Implementation of an in-house quantitative real-time polymerase chain reaction method for Hepatitis B virus quantification in West African countries.

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. HBV infection is diagnosed by serological tests, while real-time polymerase chain reaction (qRT-PCR) assays are used to quantify viral load, which is a crucial parameter to determine viral replication and to monitor antiviral treatments. However, measuring viral load in resource-limited countries remains nonsystematic, due to the high cost of commercial kits. Here, we describe the development, validation and implementation of a low-cost, in-house qRT-PCR assay to monitor HBV viral load in chronic carriers enrolled in the PROLIFICA programme in the Gambia and Senegal. Over 1500 HBsAg-positive patients, including 210 chronically infected HBV patients, who were given antiviral treatment (tenofovir), were monitored by qRT-PCR using the SYBR Green- and HBV-specific primers. Twenty-four tenofovir-treated patients were followed up and their viral load was tested every 3 months over the 12-month experimental time course. Compared to commercial assays, our in-house assay was shown to be (i) highly reliable, with good intra- and interassay reproducibility over a wide range (45-4.5 × 108 copies mL-1 ), (ii) very similar in the viral loads detected (R2  = .90), (iii) highly sensitive, as it detected loads as low as 30 copies mL-1 (~5 IU mL-1 ), (iv) cheaper (2- to 3-fold), (v) easier to implement and (vi) more rapid. Based on our experience, we recommend this assay as a reliable alternative to commercial assays, for monitoring HBV viraemia in resource-limited, highly endemic countries to reduce the cost and technical obstacles associated with commercial kits.

Recommendations for ICT use in Alzheimer's disease assessment: Monaco CTAD Expert Meeting.

Alzheimer disease (AD) and other related dementia represent a major challenge for health care systems within the aging population. It is therefore important to develop better instruments for assessing disease severity and disease progression to optimize patient's care and support to care providers, and also provide better tools for clinical research. In this area, Information and Communication Technologies (ICT) are of particular interest. Such techniques enable accurate and standardized assessments of patients' performance and actions in real time and real life situations. The aim of this article is to provide basic recommendation concerning the development and the use of ICT for Alzheimer's disease and related disorders. During he ICT and Mental Health workshop (CTAD meeting held in Monaco on the 30th October 2012) an expert panel was set up to prepare the first recommendations for the use of ICT in dementia research. The expert panel included geriatrician, epidemiologist, neurologist, psychiatrist, psychologist, ICT engineers, representatives from the industry and patient association. The recommendations are divided into three sections corresponding to 1/ the clinical targets of interest for the use of ICT, 2/ the conditions, the type of sensors and the outputs (scores) that could be used and obtained, 3/ finally the last section concerns specifically the use of ICT within clinical trials.

Distribution of total DNA and cccDNA in serum and PBMCs may reflect the HBV immune status in HBsAg+ and HBsAg- patients coinfected or not with HIV or HCV.

BACKGROUND: The potential reservoir role of serum and peripheral blood mononuclear cells (PBMCs) for total HBV DNA (tDNA) and cccDNA still remains unknown. MATERIAL AND METHODS: We analyzed tDNA and cccDNA with a single sensitive and validated standardized real-time PCR method in serum and PBMCs in two populations of chronic HBV infection coinfected or not with HCV and/or HIV viruses: a retrospective cohort of 130 HBsAg-negative (HBsAg-) patients with "anti-HBc alone" or anti-HBc and anti-HBs antibodies (Ab) and a cohort of 70 HBsAg-positive patients, 16 of them being prospectively followed under treatment. RESULTS: Among HBsAg- patients, HBV DNA was detected in serum or PBMCs in about half of the cases with various distributions of tDNA and cccDNA: in HIV-negative patients with an "antiHBc alone" profile, tDNA was mostly detected in PBMCs suggesting a possible active role of PBMCs; although cccDNA was not detected in PBMCs in HIV-positive patients, tDNA and cccDNA were mostly observed in serum, suggesting a specific pattern of more "persistent" than "occult" infection in this population. Patients with anti-HBc and anti-HBs Ab harbored tDNA in serum or in PBMCs, regardless of their HIV or HCV status, raising the question of a viral reactivation risk during immunosupression in these patients. Among HBsAg+ patients, tDNA was detected in serum and PBMCs of 88.5% of the cases and cccDNA in 22%. Levels of tDNA in both compartments were highly correlated during treatment, suggesting a passive reservoir role for PBMCs. CONCLUSION: The respective distribution of tDNA and cccDNA in serum and PBMCs may reflect the different immune statuses of the host in HBsAg+ and HBsAg- patients. The frequency of HBV DNA in PBMCs from AgHBs- patients suggests a viral reactivation risk during immunodepression in those patients.

Hepatitis B virus induced hepatocellular carcinoma.

A number of risk factors appear to play a role in Hepatocellularcinoma (HCC), HBV infection being one of the most important. Chronic inflammation and cytokines are key determinants in the development of fibrosis and liver cell proliferation. HBV DNA integration and/or expression of HBV proteins may have a direct effect on cellular functions. Occult hepatitis B virus infection is characterized by persistence of HBV DNA in hepatitis B surface antigen-negative individuals. There are evidences that occult HBV is a risk factor for the development of HCC and that the potential mechanisms whereby overt HBV might induce tumour formation are mostly maintained.

Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma.

Dysregulation of growth factors and their receptors is central to human hepatocellular carcinoma (HCC). We previously demonstrated that the Frizzled-7 membrane receptor mediating the Wnt signalling can activate the beta-catenin pathway and promotes malignancy in human hepatitis B virus-related HCCs. Expression patterns of all the 10 Frizzled receptors, and their extracellular soluble autoparacrine regulators (19 Wnt activators and 4 sFRP inhibitors) were assessed by real-time RT-PCR in 62 human HCC of different etiologies and their matched peritumorous areas. Immunostaining was performed to localise Frizzled on cell types in liver tissues. Regulation of three known Frizzled-dependent pathways (beta-catenin, protein kinase C, and C-Jun NH(2)-terminal kinase) was measured in tissues by western blot. We found that eight Frizzled-potentially activating events were pleiotropically dysregulated in 95% HCC and 68% peritumours as compared to normal livers (upregulations of Frizzled-3/6/7 and Wnt3/4/5a, or downregulation of sFRP1/5), accumulating gradually with severity of fibrosis in peritumours and loss of differentiation status in tumours. The hepatocytes supported the Wnt/Frizzled signalling since specifically overexpressing Frizzled receptors in liver tissues. Dysregulation of the eight Frizzled-potentially activating events was associated with differential activation of the three known Frizzled-dependent pathways. This study provides an extensive analysis of the Wnt/Frizzled receptor elements and reveals that the dysregulation may be one of the most common and earliest events described thus far during hepatocarcinogenesis.

Occult HBV infection may represent a major risk factor of non-response to antiviral therapy of chronic hepatitis C.

Occult hepatitis B virus (HBV) infection is common in chronic hepatitis C patient. However, its significance and consequences are still unclear. The aim of this study was to evaluate the prevalence of occult HBV among HCV chronic carriers in France and to assess its impact on liver histology and response to antiviral therapy. To this end a cohort of 203 patients with chronic hepatitis C without hepatitis B surface antigen (HBsAg) has been examined. Serum HBV-DNA was detected using a highly sensitive PCR with primers located in the S and X genes. HBV viraemia levels were further determined by real-time PCR. Results showed that 47 of 203 (23%) patients had occult HBV infection with a low HBV load (10(2)-10(4) copies/ml) but significantly higher HCV-RNA titers (P < 0.05). No significant difference in age, gender, serum ALT level, HCV genotypes, and the presence of anti-HBc was observed between patients with or without HBV-DNA. When compared histologically, patients with occult HBV infection had higher activity (A2-A3 in 53% vs. 38%, P < 0.01) and more advanced fibrosis (60% vs. 33%, P < 0.001) than HBV-DNA negative cases. Sustained response to combination therapy against Chronic hepatitis C was achieved in 11 (28%) of 40 HBV-DNA positive cases, compared with 65 (45%) of the 144 HBV-DNA negative cases (P < 0.05). Among the 144 HBV-DNA negative HCV patients those with genotype 1 responded less frequently to therapy as compared to other genotypes infected patients (38% vs. 55%, P < 0.05). Surprisingly, when considering all patients studied, irrespective to the HBV-DNA status no significant difference was observed in response to combination therapy regarding HCV genotypes (39% vs. 44%, P > 0.05). In conclusion, HBV-DNA is found in 1/4 of French chronic hepatitis C patients regardless of the presence of anti-HBc. Such an occult HBV co-infection is associated with more severe liver disease, higher HCV viral load and decreased response to antiviral therapy irrespective of HCV genotypes.

Double hepatitis B virus infection in a patient with HIV/hepatitis C virus coinfection and 'anti-HBc alone' as serological pattern.

Reported here is a case of double hepatitis B virus (HBV) infection in an HIV/hepatitis C virus (HCV)-coinfected patient with antibodies against hepatitis B core antigen as the only serological marker (anti-HBc alone). Two different HBV genotypes were identified in this patient. A search of the medical literature indicated this report is the first to describe a multiple silent HBV infection in an HIV/HCV-coinfected-patient. The elevated incidence of the anti-HBc alone pattern in HIV-positive patients and the increasing number of silent HBV infections detected in those patients demonstrate the need to carefully examine HIV-positive patients for occult HBV infection. In addition, it appears necessary to thoroughly study such patients in order to evaluate the impact of mixed HBV infection and triple HIV/HCV/HBV infection on morbidity.

Analysis of HCV co-infection with occult hepatitis B virus in patients undergoing IFN therapy.

BACKGROUND/AIM: Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA in the absence of hepatitis B surface antigen (HBsAg) in the patient serum. Although such infections have been identified in patients with chronic hepatitis C, the clinical significance of those co-infections is still not understood. Our aim was, therefore, to assess the prevalence and clinical consequences of occult HBV infection in chronic hepatitis C patients undergoing antiviral therapy. METHODS: The study population consisted of 53 HBsAg-negative patients with chronic hepatitis C treated with IFN/ribavirin or IFN/ribavirin/amantadine. Nine patients experienced a viral breakthrough (BT), 30 were non-responders (NR) and 14 were responders (R). HBV-DNA detection by PCR was performed using primers specific for the S region of the HBV genome and HCV-RNA detection by PCR with primers localised in both the 5'NC and core region of HCV genome, before, during and after treatment. Viral genome sequences were also studied. RESULTS: Occult HBV genomes were found in the serum of four of 53 (7.5%) patients, unrelated to anti-HBc status. No significant differences in biochemical, virological, or histological markers, age, duration of infection, were observed in patients with or without HBV DNA. There was an inverse correlation in the evolution of HBV DNA and HCV RNA levels. Direct sequencing showed that S gene of occult HBV presented mutations in the "a" determinant while no specific mutation in the core region of HCV was observed. None of the four patients co-infected with HBV and HCV were responders to anti-HCV therapy. CONCLUSION: In our clinical setting, the prevalence of occult HBV co-infection among patients with chronic hepatitis C was low and independent of the presence of markers of previous HBV infection. Further studies in larger cohort of patients are warranted to determine if occult HBV co-infection may be involved in HCV resistance to combination therapy.

Virtual reality therapy versus cognitive behavior therapy for social phobia: a preliminary controlled study.

Social phobia is one of the most frequent mental disorders and is accessible to two forms of scientifically validated treatments: anti-depressant drugs and cognitive behavior therapies (CBT). In this last case, graded exposure to feared social situations is one of the fundamental therapeutic ingredients. Virtual reality technologies are an interesting alternative to the standard exposure in social phobia, especially since studies have shown its usefulness for the fear of public speaking. This paper reports a preliminary study in which a virtual reality therapy (VRT), based on exposure to virtual environments, was used to treat social phobia. The sample consisted of 36 participants diagnosed with social phobia assigned to either VRT or a group-CBT (control condition). The virtual environments used in the treatment recreate four situations dealing with social anxiety: performance, intimacy, scrutiny, and assertiveness. With the help of the therapist, the patient learns adapted cognitions and behaviors in order to reduce anxiety in the corresponding real situations. Both treatments lasted 12 weeks, and sessions were delivered according to a treatment manual. Results showed statistically and clinically significant improvement in both conditions. The effect-sizes comparing the efficacy of VRT to the control traditional group-CBT revealed that the differences between the two treatments are trivial.

Clinical impact of occult HBV infections.

HBV infection in the absence of HBsAg has been a matter of debate for years, but its existence and clinical relevance are now supported by many publications, editorials and reviews. HBV DNA without HBs antigenemia was detected in the following clinical situations: (1) Chronic, presumably viral, hepatitis unrelated to HCV, atypical alcoholic hepatitis and hepatocellular carcinoma (HCC); (2) viral reactivation following immunosuppression; (3) Transmission through transplantation, transfusion or experimental transmission to chimpanzees. Occult HBV infections are not restricted to areas of high HBV endemicity. Indeed, such cases have been described in Western countries including France. It is now established that occult HBV infection among non-HCV patients suffering from chronic hepatitis varies from 20% to 30% in Europe, and in the context of HCV infection it varies from 20% in France up to 80% in Japan. The percentage of occult HBV infections among non A-E cases depends on several parameters: (1) The method of detection, including PCR primer selection; (2) patient recruitment; (3) patients from countries highly endemic for HBV are more likely to develop occult HBV infections; (4) prevalence may also vary depending on the nature of biological material tested, with a higher proportion for liver compared to serum specimen. The mechanisms leading to HCC in occult HBV infection seem similar to those overt cases, patients with low-grade but diagnosable HBV replication that retains its pro-oncogenic properties. During the course of HCV infection, occult HBV infection may worsen liver damage induced by HCV and reduce the response to HCV antiviral treatment. Occult HBV infection is a frequent phenomenon and HBV DNA testing with highly sensitive PCR in the clinical setting is therefore becoming of paramount importance.

Definition of a VR-based protocol to treat social phobia.

Social phobia is an anxiety disorder that is accessible to two forms of treatment yielding scientifically validated results: drugs and cognitive-behavioral therapies. Graded exposure to feared social situations is fundamental to obtain an improvement of the anxious symptoms. Traditionally, exposure therapies are done either in vivo or by imagining the situations. In vivo exposure is sometimes difficult to control and many patients have some difficulties in using imagination. Virtual reality (VR) seems to bring significant advantages. It allows exposures to numerous and varied situations. This paper reports the definition of a clinical protocol whose purpose is to assess the efficiency of a VR therapy compared to a CBT and to the absence of treatment for social phobic patients. It explains the illness' diagnosis and its usual treatments. It exposes all the architecture of the study, the assessment tools, the content and unfold of the therapy sessions. It finally reports first results of a clinical trial in a between-group design in 10 patients suffering from social phobia. The virtual environments used in the treatment reproduce four situations that social phobics feel the most threatening: performance, intimacy, scrutiny and assertiveness. With the help of the therapist, the patient learns adapted cognitions and behaviors with the aim of reducing her or his anxiety in the corresponding real situations. The novelty of our work is to address a group of situations that the phobic patient is most likely to experience and to treat patients according to a precise protocol.

Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-) liver disease.

Despite a number of studies documenting hepatitis B virus (HBV) infection in the absence of hepatitis B surface antigen (HBsAg) a causal relationship between silent HBV infection and liver disease remain difficult to establish. In particular, both the prevalence and clinical significance of this observation are poorly understood. Why is HBV replication apparently so low in these patients? A number of studies have tried to elucidate the mechanism of HBsAg negative infections, and considerable data documenting HBV infectivity or reinfection in the absence of detectable HBsAg support the hypothesis that in some of these cases, HBV is undergoing low-level replication in the liver and this, in several situations including: (1) chronic liver disease, alcoholic liver disease, hepatocellular carcinoma; (2) viral reactivation following cancer chemotherapy or immunosuppression and (3) transmission via transfusion or from human serum to chimpanzees. In a recent study including 50 patients with chronic liver disease of unknown etiology we could detect serum HBV DNA by nested polymerase chain reaction (PCR) in 15/50 patients (50% at the cirrhosis stage) in the absence of HBsAg; in the liver of the 15 patients both HBcAg and/or HBsAg can be detected at very low-level. Viral host factors allowing HBV persistence in the absence of HBsAg can depend on several mechanisms. Coinfections with HCV can explain only a proportion of HBsAg(-) HBV infections. Secondly, HBV mutations in the core promotor region leading to a minimal viral replication, or mutations in the HBsAg-encoding region might explain the absence of serological recognition. Finally, it is possible that in some cases host immune mechanisms can maintain HBV infection in a latent state until transmission to another individual who subsequently develops a more active infection especially when immunosuppressive therapy is employed. Existence of HBsAg(-) HBV infections should be taken into account by the use of sensitive PCR tests for prevention of viral transmission in the settings of blood donations and organ transplants.

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

High incidence of hepatitis B infections among chronic hepatitis cases of unknown aetiology.

BACKGROUND/AIMS: In approximately 5% of chronic liver disease cases, no aetiology can be identified. We selected sera from 50 patients with chronic hepatitis of unknown aetiology who were enrolled in this follow-up study whose aim is to gain insight into the possible role of viruses and to define potential clinical outcomes. METHODS: Patients' sera were screened with highly sensitive polymerase chain reaction assays for hepatitis B (HBV), C, D, and G viruses and TT virus. Sera were also retested for antibodies against the core antigen of HBV. RESULTS: Surprisingly, HBV DNA was detected in both serum and liver in 15/50 (30%) patients. Immunostaining for HBV antigens on biopsies from patients positive for HBV DNA showed HBcAg and/or HBsAg expression at low levels in 9/15 samples. Eleven of the fifteen patients were anti-HBc positive. With one exception, all patients carried HBV genomes at low levels (10(4) copies/ml or less). Histological signs of chronic liver disease were observed in all patients. CONCLUSION: Unrecognised HBV infections may account for a high proportion of chronic hepatitis cases of unknown aetiology. Improved HBV detection tests, which appear mandatory for the diagnosis and management of non-A non-E hepatitis as well as for improved safety of transfusions and transplantations are needed.

Environmental and genetic determinants of aflatoxin-albumin adducts in the Gambia.

Aflatoxins together with chronic hepatitis B virus (HBV) infection contribute to the high incidence of hepatocellular carcinoma in developing countries. An understanding of the mechanism of interaction between these factors would provide a strong rationale for developing effective prevention strategies. In this study in The Gambia we examined the effect of environmental (place of residence and timing of sample collection) and host factors (age, sex, HBV status and interindividual variations in carcinogen metabolising enzymes) in determining blood aflatoxin-albumin adduct levels in 357 individuals of whom 181 were chronic HBV carriers. Samples were analysed for aflatoxin-albumin adducts, HBV status and genotypes of glutathione S-transferase (GST) M1, GSTT1, GSTP1 and epoxide hydrolase (EPXH). Urine samples were analysed for 6beta-hydroxycortisol:cortisol ratio as a marker of cytochrome P450 (CYP) 3A4 activity. Adduct levels were significantly higher in subjects resident in rural [geometric mean adduct level 34.9 pg aflatoxin B1-lysine equivalent (28.5-42.8; 95%CI)/mg albumin] than in periurban areas [22.2 pg (14.9-33.4)/mg] and were approximately twice as high in the dry season [mid-February to March; 83.2 pg (53.3-130.8)/mg] than the wet [July to August; 34.9 pg (28.5-42.8)/mg]. In contrast, HBV status, CYP3A4 phenotype, GSTT1, GSTP1 and EPXH genotypes were not associated with aflatoxin-albumin adduct level. However, mean adduct levels were significantly higher in non-HBV infected subjects with GSTM1 null genotype. The main factors which affect aflatoxin-albumin adduct levels in this population are environmental, notably place of residence and timing of sample collection. This study further emphasises the priority to reduce aflatoxin exposure in these communities by primary prevention measures.

[The TT virus: review of the literature]. / Le virus TT: revue de la littérature.

OBJECTIVE: To review the literature on the TT virus. METHODS: The literature review was based on articles identified through MEDLINE between Jan. 1, 1997, and August 15, 1999. RESULTS: In 1997, a new DNA virus, designated TTV, was isolated and seemed to be associated with non A-G post-transfusion hepatitis. The virus was identified using a polymerase chain reaction (PCR) because serology was not routinely available. At least 16 genotypes were identified. Depending on the PCR technique used, the prevalence of infection ranged from 17% to 71% in a group of sera tested. The prevalence rate ranged from 1.2% to 62% among blood donors, from 0.5% to 83% among hemophiliacs and from 1% to 71% in cases of chronic hepatitis. The current hypothesis is that routes of infection were parenteral and orofecal. The pathogenesis of this virus, if it really exists, is not yet clearly established. It has been postulated that some interaction may exist between the TT virus and the hepatitis C virus. The use of interferon seems to decrease the TT viremia, according to results obtained outside the context of clinical trials. CONCLUSION: The pathogenesis of the TT virus needs to be rapidly established for transmission prevention and therapeutic intervention.

Altered expression of hepatic carcinogen metabolizing enzymes with liver injury in HBV transgenic mouse lineages expressing various amounts of hepatitis B surface antigen.

AIMS/BACKGROUND: The objective of this work was to evaluate the possible modulation of carcinogen metabolizing enzymes in relation to chronic infection by hepatitis B virus (HBV). This was to test whether enzyme level is altered in association with HBV gene expression per se or only when that expression was associated with an induction of liver injury. METHODS: For this purpose, we studied four different HBV transgenic mouse lineages (23.3, 45.2, 50.4 and 107.5) that express the transgene encoding for the large envelope protein (HBsAg) at different levels. These lineages exhibit an associated liver injury which progresses with age and is positively correlated with the degree of accumulation of HBsAg in the hepatocytes. The modulation of levels of cytochrome P450 (1a, 2a-5, 2b, 2c, 3A4 and 2E1) and glutathione S-transferases (GST alpha and pi) involved in carcinogen metabolism was examined by immunohistochemistry in these lineages. RESULTS: While we observed an increase in staining intensity of P450s 1-a and 2a-5 in lineages expressing cytopathic amounts of HBsAg (lineages 50.4 and 45.2), we only observed minor changes or no changes at all for the other lineages (23.3 and 107.5). Staining with antibodies to cytosolic pi class GST demonstrated an increase in older mice, although no major alterations were observed for GST alpha. CONCLUSIONS: These results suggest that liver cell injury induced by accumulation of HBV antigens can result in the induction of some carcinogen metabolizing enzymes and this may be one mechanism of chemical-viral interaction in hepatocarcinogenesis.

Liver-directed gene transfer: a linear polyethlenimine derivative mediates highly efficient DNA delivery to primary hepatocytes in vitro and in vivo.

Efficient DNA delivery is a prerequisite for the successful implementation of molecular antiviral strategies against chronic viral hepatitis and gene therapy in general. The cationic polymer polyethylenimine (PEI) has recently been explored as a gene transfer vector in various cell types in vitro and in vivo. In this study, we evaluated a linear PEI derivative (lPEI) as a vector for gene and oligodeoxynucleotide transfer into hepatocytes in vitro and in vivo. A simple protocol was developed that allowed transfection of up to 50% of primary hepatocytes in vitro. In addition, fluorescent oligodeoxynucleotides were efficiently delivered to the liver in vivo after intravenous injection into Pekin ducks. Thus, lPEI mediates highly efficient gene and oligodeoxynucleotide transfer into primary hepatocytes and is potentially useful for DNA delivery in vivo.

Differential induction of carcinogen metabolizing enzymes in a transgenic mouse model of fulminant hepatitis.

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S-transferases [GST]) in relation to hepatitis B virus (HBV)-associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg-specific, major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post-CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as in vitro metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST alpha). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, alpha) showed that while GST mu was not changed in the CTL-injected HBV transgenic mice, GST pi and, to a lesser extent, alpha were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant-like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse.

Induction of specific cytochrome P450s involved in aflatoxin B1 metabolism in hepatitis B virus transgenic mice.

The relative roles of hepatitis B virus (HBV) and aflatoxin and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen-metabolizing enzymes. We tested this hypothesis in an HBV-transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278-1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a-5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an alpha-class glutathione S-transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a-5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV-transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV-transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver-damaging agents.