Steatosis: Co-factor in other liver diseases

The prevalence of fatty liver is rising in association with the global increase in obesity and type 2 diabetes. In the past, simple steatosis was regarded as benign, but the presence of another liver disease may provide a synergistic combination of steatosis, cellular adaptation, and oxidative damage that aggravates liver injury. In this review, a major focus is on the role of steatosis as a co-factor in chronic hepatitis C (HCV), where the mechanisms promoting fibrosis and the effect of weight reduction in minimizing liver injury have been most widely studied. Steatosis, obesity, and associated metabolic factors may also modulate the response to alcohol- and drug-induced liver disease and may be risk factors for the development of hepatocellular cancer. The pathogenesis of injury in obesity-related fatty liver disease involves a number of pathways, which are currently under investigation. Enhanced oxidative stress, increased susceptibility to apoptosis, and a dysregulated response to cellular injury have been implicated, and other components of the metabolic syndrome such as hyperinsulinernia and hyperglycemia are likely to have a role. Fibrosis also may be increased as a by-product of altered hepatocyte regeneration and activation of bipotential hepatic progenitor cells. In conclusion, active management of obesity and a reduction in steatosis may improve liver injury and decrease the progression of fibrosis.

Caveolin, cholesterol, and lipid bodies

In mammalian cells a complex interplay regulates the distribution of cholesterol between intracellular membrane compartments. One important aspect of cholesterol regulation is intracellular cholesterol storage in neutral lipid storage organelles called lipid droplets or lipid bodies (LBs). Recent work has thrust the LB into the limelight as a complex and dynamic cellular organelle. LBs play a crucial role in maintaining the cellular levels of cholesterol by regulating the interplay between lipid storage, hydrolysis and traffickin,-. Studies of caveolins, caveolar membrane proteins linked to lipid regulation, are providing new insights into the role of LBs in regulating cholesterol balance. (c) 2005 Elsevier Ltd. All rights reserved.

Alcohol use and risk taking among regular ecstasy users

We examine alcohol use in conjunction with ecstasy use and risk-taking behaviors among regular ecstasy users in every capital city in Australia. Data on drug use and risks were collected in 2004 from a national sample of 852 regular ecstasy users (persons who had used ecstasy at least monthly in the preceding 6 months). Users were grouped according to their typical alcohol use when using ecstasy: no use, consumption of between one and five standard drinks, and consumption of more than five drinks (binge alcohol use). The sample was young, well educated, and mainly working or studying. Approximately two thirds (65%) of the regular ecstasy users reported drinking alcohol when taking ecstasy. Of these, 69% reported usually consuming more than five standard drinks. Those who did not drink alcohol were more disadvantaged, with greater levels of unemployment, less education, higher rates of drug user treatment, and prison history. They were also more likely than those who drank alcohol when using ecstasy to be drug injectors and to be hepatitis C positive. Excluding alcohol, drug use patterns were similar between groups, although the no alcohol group used cannabis and methamphetamine more frequently. Binge drinkers were more likely to report having had three or more sexual partners in the past 6 months and were less likely to report having safe sex with casual partners while under the influence of drugs. Despite some evidence that the no alcohol group were more entrenched drug users, those who typically drank alcohol when taking ecstasy were as likely to report risks and problems associated with their drug use. It appears that regular ecstasy users who binge drink are placing themselves at increased sexual risk when under the influence of drugs. Safe sex messages should address the sexual risk associated with substance use and should be tailored to reducing alcohol consumption, particularly targeting heavy alcohol users. The study's limitations are noted.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.

Delipidation of a hepadnavirus: Viral inactivation and vaccine development

Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the “correct” functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p < 0.001). Delipidated DHBV was then used to vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p < 0.05). Although this solvent system completely inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.

Prevalence of injecting drug use and associated risk behavior among regular ecstasy users in Australia

Background: The aim of the study was to investigate the prevalence of injecting drug use and associated risk behaviour among a sentinel sample of ecstasy users. Methods: Cross-sectional surveys were conducted with regular ecstasy users as part of an annual monitoring study of ecstasy and related drug markets in all Australian capital cities. Results: Twenty-three percent of the sample reported having ever injected a drug and 15% reported injecting in the 6 months preceding interview. Independent predictors of lifetime injection were older age, unemployment and having ever been in prison. Completion of secondary school and identifying as heterosexual was associated with a lower likelihood of having ever injected. Participants who had recently injected typically did so infrequently; only 9% reported daily injecting. Methamphetamine was the most commonly injected drug. Prevalence of needle sharing was low (6%), although half (47%) reported sharing other injecting equipment in the preceding 6 months. Conclusions: Ecstasy users who report having injected a drug at some time appear to be demographically different to ecstasy users who have not injected although neither are they typical of other drug injectors. The current investigation suggests that ongoing monitoring of injecting among regular ecstasy users is warranted. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Transcriptome analysis of a cnidarian-dinoflagellate mutualism reveals complex modulation of host gene expression

Background: Cnidarian - dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian - algal symbiosis. Results: We detected statistically significant differences in host gene expression profiles between sea anemones ( Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress. Conclusion: Our data do not support the existence of symbiosis- specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian - dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Using cohort studies to estimate mortality among injecting drug users that is not attributable to AIDS

Background: Injecting drug use (IDU) and associated mortality appear to be increasing in many parts of the world. IDU is an important factor in HIV transmission. In estimating AIDS mortality attributable to IDU, it is important to take account of premature mortality rates from other causes to ensure that AIDS related mortality among injecting drug users (IDUs) is not overestimated. The current review provides estimates of the excess non-AIDS mortality among IDUs. Method: Searches were conducted with Medline, PsycINFO, and the Web of Science. The authors also searched reference lists of identified papers and an earlier literature review by English et al (1995). Crude. mortality rates (CMRs) were derived from data on the number of deaths, period of follow UP, and number of participants. In estimating the all-cause mortality, two rates were calculated: one that included all cohort studies identified in the search, and one that only included studies that reported on AIDS deaths in their cohort. This provided lower and upper mortality rates, respectively. Results: The current paper derived weighted mortality rates based upon cohort studies that included 179 885 participants, 1 219 422 person-years of observation, and 16 593 deaths. The weighted crude AIDS mortality rate from studies that reported AIDS deaths was approximately 0.78% per annum. The median estimated non-AIDS mortality rate was 1.08% per annum. Conclusions: Illicit drug users have a greatly increased risk of premature death and mortality due to AIDS forms a significant part of that increased risk; it is, however, only part of that risk. Future work needs to examine mortality rates among IDUs in developing countries, and collect data on the relation between HIV and increased mortality due to all causes among this group.

Prevalence of HBsAg mutants and impact of hepatitis B infant immunisation in four Pacific Island countries

The prevalence rate of hepatitis B virus (HBV) infection in Pacific Island countries is amongst the highest in the world. Hepatitis B immunisation has been incorporated into national programmes at various times, often with erratic supply and coverage, until a regionally co-ordinated programme, which commenced in 1995 ensured adequate supply. The effectiveness of these programmes was recently evaluated in four countries, Vanuatu and Fiji in Melanesia, Tonga in Polynesia and Kiribati in Micronesia. That evaluation established that the programmes had a substantial beneficial impact in preventing chronic hepatitis B infection [Vaccine 18 (2000) 3059]. Several studies of hepatitis B vaccination programmes in endemic countries have identified the potential significance of surface gene mutants as a cause for failure of immunisation. In the study outlined in this paper, we screened infected children and their mothers for the emergence and prevalence of these variants in specimens collected from the four country evaluation. Although the opportunity for the emergence of HBV vaccine escape mutants in these populations was high due to the presence of a considerable amount of the virus in the population and the selection pressure from vaccine use, there were no a determinant vaccine escape mutants found. This suggests that vaccine escape variants are not an important cause for failure to prevent HBV transmission in this setting. Other HBsAg variants were detected, but their functional significance remains to be determined. The failure to provide satisfactory protection during such immunisation programmes reflects the need for achieving and sustaining high vaccine coverage, improving the timeliness of doses as well as improving 'cold-chain' support, rather than the selection of vaccine-escape mutants of HBV. (C) 2004 Elsevier Ltd. All rights reserved.

Chronic hepatitis B: A critical appraisal of current approaches to therapy

Background & Aims: Treatment of chronic hepatitis B (CHB) involves a number of complex and controversial issues. Expert opinions may differ from those of practicing hepatologists and gastroenterologists. We aimed to explore this issue further after a critical review of the literature. Methods: A panel of 14 international experts graded the strength of evidence for 16 statements addressing 3 content areas: patient selection, therapeutic end points, and treatment options. Available data relating to the statements were reviewed critically in 3 small work groups. After discussion of each statement with the entire panel, the experts voted anonymously to accept or reject statements based on the strength of evidence and their experience. A total of 241 members of the American Association for the Study of Liver Diseases (AASLD) responded to the same statements and their responses were compared with those of the experts. A discordant response was defined as a difference of more than 20% in any of the 5 graded levels of response (accept or reject) between the 2 groups. Results: With the exception of 2 statements, the experts’ responses were relatively uniform. However, the responses of the AASLD members were discordant from the experts in 12 statements, spanning all 3 content areas. Conclusions: Several areas of disagreement on the management of CHB exist between experts and AASLD members. Our results indicate a potential knowledge gap among practicing hepatologists. Better educational efforts are needed to meet the challenge of managing this complex disorder in which even expert opinion occasionally may disagree.

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

Aim: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Methods: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. Results: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/ mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/ mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples, Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Conclusion: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA. © 2006 The WJG Press. All rights reserved.