importance of coinfection with hepatitis C virus (HCV) and HBV has not been well documented. In the present study, the clinical and pathological severity of liver disease among patients with hepatitis resulting from multiple viruses was examined and an open trial of the efficacy of interferon-alpha 2b (IFN-alpha) treatment was conducted. Nineteen patients with chronic HBV and HCV infection and 17 with HBV, HCV and HDV infection were studied; 12 in each group underwent liver biopsy. For each coinfected patient, two patients infected with HCV alone were selected as controls, and these were matched for age and risk factor and were estimated to have been infected for a similar duration. Coinfection with HBV and HCV or HBV, HCV and HDV was associated with more severe liver disease than HCV alone (P < 0.01); total Scheuer score, portal and lobular inflammation and fibrosis were all worse in coinfected subjects. Eight patients with chronic HBV and HCV were treated with recombinant IFN-alpha 2b [3 million units (MU), thrice weekly for 6 months]. Liver function tests normalized in two patients and one lost hepatitis B surface antigen (HBsAg). Seven patients with hepatitis B, C and delta coinfection were treated with the same regimen and only one normalized serum alanine aminotransferase (ALT) during (and after) treatment. It is concluded that coinfection with multiple hepatitis viruses is associated with histologically more severe liver disease than HCV alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocellular carcinoma in western Sydney. Aetiology, changes in incidence, and opportunities for better outcomes.

OBJECTIVES: To examine the incidence of hepatocellular carcinoma (HCC) in western Sydney over the last 14 years, to assess risk factors for the disease among ethnic groups of Australian residents, and to consider the opportunities for improving its usually poor outcome. DESIGN AND SUBJECTS: Retrospective case-record review of clinical features in all (122) patients discharged from a 900-bed tertiary-referral teaching hospital with a diagnosis of HCC from January 1979 to March 1993. MAIN OUTCOME MEASURES: Annual number of new cases; risk factors according to birthplace; surgical resectability of tumours. RESULTS: New cases admitted each year at least doubled between 1979-1985 and 1986-1992. This apparent increase involved individuals born in Australia (50% of all patients) as well as immigrants. Cirrhosis was found in 93% at liver biopsy or autopsy. Excessive alcohol intake was an associated risk factor for 46% of Australian-born patients and for 13% of those born overseas. Among the latter, HCC was associated with markers of hepatitis B virus infection in 64%. Since hepatitis C virus (HCV) tests became available in 1990, five of nine patients tested were anti-HCV positive. Surveillance screening of patients known to have cirrhosis detected eight cases of early HCC. Seven of these had surgical resection and all are alive. CONCLUSIONS: New diagnoses of HCC have increased recently, irrespective of country of birth. In Australian-born patients alcoholic liver disease remains a major aetiological factor but the role of HCV requires further evaluation. Among immigrants, cirrhosis from chronic viral hepatitis accounts for most cases. We propose that prevention of cirrhosis caused by chronic viral hepatitis should have the greatest long-term impact on prevention of HCC in Australia. The role of surveillance of people with cirrhosis to detect small and potentially resectable tumours should be explored.

Role of the oestrogen receptor in liver regeneration in the male rat.

Partial hepatectomy in male rats results in raised serum oestrogen levels, nuclear binding of oestrogen receptor (ER) and feminization of certain aspects of hepatic metabolism. It has been proposed that these changes may have an important role in liver regeneration. The present study was performed to ascertain the effects of the oestrogen agonist diethylstilbestrol (DES), 2 mg/kg, and the oestrogen antagonist tamoxifen (TAM), 2 mg/kg, on liver regeneration induced by partial hepatectomy in the male rat. Regenerative activity was determined by incorporation of [3H]-thymidine into hepatic DNA as well as by measurement of liver remnant weight. Following partial hepatectomy, there was a trend towards an increase in liver remnant weight at 24 h in rats treated with DES (DES, 5.95 +/- 1.52 g; vehicle, 4.87 +/- 0.66 g; P = 0.06) though by 48 h no effect was found. Tamoxifen treatment did not significantly affect liver weight at 24 h but by 48 h there was a highly significant reduction in liver remnant weight (TAM, 5.41 +/- 0.85 g; vehicle, 7.31 +/- 1.43 g; P < 0.001). Neither DES nor TAM treatment influenced liver regeneration as determined by [3H]-thymidine incorporation into hepatic DNA. We conclude that pharmacologic manipulation of oestrogens does not influence the initiation of the regenerative process but that oestrogen may facilitate later phases of hepatic growth.