Search

Hepatitis B virus (HBV) persists with global and virus-specific T-cell dysfunction, without T-cell based correlates of outcomes. To determine if γδT-cells are altered in HBV infection relative to clinical status, we examined the frequency, phenotype and function of peripheral blood Vδ1(+) and Vδ2(+)γδT-cells by multi-parameter cytometry in a clinically diverse North American cohort of chronic hepatitis B (CHB), acute hepatitis B (AHB) and uninfected control subjects. We show that circulating γδT-cells were comprised predominantly of CD3(hi)CD4(-) Vδ2(+)γδT-cells with frequencies that were 2–3 fold higher among Asian than non-Asian Americans and inversely correlated with age, but without differences between CHB, AHB and control subjects. However, compared to control subjects, CHB was associated with increased Tbet(hi)Eomes(dim) phenotype in Vδ2(+)γδT-cells whereas AHB was associated with increased Tbet(hi)Eomes(dim) phenotype in Vδ1(+)γδT-cells, with significant correlations between Tbet/Eomes expression in γδT-cells with their expression of NK and T-cell activation and regulatory markers. As for effector functions, IFNγ/TNF responses to phosphoantigens or PMA/Ionomycin in Vδ2(+)γδT-cells were weaker in AHB but preserved in CHB, without significant differences for Vδ1(+)γδT-cells. Furthermore, early IFNγ/TNF responses in Vδ2(+) γδT-cells to brief PMA/Ionomycin stimulation correlated inversely with serum ALT but not HBV DNA. Accordingly, IFNγ/TNF responses in Vδ2(+)γδT-cells were weaker in patients with CHB with hepatitis flare compared to those without hepatitis flares, and this functional deficit persisted beyond clinical resolution of CHB flare. We conclude that circulating γδT-cells show distinct activation and differentiatiation in acute and chronic HBV infection as part of lymphoid stress surveillance with potential role in clinical outcomes.

OBJECTIVE Metabolic syndrome (MS) is prevalent and is associated with adverse outcomes of liver disease. We evaluated the prevalence of MS and its influence on alanine aminotransferase (ALT) levels and fibrosis, as estimated by the aspartate aminotransferase–to–platelet ratio index (APRI), in a large, multiethnic North American cohort with chronic hepatitis B (HBV) infection. RESEARCH DESIGN AND METHODS Adults with chronic HBV from 21 centers within the U.S. and Canada were evaluated at baseline and for up to 5 years (median 3.7 years) of follow-up. MS was defined as the presence of at least three of five criteria including waist circumference, blood pressure, glucose, triglyceride, and HDL levels. RESULTS Analysis included 777 participants, of whom 171 (22%) had MS. Participants with MS (vs. those without MS) were older (median age 54.4 vs. 40.2 years), more often male (61% vs. 51%), and born in the U.S./Canada or had immigrated >20 years ago (60% vs. 43%). MS was not associated with ALT or APRI at baseline. Upon adjusted multivariable analysis of serial ALT values, ALT was significantly higher (mean 12%; P = 0.02) among those with MS at baseline and even higher (mean 19%; P = 0.003) among those with persistent MS compared with those with persistent absence of MS. MS was not associated with serial APRI on follow-up. CONCLUSIONS MS was prevalent in this HBV cohort and was independently associated with higher ALT levels longitudinally. These findings highlight the importance of screening for MS and the potential for MS to influence ALT and its interpretation in the context of HBV treatment decisions.

BACKGROUND & AIMS: The incidence and outcomes of alanine aminotransferase (ALT) flares during the natural history of chronic HBV infection has not been determined in a large, racially heterogeneous group of patients in North America. METHODS: We collected data from the Hepatitis B Research Network—an observational cohort study of untreated adults with chronic HBV infection enrolled at 21 sites in the United States and Canada. Clinical and laboratory data were collected from 1587 participants (49.9% male, 73.7% Asian, 35.2% genotype B infection, mean age of 42.6 years) at enrollment, at weeks 12 and 24, and every 24 weeks thereafter for a planned 5 years of follow up (from January 2011 through May 2016). Participants were excluded if they had a history of hepatic decompensation, hepatocellular carcinoma, solid organ or bone marrow transplantation, chronic immune suppression, or antiviral therapy within 6 months before enrollment. Levels of ALT were measured in serum samples and flares were defined as at least 10 times the upper limit of normal (300 U/L in males and 200 U/L in females). RESULTS: ALT flares occurred in 102 participants (6%), with 31 flares (30%) occurring at baseline. The 4-year cumulative incidence of ALT flares was 5.7%. The median peak level of ALT was 450 U/L (25th–75th percentile, 330 U/L to 747 U/L) with a maximum of 2578 U/L. In multivariable analysis, factors associated with the occurrence of an ALT flares were: male sex (odds ratio [OR], 3.02; P=.0007), higher baseline HBV DNA values (OR per log10, 1.41; P

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Sulforaphane (SFN), is an effective in vitro antagonist of ligand activation of the human pregnane and xenobiotic receptor (PXR). PXR mediated CYP3A4 up-regulation is implicated in adverse drug-drug interactions making identification of small molecule antagonists a desirable therapeutic goal. SFN is not an antagonist to mouse or rat PXR in vitro; thus, normal rodent species are not suitable as in vivo models for human response. To evaluate whether SFN can effectively antagonize ligand activation of human PXR in vivo, a three-armed, randomized, crossover trial was conducted with 24 healthy adults. The potent PXR ligand – rifampicin (300 mg/d) was given alone for 7 days in arm 1, or in daily combination with 450 µmoles SFN (Broccoli Sprout extract) in arm 2; SFN was given alone in arm 3. Midazolam as an in vivo phenotype marker of CYP3A was administered before and after each treatment arm. Rifampicin alone decreased midazolam AUC by 70%, indicative of the expected increase in CYP3A4 activity. Co-treatment with SFN did not reduce CYP3A4 induction. Treatment with SFN alone also did not affect CYP3A4 activity in the cohort as a whole, although in the subset with the highest basal CYP3A4 activity there was a statistically significant increase in midazolam AUC (i.e., decrease in CYP3A4 activity). A parallel study in humanized PXR mice yielded similar results. The parallel effects of SFN between humanized PXR mice and human subjects demonstrate the predictive value of humanized mouse models in situations where species differences in ligand-receptor interactions preclude the use of a native mouse model for studying human ligand-receptor pharmacology.

Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.

Oxidative catabolism of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)(2)D(3) and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)(2)D(3) treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)(2)D(3) and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6',7'-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)(2)D(3) clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)(2)D(3) disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)(2)D(3) and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.

Aliment Pharmacol Ther 2011; 33: 566–577 Summary Background The AST to platelet ratio index (APRI), a non-invasive marker of liver fibrosis, has not been well studied in HCV/HIV (hepatitis C virus/human immunodeficiency virus) co-infected patients with advanced HIV. Aim To compare the accuracy of APRI in HCV/HIV co-infected patients to that in HCV mono-infected patients and to determine the impact of CD4+ T-cell counts on its performance. Methods We identified 106 consecutive HCV/HIV co-infected patients and 105 matched HCV mono-infected patients who underwent liver biopsy at Harborview Medical Center over a 5-year period. Performance characteristics were calculated and receiver operating characteristic (ROC) analysis conducted. Results The area under the ROC curve (AUROC) of APRI for predicting significant fibrosis was similar when comparing those with and without HIV co-infection (0.77 vs. 0.86, P = 0.18), but was lower in HIV co-infected patients with CD4 counts

Background Risk factors for hepatocellular carcinoma (HCC) have not been well characterized among African immigrants with chronic hepatitis B virus (HBV) infection. We conducted a case-control study to identify demographic and clinical factors associated with HCC among a diverse cohort of patients with chronic HBV infection seen in a large academic health setting. Methods We identified a total of 278 patients with HCC and chronic HBV seen at two medical centers in a 14-year span from January 2002 to December 2015. These cases were age- and sex-matched in a 1:3 ratio with 823 non-cancer control subjects with chronic HBV. Conditional logistic regression was used to estimate the odds of HCC by race, with black race stratified by African-born status, after adjusting for diabetes, HIV or HCV coinfection, alcohol misuse and cirrhosis. Results Of the 278 HCC cases, 67% were 60 years of age or older, 78% were male, 87% had cirrhosis and 72% were Asian. HIV infection was present in 6% of cases. Only 7% (19 of 278) of HCC cases were black, of whom 14 were African immigrants. The median age at HCC diagnosis was 44 years in Africans. Notably, nearly all (93%) of the African-born patients with HCC were diagnosed at an age younger than 60 years compared with 52% of Asian cases (P

Background and Aims: Hepatitis C virus (HCV)-induced chronic inflammation may induce oxidative stress which could compromise the repair of damaged DNA, rendering cells more susceptible to spontaneous or mutagen-induced alterations, the underlying cause of liver cirrhosis and hepatocellular carcinoma. In the current study we examined the induction of reactive oxygen species (ROS) resulting from HCV infection and evaluated its effect on the host DNA damage and repair machinery. Methods: HCV infected human hepatoma cells were analyzed to determine (i) ROS, (ii) 8-oxoG and (iii) DNA glycosylases NEIL1, NEIL2, OGG1. Liver biopsies were analyzed for NEIL1. Results: Human hepatoma cells infected with HCV JFH-1 showed 30–60-fold increases in ROS levels compared to uninfected cells. Levels of the oxidatively modified guanosine base 8-oxoguanine (8-oxoG) were significantly increased sixfold in the HCV-infected cells. Because DNA glycosylases are the enzymes that remove oxidized nucleotides, their expression in HCV-infected cells was analyzed. NEIL1 but not OGG1 or NEIL2 gene expression was impaired in HCV-infected cells. In accordance, we found reduced glycosylase (NEIL1-specific) activity in HCV-infected cells. The antioxidant N-acetyl cystein (NAC) efficiently reversed the NEIL1 repression by inhibiting ROS induction by HCV. NEIL1 expression was also partly restored when virus-infected cells were treated with interferon (IFN). HCV core and to a lesser extent NS3-4a and NS5A induced ROS, and downregulated NEIL1 expression. Liver biopsy specimens showed significant impairment of NEIL1 levels in HCV-infected patients with advanced liver disease compared to patients with no disease. Conclusion: Collectively, the data indicate that HCV induction of ROS and perturbation of NEIL1 expression may be mechanistically involved in progression of liver disease and suggest that antioxidant and antiviral therapies can reverse these deleterious effects of HCV in part by restoring function of the DNA repair enzyme/s.

The small intestine and liver express high levels of cytochrome P450 3A (CYP3A), an enzyme subfamily that contributes significantly to drug metabolism. In patients with cirrhosis, reduced metabolism of drugs is typically attributed to decreased liver function, but it is unclear whether drug metabolism in the intestine is also compromised. In this study, we compared CYP3A protein expression and in vitro midazolam hydroxylation in duodenal mucosal biopsies from subjects with normal liver function (controls; n = 20) and subjects with various levels of severity of cirrhosis (n = 23). In samples from subjects with cirrhosis, duodenal CYP3A expression and total midazolam hydroxylation were lower by 47 and 34%, respectively, as compared with samples from controls. Greater decreases in CYP3A expression were seen in subjects with more severe cirrhosis. Therefore, patients with advanced cirrhosis may have greater drug exposure following oral dosing as a result of both impaired liver function and decreased intestinal CYP3A expression and activity.

Goal: This study examines the prevalence and correlates of significant liver fibrosis among patients with immunotolerant hepatitis B. Background: Adults with chronic hepatitis B infection acquired early in life often have normal serum alanine aminotransferase (ALT) activity and high serum hepatitis B virus deoxyribonucleic acid loads (HBV DNA), known as "immunotolerant" hepatitis B. Study: We conducted a cross-sectional study of 28 hepatitis B patients with serum HBV DNA titer > 10 6 copies/mL, positive hepatitis B envelope antigen, and persistently normal serum ALT in a tertiary care setting. Liver biopsies were reviewed by a single pathologist who was blinded to other data. The prevalence of significant hepatic fibrosis was determined using the hospital-defined upper limit of normal for ALT and 2 more stringent criteria proposed by recent studies. Statistical analyses were conducted to identify factors associated with hepatic fibrosis. Results: The prevalence of stage 2 fibrosis using the hospital laboratory, more stringent, and most stringent definitions of normal serum ALT, was 32%, 32%, and 13%, respectively, corresponding to negative predictive values of 68%, 68%, and 88%, respectively. Age greater than 30 years (P = 0.035), grade 2 liver inflammation (P = 0.005), and lower serum HBV DNA level (mean 7.45 vs. 8.42 log 10 copies/mL, P

The biological effects of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH) 2 D 3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D 3 . Among 12 different UGT isozymes tested, only UGT1A4 ≫ 2B4 and 2B7 supported the reaction. Two different 1,25(OH) 2 D 3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25- O -glucuronide isomer. The formation of 25- O -glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective K m and V max values of 7.3 and 11.2 μM and 33.7 ± 1.4 and 32.9 ± 1.9 pmol/min/mg protein. The calculated intrinsic 25- O -glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25- O -glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH) 2 D 3 represents a significant component of intestinal and systemic 1,25(OH) 2 D 3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.

We describe repeated episodes of hepatitis C (HCV) activation associated with initiation of highly active antiretroviral therapy (HAART) in two HIV/HCV coinfected individuals with undetectable serum HCV RNA. Both patients developed high HCV viremia (>1 million IU/mL) and elevations in aminotransferases >10 times upper limit of normal) within 4 months of starting HAART. This is the first report of clinically significant HCV activation in HCV-seropositive patients with initially undetectable HCV viremia. These observations suggest that flares of hepatitis C in the setting of the immune reconstitution inflammatory syndrome can occur even in those patients who have undetectable serum HCV levels prior to HAART initiation.

Background. Human immunodeficiency virus (HIV) coinfection and low peripheral blood CD4 + T cell counts are associated with increased hepatitis C liver disease.Methods. Hepatitis C virus (HCV)-specific CD4 + T cell responses were assessed using interferon (IFN)-γ enzyme-linked immunospot assays on peripheral blood mononuclear cells and expanded liver lymphocytes from HCV-monoinfected and HCV/HIV-coinfected subjects. Cell frequencies were determined using flow cytometry.Results. HIV coinfection was associated with decreased CD4 + T cell percentages in both peripheral blood (21% vs. 48%; P < .0001) and liver (15% vs. 36%; P < .0001) and with reduced responsiveness of peripheral CD4 + T cells to HCV antigens compared with HCV monoinfection (22% vs. 45%; P = .021). However, intrahepatic HCV-specific responses were maintained in HCV/HIV coinfection, compared with HCV monoinfection (38% vs. 32%; P = .7). Notably, the presence of HCV-specific responses was not related to the frequency of liver CD4 + T cells (P = .4). Circulating and liver CD4 + T cell percentages were correlated (r = 0.58; P < .0001). Circulating percentages were also inversely associated with liver fibrosis stage among HCV/HIV-coinfected subjects (P = .029). Neither hepatic CD4 + T cell percentages nor HCV-specific IFN-γ responses in the liver or periphery predicted stage.Conclusions. Despite decreases in peripheral blood HCV-specific CD4 + T cell responses and intrahepatic CD4 + T cell percentages, intrahepatic HCV-specific CD4 + IFN-γ responses were preserved in HCV/HIV coinfection.

Background & Aims: Chronic hepatitis C is a serious global medical problem necessitating effective treatment. Because standard of care with pegylated interferon plus ribavirin therapy is costly, has significant side effects, and fails to cure about half of all infections, many patients seek complementary and alternative medicine to improve their health, such as Silymarin, derived from milk thistle ( Silybum marianum ). Milk thistle's clinical benefits for chronic hepatitis C are unsettled due to variability in standardization of the herbal product. Methods: In the current study, we focused on the anti-inflammatory and antiviral properties of a standardized Silymarin extract (MK-001). Results: MK-001 inhibited expression of tumor necrosis factor-alpha in anti-CD3 stimulated human peripheral blood mononuclear cells and nuclear factor kappa B-dependent transcription in human hepatoma Huh7 cells. Moreover, MK-001 dose dependently inhibited infection of Huh7 and Huh7.5.1 cells by JFH-1 virus. MK-001 displayed both prophylactic and therapeutic effects against HCV infection, and when combined with interferon-α, inhibited HCV replication more than interferon-α alone. Commercial preparations of Silymarin also displayed antiviral activity, although the effects were not as potent as MK-001. Antiviral effects of the extract were attributable in part to induction of Stat1 phosphorylation, while interferon-independent mechanisms were suggested when the extract was biochemically fractionated by high-performance liquid chromatography. Silybin A, silybin B, and isosilybin A, isosilybin B elicited the strongest anti-NF-κB and anti-HCV actions. These effects were independent of MK-001-induced cytotoxicity. Conclusions: The data indicate that Silymarin exerts anti-inflammatory and antiviral effects, and suggest that complementary and alternative medicine-based approaches may assist in the management of patients with chronic hepatitis C.

Coinfection with hepatitis C virus (HCV) and HIV is common. Studies conducted prior to the advent of highly active antiretroviral therapy reported increased HCV liver disease severity in coinfected persons compared with those infected with HCV alone. In the current era, the influences of HIV and highly active antiretroviral therapy on the natural history of HCV liver disease are less well defined. Peginterferon alfa and ribavirin are the current standard of care in treating HCV, but HIV coinfection is associated with lower response rates to treatment. Further research is needed to enhance our understanding of the natural history and pathogenesis of HCV liver disease in the setting of HIV coinfection and to identify more effective HCV treatments.

Gene expression profiling was performed on liver biopsies from 28 patients (12 HCV and 16 HCV/HIV infected) in an attempt to understand the mechanisms of HCV liver disease in the presence and absence of HIV coinfection. The data were compared with clinical observations and a gene expression database obtained for transplant HCV-infected samples. This is the first report of functional genomics being used to compare intrahepatic gene expression profiles of HCV- and HCV/HIV-infected individuals. Significantly, the intrahepatic global gene expression profiles do not differ between HCV- and HCV/HIV-infected individuals. However, a subset of patients was identified who share a specific pattern of gene expression, termed the enhanced gene expression (EGE) pattern. Specifically, the EGE (+) patients show a dramatic decreased expression of multiple genes associated with the FAS-apoptosis pathway and increased expression of lymphocyte adhesion molecules and lymphocyte-specific genes. The EGE (+) patients also have partially impaired Type I and II IFN-mediated antiviral responses, including a lack of induction of the anti-fibrogenic cytokine IFN-γ. Importantly, the pattern of gene expression observed in EGE (+) patients has similarities to patients who developed fibrosis within 1 year of receiving a liver transplant.

BACKGROUND Hepatitis C virus (HCV) quasispecies variability has been associated with liver disease progression. The effects of human immunodeficiency virus (HIV) coinfection and highly active antiretroviral therapy (HAART) on HCV quasispecies variability have not been firmly established. METHODS We determined HCV quasispecies complexity and diversity in 69 subjects, 28 of whom were HIV infected, using clonal frequency analysis via heteroduplex mobility analysis of the second envelope gene hypervariable region. Nucleotide sequencing was performed for a small subset of subjects. RESULTS HIV-positive, HAART-naive subjects had significantly lower HCV quasispecies complexity and diversity than did both HIV-negative and HIV-positive HAART-treated subjects. In multivariate analysis, HIV infection predicted decreased complexity (P < .0001) and diversity (P = .001) of HCV quasispecies, whereas HAART predicted increased complexity (P = .013) and diversity (P = .026). For 4 of 6 patients, sequence analysis yielded data supporting the model that positive host pressure drives HCV quasispecies heterogeneity, although data favoring the hypothesis of selective outgrowth of the most fit variants were also observed. CONCLUSION HIV coinfection is associated with decreased HCV quasispecies variability, which appears to be reversed by effective HAART. Although HIV- and HAART-related effects on host immune pressure are likely to play a role in the observed differences in HCV genetic heterogeneity, other mechanisms may be operative.

The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration ( P = 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease ( P = 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation ( P < 0.001), liver fibrosis ( P < 0.001), and serum alanine aminotransferase levels ( P = 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens ( P = 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.

Prior studies have suggested that natural killer (NK) cell function might be impaired in chronic hepatitis C virus (HCV) infection. Circulating NK cell frequency and cytolytic activity were examined freshly ex vivo in HCV-infected and uninfected subjects. Surprisingly, the intrinsic cytolytic activity of peripheral blood NK-enriched cells was similar between HCV-infected and uninfected groups (P = .91). Although the percentage of circulating CD3- CD16/56+ NK cells was 30% lower in HCV-infected compared with uninfected subjects (P = .02) paralleled by a decrease of CD56(dim) cytolytic NK cells (P = .02), overall K562 cytolysis by unfractionated peripheral blood mononuclear cells was not affected (P = .29). Analysis of the relationships between NK cytolytic activity and other clinical information revealed an inverse association with liver fibrosis stage (P = .035). In conclusion, NK cell cytolytic function does not appear to be impaired in chronic hepatitis C, but higher levels of NK cell cytolysis are associated with less liver fibrosis.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Hepatitis C virus (HCV), an RNA virus classified within the Flaviviridae (12), has a remarkable propensity for persistence in the human host following acute infection. Infections spontaneously resolve in 20-30% of infected individuals, while 70-80% of infections result in long-term persistent viremia. Chronically infected individuals are at an increased risk of developing hepatocellular injury compared to subjects with acute resolved infections (25), with manifestations progressing from mild to severe (bridging) fibrosis, and, ultimately cirrhosis, in 10-30% of chronic infections. Cirrhosis underlies life-threatening complications of end stage liver disease and/or hepatocellular carcinoma, after a long and extremely variable disease incubation period (25, 38). It is presently impossible to predict which persons with chronic hepatitis C are at greatest risk for disease progression, and, likewise, host-virus relationships are most important for driving chronic hepatitis C disease progression have not been defined. In vitro evidence supporting the concept of virusmediated liver injury has recently been reported, including isolation of cytopathic derivatives of HCV infectious clone JFH1 (27), and also in the chimeric mouse (immune deficient mouse with humanized liver) hepatitis C model (18). Based on experimental data from acute liver injury rodent model systems, where massive hepatic necrosis is experimentally induced by toxins such as carbon tetrachloride (29), the master mediators of hepatic fibrogenic processes are Transforming Growth Factor beta (TGFbeta) and Platelet Derived Growth factor (PDGF) (17). Hepatic Stellate Cells (HSCs) and macrophages appear to be the major cell types regulating hepatic fibrosis (17, 46). HSCs, normally quiescent in the liver, and potentially derived from hepatic oval cells (45), respond to injury by proliferation and secretion of large amounts of extracellular matrix proteins, in addition to pro-fibrogenic cytokines including TGFbeta. During the process, HSCs are transformed into fibrogenic myofibroblasts. A well characterized liver cirrhosis

Seven patients were treated with recombinant human tissue plasminogen activator (tPA) for severe hepatic venocclusive disease (VOD) that developed after bone marrow transplantation for hematologic malignancy. Recombinant human tPA (10 mg/d x 2 days) and heparin (1,000 U bolus followed by continuous intravenous infusion of 150 U/kg/d x 10 days) were begun a median of 9 days (range, 4 to 18 days) posttransplant. The median total serum bilirubin and percent weight gain from baseline were 19.4 mg/dL (range, 14.6 to 34.9 mg/dL) and 9.1% (range, 1% to 18.5%), respectively, at the start of tPA administration. Five patients responded to therapy with prompt reduction in total serum bilirubin within 96 hours of starting tPA. Three patients are alive 178 to 379 days posttransplant without evidence of VOD. No patient had significant hemorrhagic complications with tPA. We conclude that recombinant human tPA can be administered to patients with severe VOD at the dosage described. Whereas preliminary data suggests that recombinant human tPA can alter the natural history of severe VOD, further study is necessary to determine its efficacy.

Background & Aims Silymarin, an extract from the seeds of the milk thistle plant Silybum marianum, has been used for centuries for the treatment of chronic liver diseases. Despite common use by patients with hepatitis C in the United States, its clinical efficacy remains uncertain. The goal of this study was to determine whether silymarin has in vitro effects on immune function that might have implications for its potential effect on hepatitis C virus (HCV)-induced liver disease. Methods Freshly isolated peripheral blood mononuclear cells (PBMC) and T cells from HCV-infected and uninfected subjects were tested in vitro for responses to nonspecific and antigenic stimulation in the presence and absence of a standardized preparation of silymarin (MK001). Results Minimal MK001 toxicity on PBMC was found at concentrations between 5 and 40 μg/mL. MK001 dose dependently inhibited the proliferation and secretion of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2 by PBMC stimulated with anti-CD3. In addition, MK001 inhibited proliferation by CD4+ T cells to HCV, Candida, and tetanus protein antigens and by HLA-A2/HCV 1406–1415-specific CD8+ T cells to allogeneic stimulation. MK001 inhibited T-cell TNF-α and IFN-γ cytokine secretion to tetanus and Candida protein antigens. Finally, MK001 inhibited nuclear factor-κB transcriptional activation after T-cell receptor-mediated stimulation of Jurkat T cells, consistent with its ability to inhibit Jurkat T-cell proliferation and secretion of IL-2. Conclusions Silymarin's ability to inhibit the proliferation and proinflammatory cytokine secretion of T cells, combined with its previously described antiviral effect, suggests a possible mechanism of action that could lead to clinical benefit during HCV infection.

We describe repeated episodes of hepatitis C (HCV) activation associated with initiation of highly active antiretroviral therapy (HAART) in two HIV/HCV coinfected individuals with undetectable serum HCV RNA. Both patients developed high HCV viremia (1 million IU/mL) and elevations in aminotransferases10 times upper limit of normal) within 4 months of starting HAART. This is the first report of clinically significant HCV activation in HCV-seropositive patients with initially undetectable HCV viremia. These observations suggest that flares of hepatitis C in the setting of the immune reconstitution inflammatory syndrome can occur even in those patients who have undetectable serum HCV levels prior to HAART initiation.

Prevention and Management of Gastroesophageal Varices and Variceal Hemorrhage in Cirrhosis Guadalupe Garcia-Tsao, M.D.,1 Arun J. Sanyal, M.D.,2 Norman D. Grace, M.D., FACG,3 William D. Carey, M.D., MACG,4 the Practice Guidelines Committee of the American Association for the Study of Liver Diseases and the Practice Parameters Committee of the American College of Gastroenterology 1Section of Digestive Diseases, Yale University School of Medicine and VA-CT Healthcare System, New Haven, Connecticut; 2Division of Gastroenterology, Virginia Commonwealth University Medical Center, Richmond, Virginia; 3Division of Gastroenterology, Brigham and Women’s Hospital, Boston, Massachusetts; 4The Cleveland Clinic, Cleveland, Ohio

Background Hepatitis C virus (HCV) circulates as quasispecies (QS), whose evolution is associated with pathogenesis. Previous studies have suggested that the use of thermostable polymerases without proofreading function may contribute to inaccurate assessment of HCV QS. In this report, we compared non-proofreading (Taq) with proofreading (Advantage High Fidelity-2; HF-2) polymerases in the sensitivity, robustness, and HCV QS diversity and complexity in the second envelope glycoprotein gene hypervariable region 1 (E2-HVR1) on baseline specimens from 20 patients in the HALT-C trial and in a small cohort of 12 HCV/HIV co-infected patients. QS diversity and complexity were quantified using heteroduplex mobility assays (HMA). Results The sensitivities of both enzymes were comparable at 50 IU/ml, although HF-2 was more robust and slightly more sensitive than Taq. Both enzymes generated QS diversity and complexity scores that were correlated (r = 0.68; p < 0.0001, and r = 0.47; p < 0.01; Spearman's rank correlation). QS diversity was similar for both Taq and HF-2 enzymes, although there was a trend for higher diversity in samples amplified by Taq (p = 0.126). Taq amplified samples yielded complexity scores that were significantly higher than HF-2 samples (p = 0.033). HALT-C patients who were HCV positive or negative following 20 weeks of pegylated IFN plus ribavirin therapy had similar QS diversity scores for Taq and HF-2 samples, and there was a trend for higher complexity scores from Taq as compared with HF-2 samples. Among patients with HCV and HIV co-infection, HAART increased HCV QS diversity and complexity as compared with patients not receiving therapy, suggesting that immune reconstitution drives HCV QS evolution. However, diversity and complexity scores were similar for both HF-2 and Taq amplified specimens. Conclusion The data suggest that while Taq may overestimate HCV QS complexity, its use does not significantly affect results in cohort-based studies of HCV QS analyzed by HMA. However, the use of proofreading enzymes such as HF-2 is recommended for more accurate characterization of HCV QS in vivo.

Background & Aims: Liver transplant recipients infected with hepatitis C virus (HCV) develop recurrent hepatitis soon after transplantation and, in some cases, progress to fibrosis within the first 2 years. Our goals were to identify molecular processes influencing the liver disease progression and to find potential gene markers of early fibrosis. Methods: We performed gene expression profiling on serial liver biopsy specimens obtained from 13 (11 infected and 2 uninfected) transplant recipients within the first year after transplantation at 0, 3, 6, and 12 months. The data were compared with clinical observations and with a gene expression database obtained for 55 nontransplant HCV-infected and uninfected liver samples. Results: We identified several specific gene expression patterns. The first pattern was unique for the transplant recipients regardless of their infection status. The corresponding genes encoded stress response proteins and blood proteins involved in coagulation that were differentially expressed in response to posttransplantation graft recovery. The second pattern was specific to HCV-infected samples and included up-regulation of genes encoding components of the interferon-mediated antiviral response and immune system (antigen presentation, cytotoxic response). This up-regulation pattern was absent or suppressed in the patients who developed early fibrosis, indicating that the disease progression might result from an impaired liver response to infection. Finally, we identified gene expression patterns that were specific for 12-month biopsy specimens in all 4 HCV-infected patients who developed early fibrosis. Conclusions: The identified gene expression patterns may prove useful for diagnostic and prognostic applications in HCV-infected patients, including predicting early progression to fibrosis.

Alcoholism and chronic hepatitis C (HCV) infection are common causes of liver disease worldwide. Hepatitis C virus and alcohol use frequently coexist, and together lead to more rapid progression of liver disease.To critically review the literature pertaining to the combined effects of alcohol and HCV, focusing primarily on how alcohol influences the natural history, pathogenesis, and treatment of HCV liver disease.A thorough review of the English literature was conducted, using a MEDLINE-based computerized literature search and review of cited references.Hepatitis C virus is prevalent in unselected alcoholic populations (14-36%) and in alcoholic individuals with liver disease (or =51%). Hepatitis C virus-infected individuals who drink alcohol in excess have more severe histologic injury, more rapid disease progression, and a higher frequency of cirrhosis and hepatocellular carcinoma. Alcohol use also appears to decrease response rates to interferon therapy. The mechanisms of interaction between alcohol and HCV are not fully understood, but they likely include the effects of alcohol on the host immune system and the virus and on other factors possibly related to HCV liver disease and hepatic carcinogenesis.Alcohol use and HCV infection frequently coexist. Although there is ample evidence that alcohol use adversely affects the natural history of HCV liver disease, how the two interact is not well understood. Patients with chronic HCV should be encouraged to avoid alcohol; however, the threshold above which alcohol results in accelerated liver disease remains to be determined.

cant fibrosis according to CD4 count. Although a slightly better performance of the lower cutoff of APRI was observed in patients with CD4 count >250, a lower CD4 count had no major impact on the diagnostic performance of the upper cutoffs of both APRI and FIB-4 models. In addition, a similar proportion of patients were correctly classified, regardless of CD4 count (£250 or >250) by the APRI (67% vs. 76%) and by the FIB-4 (91% vs. 88%). In conclusion, in our cohort of co-infected patients, performance of APRI and FIB-4 models for detecting significant fibrosis were not significantly influenced by CD4 count.

Bile leaks after T-tube removal are a frequent cause of morbidity in orthotopic liver transplant recipients. The aim of this study was to determine factors that predict the development of these leaks in liver transplant recipients. Records of all patients who had undergone liver transplantation at the University of Washington Medical Center between January 1990 and September 1993 were reviewed. The following were excluded: patients with a Roux-en-Y anastomosis or inadvertent early T-tube removal and patients who died or underwent retransplantation before T-tube removal. All T-tube cholangiograms were reviewed blindly by two authors. Using logistic regression, several variables were assessed for possible association with bile leaks after T-tube removal; these included patient demographics, intraoperative variables, and clinical and cholangiographic variables related to T-tube removal. Of the 166 liver transplants performed in 150 patients, 99 transplants in 97 patients were evaluable for bile leak after T-tube removal. Thirty-three patients developed symptomatic bile leaks, and 21 underwent endoscopic or operative intervention for persistent symptoms. Only duct mural irregularities on the final cholangiogram were strongly associated with the development of a bile leak after T-tube removal (P = 0.001). In conclusion, bile leaks after T-tube removal occurred in one-third of patients undergoing orthotopic liver transplantation; the majority of these patients required some intervention. Duct mural irregularities were associated with bile leaks.

Hepatitis frequently recurs after liver transplantation for hepatitis C. However, the histological progression of disease, predictors of recurrence and disease severity, and patient survival remain uncertain. Fifty-five patients with cirrhosis caused by chronic hepatitis C underwent liver transplantation between January 1990 and December 1993. Hepatitis C genotype was determined, and liver biopsies were performed at frequent intervals posttransplantation. The median follow-up time was 40.4 months. The cumulative rate of survival was no different in liver transplant recipients for hepatitis C than in liver transplant recipients for other chronic liver diseases (P = .62). Histological recurrent hepatitis C developed in 33 of 50 patients assessable for disease recurrence; the median recurrence-free survival time was 13.4 months. Histological activity and stage were mild in most cases. Only 2 patients developed cirrhosis, and no patient required a second transplantation for recurrent disease. Patients with acute cellular rejection had a shorter recurrence-free survival (P = .0141). In patients with recurrent hepatitis, rejection also was correlated with increased histological grade 2 years after transplantation (P = .0061). Recurrence-free survival was decreased in patients infected with genotype 1 (1a and 1b combined) compared with genotypes 2 and 3 combined (P = .02), whereas there was no difference between genotypes 1a and 1b (P > .80). Only patients infected with genotype 1a or 1b developed bridging fibrosis or cirrhosis. In addition, patients who had an early recurrence had a greater risk of progressing to bridging fibrosis or cirrhosis (hazard ratio, 5.1; P = .0473). In our experience, recurrent hepatitis C after liver transplantation in most cases is mild and survival is unaffected. Both acute cellular rejection and infection with genotype 1 are independent risk factors for reduced recurrence-free survival, and early recurrence is associated with a higher risk of disease progression. (Hepatology 1997 Dec;26(6):1646-52)

The present study describes natural genetic heterogeneity of hepatitis C virus (HCV) p7 protein, the ion channel that plays a critical role in assembly and release of HCV, within 299 variants isolated from serum specimens of 27 chronically infected patients, 12 of whom with human immunodeficiency virus (HIV) co-infection. Liver fibrosis stage was inversely correlated with p7 synonymous substitutions (dS) (p=0.033), and indices of p7 genetic diversity were significantly higher in HIV-negative subjects compared to HIV-positive subjects (dS, p=0.005; non-synonymous substitutions (dN), p=0.002; dN/dS ratio, p=0.024; amino acid distances, p=0.007). Six p7 genes with naturally occurring unique amino acid variations were selected for in vitro study. The variants demonstrated diversified functional heterogeneity in vitro, with one variant from a subject with severe liver disease displaying hyperactive ion channel function, as well as other variants presenting altered pH-activated channel gating activities.